POINT OF CARE (POC) DEVICE FOR FACILITATING NUCLEIC ACID BASED TESTING AND METHOD THEREOF

Abstract

A generic point of care based portable device and method thereof as a platform technology for detecting pathogenic infection via nucleic acid based testing achieving sample-to-result integration, comprising the following interconnected stand-alone modules: a thermal unit for executing piece-wise isothermal reactions in a pre-programmable concomitant fashion without necessitating in-between operative intervention; a colorimetric detection unit seamlessly interfaced with smartphone-app based analytics for detecting the target analyte. The said platform technology is thus capable of detecting targeted pathogen-associated RNA by coupling additional complementary DNA probe hybridization combined with isothermal reaction purposed for reverse transcription of RNA followed by amplification of the resulting c-DNA as well as subsequent specific binding of the same in a single user-step in a concomitant fashion and its smartphone-enabled interpretation, in a generic modular format that renders operative suitability outside controlled laboratory environment in a user-friendly manner, with predictive accuracy favorably comparable with gold standard RT-PCR tests.

Claims

1. A microfluidic paper substrate (7) based colorimetric detector suitable for POC-based device for the detection of pathogenic infection via nucleic acid based testing comprising selectively functionalized microfluidic paper strip for sequential executing surface plasmon resonating nanomaterial conjugated complementary analyte binding/hybridization reaction enabling ready on site colorimetric detection of analyte following pre-set programmed interpretation protocol.

2. The microfluidic paper substrate based colorimetric detector as claimed in claim 1 comprising sequentially: (a) a sample introducing chamber integrated with a channel body of nitrocellulose filter paper; (b) binding section immobilized with colloidal nanomaterials conjugated with primary target analytes in channel made of glass fibre material; (c) detection area on a membrane base including a reaction area adapted for immobilization of specific reaction analytes including a control line for secondary binding; and (d) waste absorbing section comprising of blotting paper/absorbing material with high absorption quality.

3. The microfluidic paper substrate based colorimetric detector as claimed in claim 1 comprising: sample introducing chamber obtained of nitrocellulose filter paper or porous glass fiber; binding section obtained of glass fiber immobilized with colloidal surface plasmonic nanomaterials including preferably gold nano materials conjugated with primary antibodies; detection section including microfluidic paper platform including microcellulosic nitrocellulose membrane having corresponding specific antibody; and waste absorption section.

4. The microfluidic paper substrate based colorimetric detector (7) as claimed in claim 1 comprising: microfluidic paper substrate comprising: sample introducing chamber (a) having selectively a circular, polygonal, rectangular section or any other shapes integrated with a channel body of rectangular section made of nitrocellulose filter paper or porous glass fiber; binding section (b) immobilized with colloidal nanomaterials conjugated with primary target analytes are rectangular shaped channel made of glass fiber material; detection area (c) on a membrane base including a reaction area adapted for immobilization of specific reaction analytes including a control line for secondary binding; and waste absorbing section (d) with rectangular shaped channel comprising of blotting paper/absorbing material with high absorption quality.

5. The microfluidic paper substrate based colorimetric detector (7) as claimed in claim 1 adapted for specific detection of SARS-CoV-2 viral RNA comprising: said binding section of said the colloidal gold conjugate Anti-FAM antibody that binds with the targeted DNA, and the amplified dual labelled DNA complex further migrates downwards to the said detection section for reaction where streptavidin and anti-FAM secondary antibodies are immobilized at the test line and control lines of the strip, respectively while flowing from the sample pad, the amplified DNA complex reaches the test line and binds with the streptavidin for producing the color by the concentration of colloidal nanoparticles/nanoshells attached to the same, the free nanoparticles/nanoshells antibody conjugates bypass the test line and reach the control line to bind with the immobilized anti-FAM secondary antibodies and produce the color of colloidal nanoparticles/nanoshells.

6. Primer suitable for the desired SARS-CoV-2 detection with enhanced specificity and sensitivity including anyone or more of the selective sequences as hereunder: TABLE-US-00007 Target Primer Region Primer Sequence (5′ to 3′) Modification ORF 1b F3 GCCATTAGTGCAAAGAATAGAGC SEQ.ID No. 1 B3 GGCATGGCTCTATCACATTTAGG SEQ.ID No. 2 FIP TAGCTCCTCTAGTGGCGGCTATTGCACCG 5′-[Btn] (F1c + F2) TAGCTGGTGTCTC SEQ.ID No. 3 BIP TGTAGTAATTGGAACAAGCAAATTCTAT (B1c + B2) GGTGGCCAACCCATAAGGTGAGGG SEQ.ID No. 4 Loop F TTTTTGATGAAACTGTCTATTGGTCATAG SEQ.ID TACTACAG No. 5 Loop B GGCACAACATGTTAAAAACTGTTTATAGT SEQ.ID GATGTAG No. 6 BLP Probe TTGGCACAACATGTTAAAAACTGTTTATA 5′-[6FAM] SEQ.ID GTGATG No. 7 BLP 3′- TTGGCACAACATGTTAAAAACTGTTTATA 5′-[6FAM], mod Probe GTGATG 3′-[3d_G] SEQ.ID No. 8 FLP Probe GCGGCTATTGATTTCAATAATTTTTGATG 5′-[6FAM] SEQ.ID AAAC No. 9 N gene F3 ACAATGTAACACAAGCTTTCG SEQ.ID No. 10 B3 TTGGATCTTTGTCATCCAATT SEQ.ID No. 11 FIP GGCCAATGTTTGTAATCAGTTCCTTAGAC 5′-[Btn] (F1c + F2) GTGGTCCAGAACAA SEQ.ID No. 12 BIP GCTTCAGCGTTCTTCGGAATCACCTGTGT (B1c + B2) AGGTCAACC SEQ.ID No. 13 Loop F TGGTCCCCAAAATTTCCTTGG SEQ.ID No. 14 Loop B CGCGCATTGGCATGGAAGT SEQ.ID No. 15 BLP Probe TTGGCATGGAAGTCACACCTTC 5′-[6FAM] SEQ.ID No. 16 BLP 3′- TTGGCATGGAAGTCACACCTTC 5′-[6FAM], mod Probe 3′-[3d_C] SEQ.ID No. 17 FLP Probe GATTAGTTCCTGGTCCCCAAAATTTCC 5′-[6FAM] SEQ.ID No. 18 E gene F3 TTGTAAGCACAAGCTGATG SEQ.ID No. 19 B3 AGAGTAAACGTAAAAAGAAGGTT SEQ.ID No. 20 FIP CGAAAGCAAGAAAAAGAAGTACGCTAGT 5′-[Btn] (F1c + F2) ACGAACTTATGTACTCATTCG SEQ.ID No. 21 BIP GGTATTCTTGCTAGTTACACTAGCCAAGA (B1c + B2) CTCACGTTAACAATATTGC SEQ.ID No. 22 Loop F ATTAACGTACCTGTCTCTTCCGAAA SEQ.ID No. 23 Loop B ATCCTTACTGCGCTTCGATTGTGTG SEQ.ID No. 24 BLP Probe ATCCTTACTGCGCTTCGATTGTGTG 5′-[6FAM] SEQ.ID No. 25 BLP 3′- ATCCTTACTGCGCTTCGATTGTGTG 5′-[6FAM], mod Probe 3′-[3d_G] SEQ.ID No. 26 FLP Probe ATTAACTATTAACGTACCTGTCTCTTCC 5′-[6FAM] SEQ.ID No. 27 RNaseP F3 TTGATGAGCTGGAGCCA SEQ.ID No. 28 B3 CACCCTCAATGCAGAGTC SEQ.ID No. 29 FIP GTGTGACCCTGAAGACTCGGTTTTAGCCA 5′-[Btn] (F1c + F2) CTGACTCGGATC SEQ.ID No. 30 BIP CCTCCGTGATATGGCTCTTCGTTTTTTTCT (B1c + B2) TACATGGCTCTGGTC SEQ.ID No. 31 Loop F ATGTGGATGGCTGAGTTGTT SEQ.ID No. 32 Loop B CATGCTGAGTACTGGACCTC SEQ.ID No. 33 BLP Probe CATGCTGAGTACTGGACCTCG 5′-[6FAM] SEQ.ID No. 34 FLP Probe ATGTGGATGGCTGAGTTGTT 5′-[6FAM] SEQ.ID No. 35

Description

BRIEF DESCRIPTION OF THE NON-LIMITING EXEMPLARY ACCOMPANYING FIGURE

[0091] FIG. 1 illustrates schematic drawing of the portable POC device for colorimetric detection of pathogen associated nucleic acids. Each part of the device has been marked and explained in the following figures.

[0092] FIG. 2 illustrates schematic drawing of the part (1) of the device marked in FIG. 1. This is the base cover of the device for accommodating all the components for the isothermal heating unit.

[0093] FIG. 3 illustrates functional block diagram of the isothermal heating unit comprising a heating block, micro-heater, temperature sensor, heatsink-fan assembly, microcontroller and optocoupler-relay unit.

[0094] FIG. 4 illustrates the design drawing of the middle part of the device that includes guided rail arrangement for insertion of microchamber and paper-strip. This part also accommodates the fluid dispensing unit, heating block.

[0095] FIG. 5 illustrates an embodiment of the part (2) marked in FIG. 1 with heating block and heat sink holding arrangement.

[0096] FIG. 6 illustrates an embodiment of the part (3) marked in FIG. 1 i.e. the cartridge for holding the microchamber and insert into the device through the guided rail assembly.

[0097] FIG. 7 illustrates an embodiment of the part (4) marked in FIG. 1 i.e. the microchamber for holding and isothermal amplification of DNA and RT-LAMP mixture.

[0098] FIG. 8 illustrates an embodiment of the part (6) marked in FIG. 1 i.e. the cartridge for holding the paper-strips and insert into the device through the guided rail assembly.

[0099] FIG. 9 illustrates an embodiment of the part (7) marked in FIG. 1 i.e. the paper-strip for colorimetric detection of the targeted DNA. As further illustrated in said FIG. 9, Part (a) of the strip (7) is the sample introducing section; part (b) is the conjugation section functionalized with surface plasmon nanomaterials; part (c) is the detection section of the strip made of cellulose membrane functionalized with streptavidin and anti-FAM antibodies; part (d) is the absorbent pad.

[0100] FIG. 10 illustrates an embodiment of the part(8) as marked in FIG. 1 i.e. the guided rail arrangement for insertion of the microchamber and the paper-strip in their desired location into the device for seamless dispensing of the amplified DNA onto the paper-strip without manual intervention.

[0101] FIG. 11 illustrates the functional block diagram/flow chart of the smartphone-based android app for image acquisition, analysis and result display.

[0102] FIG. 12 illustrates the functional block diagram of the generic portable point-of-care device.

[0103] FIG. 13 illustrates the overview of the generic testing methodology in accordance with the present advancement, abbreviated as piecewise isothermal nucleic acid testing (PINAT) for pathogenic infection detection in the integrated device. Followed by collection, the patient body fluid sample may be subjected to RNA extraction or alternatively to a brief initial heat treatment. The purified RNA or the heat-treated sample can be subjected to the subsequent detection protocol. The input RNA is mixed with a reaction master mix containing appropriate enzyme, labelled primers and DNA probes and dispensed to modular reaction-chambers made of soft polymer tubing for subsequent isothermal amplification process. The right inset shows the caps of the reaction-chamber for air-tight sealing. Piecewise isothermal heating of the reaction-mix occurs in the portable device unit as a single user-step process without any intermediate manual intervention. Customization of this thermal protocol for any given test is pre-programmable by using a mathematical step-function: T=f(t). Dispensing of the amplified and labelled cDNA product from reaction chamber to the LFA strip occurs via a manually operated needle valve, with potential automation at the expense of a higher cost. Colorimetric detection of the labelled c-DNA is made on the LFA strip. A smartphone camera is used for image capturing of the reaction pad of the LFA. Smartphone app-based image analysis and result display are rendered onto the mobile screen. Controlled dissemination of the test results to clinical information systems and data analytics may be accommodated via cloud integration.

[0104] FIG. 14 illustrates the step-wise operative integration of device components during its operation for detection of pathogen associated nucleic acid. Step-1: insertion of the microchamber assembly (4) filled with RNA and master mix onto the heating block (2). For the purpose, at first, the microchambers (transparent) are filled with RNA, master mix with probes, altogether, and sealed airtight, and thereafter, the thus sealed microchamber with its contents is placed onto the microchamber carrying cartridge (3). The microchamber carrying cartridge assembly is then inserted into the device through the desired guided rail (8) assembly (as shown under step-1) up to position I onto the heating block (2) for subjecting to required thermal reactions. Piece-wise isothermal processing of the reaction mix is then performed by the device at this position until all the thermal operations are completed. Step-2: Once the required thermal reaction of contents in said microchamber is completed, the microchamber (4) carrying cartridge (3) is inserted further to a second position (position-II) to enable its positioning in between the microfluidic paper substrate based colorimetric detector/paper strip (7) and fluid dispensing mechanism (5). The microfluidic paper substrate based colorimetric detector carrying cartridge (6) is inserted through another guided rail assembly (8) located on the opposite side of the microchamber carrying cartridge (3). The fluid dispensing mechanism (5(a) and 5(b)) includes a puncturing needle valve attached with a dropper holding LFA buffer. At first, the base of the dropper is pressed (shown by arrow in 5(a)) to penetrate the needle through the bottom wall of the micro chamber and puncture it. The product of thermal reactions contained in said microchamber is thus dispensed semi-automatically onto the microfluidic paper substrate based colorimetric detector/paper strip (7). Subsequently, the LFA buffer is also dispensed onto the microfluidic paper substrate based colorimetric detector/paper strip from the dropper (by pressing the side of the dropper as shown in 5(b) using arrow) attached with the dispensing mechanism. This dispensing mechanism can be made automatic using programmable instrumentation or using microfluidic valving system. Step-3: Color generation at the test line and control line. The complex of thermal reaction products and antibody-conjugate traverses through the microfluidic paper substrate based colorimetric detector/paper strip (7) and attached at the test and control line. This color generation takes place within 5-10 minutes of dispensing the products of the thermal reactions onto the LFA paper strip (part-7). Step-4: Smartphone (10) is placed onto the top window (9) of the device at the desired position and captures image of the paper strip with color generated at the test and control lines for ready analytics and dissemination of the test outcome.

[0105] FIG. 15 illustrates the optimization of the test for detecting different target genes in SARS-CoV-2 genomic RNA. (A) Schematic representation of SARS-CoV-2 genome architecture. Different open reading frames were represented with differentially colored boxes. Specific target regions are annotated with dark gray bars and designated as target 1 for RdRp/ORF1b, target 2 for N and target 3 for E genes respectively (B). The integrated reaction cum detection procedure for the testing of individual target regions of viral genomic RNA and RnaseP as internal control. Individual target RNAs were amplified through RT-LAMP reaction and analyzed through 2% agarose gel (top panel) for cross-verification. The amplified reactions were subjected to probing with 6-FMA labelled DNA probes (FLP and BLS) and analyzed through paper strip. Non-specific probing results in appearance of test line in negative control reactions are denoted with asterisks. (C) Cross reactivity of one DNA probe (BLP) towards other SARS-CoV-2 genes or human RNaseP gene. (D) Cross reactivity of individual set of primers and probes against the non-target RNA sequences. RT-LAMP primers designed for one target gene of SARS-CoV-2 showed no cross reactivity against other gene targets,

[0106] FIG. 16 illustrates the analysis of the conserved nature of the SARS CoV-2 genome. (A) Total 500 complete sequences were aligned with the reference sequence. Positions with gap in reference sequence were not considered. For each position, conservation was calculated by analyzing the positional numerical summery in Bioedit software. Conserved nature of each target regions are expanded in (B) RdRp/ORF1b, (C) N and (D) E.

[0107] FIG. 17 illustrates cross reactivity of SARS-CoV-2 primers against the genomic RNA of other viruses. (A) Cross reactivity of COVID-19 specific primers were tested using the present protocol against influenza A, influenza B and Japanese Encephalitis viral genomic RNAs isolated from virus particles. Reaction products were analyzed fragments in sextuplets, with LFA based detection. Respective SARS-CoV-2 target genes were used as positive control. (B) Cross reactivity of SARS-CoV-2 specific primers were tested against different RNA viruses in silico.

[0108] FIGS. 18A-18D illustrates that the present method shows high sensitivity and specificity for detecting SARS-CoV-2 infection in patient samples (validation tests conducted at ICMR-NICED, Kolkata, India as per strict regulatory guidelines). 18A represents the limit of detection of the method for individual gene targets. 10-fold serial dilutions of in vitro synthesized gene fragments were subjected to detection in sextuplets. Colored boxes represent positive detection while empty boxes represent negative results for individual replicates. 18B-18D represents patient sample validation results. 18B illustrates distribution of the individual samples across the range of Ct values from 25-35. Samples showing positive or negative for both of the gene targets are represented with dark grey and black circles respectively while positive for one and negative for the other gene target are represented with light grey circles. 18C represents box plots showing sensitivity of the detection method for patient samples grouped based upon the Ct values. 18D illustrates specificity of the method.

[0109] FIGS. 19A-19C represents sensitivity of the test using SARS-CoV-2 specific primers and probes. FIG. 19A represents RdRp/ORF1b specific primers; FIG. 19B represents N gene specific primers; FIG. 19C represents E gene specific primers.

[0110] The test protocols were performed with ten-fold serial dilutions of the gene fragments in sextuplets. RT-LAMP reaction products were analyzed by agarose gel electrophoresis followed by DNA probe hybridization and LFA based detection.

[0111] FIG. 20 illustrates kit packaging (upper) and transportation pathway to the test center (lower). The reaction master mixes corresponding to N and RdRp/ORF1b gene targets were reconstituted, tested for quality control and aliquoted in reaction tubes. The reaction master mixes were then transported in ice packs from Indian Institute of Technology Kharagpur to ICMR-NICED where the test with patient samples were conducted using the test method. In peak traffic hours, the nearly 150 km of road travel used to take around 4.5 hours.

[0112] FIG. 21 illustrates method applied for the detection of influenza virus RNA extracted from viruses amplified in MDCK cells. RT-LMAP products were analyzed through agarose gel electrophoresis followed by hybridization with backward or forward loop probed (BLP, FLP) and detected on lateral flow assay strip.

[0113] FIG. 22 illustrates a single step solution for detection of RNA virus infection. (A) Detection of influenza A virus genomic RNA directly from infected cells in the presence of human saliva. Human A549 cells infected with influenza A virus, or mock infected, were resuspended in PBS or PBS spiked with human saliva. The suspension was subjected to the detection procedure without prior RNA extraction procedure, but heating at a temperature of 93-98° C. preferably about 95° C. for 2-5 minutes preferably about 3 minutes. (B) Defined numbers of influenza virion particles (PFU), amplified from MDCK cells, were subjected to detection procedure (as mentioned in A) in sextuplets either in presence or absence of human saliva. (C) 10-fold serial dilutions of in vitro synthesized N gene fragment were subjected to detection process in sextuplets either in presence or absence of human saliva. (D) Simultaneous amplification and probing for detection of viral RNA in a single step. RdRp/ORF1b Gene fragment was subjected either to RT-LAMP followed by probing in a sequential manner or simultaneous RT-LAMP and probing reaction in a single step process. In both the cases, BLPs labelled with 5′-FAM and with and without 3′-ddNTP modification were used. Products, at the end of the reaction, were subjected to agarose gel electrophoresis in absence of EtBr and imaged under Cy5 channel.

[0114] FIG. 23 illustrates (A) RT-LAMP reactions were performed with in vitro transcribed SARS-CoV-2 N gene target region and corresponding primers. Products were subjected to hybridization with BLPs labelled with 5′ FAM and with or without 3′ ddNTP modification. (B) For single step amplification-cum-probing reactions, both kinds of modified BLPs were added at the beginning of RT-LAMP reaction followed by amplification, head denaturation and hybridization steps carried out without any interruptions. Products were analyzed through agarose gel electrophoresis in absence of EtBr and imaged under Cy5 channel.

[0115] FIG. 24 illustrates limit of detection of the test for detecting influenza A virus RNA directly from virus particles in presence and absence of human saliva. Known PFU of influenza virus particles were diluted in PBS or PBS spiked with human saliva. Subsequently, the sample was directly used for the test (including prior heating at a temperature of 93-98° C. preferably about 95° C. for 2-5 minutes preferably about 3 minutes) without RNA purification.

[0116] FIG. 25 illustrates Sensitivity of the test for detecting SARS-CoV-2 N-gene specific RNA from human saliva sample. 10-fold serial dilutions of the in-vitro transcribed SARS-CoV-2 N-gene RNA were prepared in PBS or PBS spiked with human saliva which were then subjected to the protocol.

[0117] FIG. 26(A) illustrates RT-LAMP reactions were performed with in vitro transcribed SARS-CoV-2 N-gene target region and the corresponding primers. Products were subjected to hybridization with BLPs labelled with 5′ FAM and with or without 3′ ddNTP modification. (B) For the integrated amplification-cum-probing step-based protocol, both kinds of modified BLPs were added at the beginning of RT-LAMP reaction followed by amplification, heat denaturation and hybridization steps carried out without any interruptions. Products were analyzed through agarose gel electrophoresis in absence of EtBr and imaged under fluorescein channel.

[0118] FIG. 27 illustrates RT-LAMP reactions were performed with in vitro transcribed SARS-CoV-2 N-gene target region and the corresponding primers. Products were subjected to hybridization with BLPs labelled with 5′ FAM and with or without 3′ ddNTP modification (left panel). For the integrated amplification-cum-probing step-based protocol, both kinds of modified BLPs were added at the beginning of RT-LAMP reaction followed by amplification, heat denaturation and hybridization steps carried out without any interruptions. Products were analyzed through LFA.

DESCRIPTION OF THE INVENTION

[0119] The present invention as stated herein before thus provides a diagnostic POC device for the detection of pathogen associated nucleic acid, producing test results compatible with acceptable gold standards. The said device and method thereof perform the nucleic acid based detection commensurate with the accuracies of the resource-intensive gold standard RT-PCR method and at the same time is cost effective, robust, user friendly. Such portable device can be installed and functionalized at locations with minimal laboratory resources or can be potentially extrapolated as a POC test in a decentralized manner. Additionally, the generic nature of the portable device unit makes it compatible of performing any similar test methods that combines isothermal reaction and/or lateral flow assay-based detection of the labelled/probed molecules.

[0120] The said simple POC device performs three major functionalities sequentially to execute the entire diagnosis process seamlessly with minimal manual intervention. The first major functionality of the device is a reverse transcription concomitantly coupled with the amplification of a tiny amount of RNA to billions of copies of cDNA for ease of detection and identification of the presence of pathogen in the sample. The said amplification process in RT-PCR machine is very complex requires and 40-50 cycles with three-step heating for fast change of temperature at 94-96° C. for denaturation (15-30 sec), at 50-60° C. for annealing (15-30 sec) and 68-72° C. for elongation (>2.5 minutes) per cycle. On the other hand, the amplification process in the device relies upon piecewise isothermal heating steps (typical temperature values range from 62° C. to 98° C.) over pre-defined temporal regimes spanning over minutes. The heating system comprises a heating block, a programmable temperature control unit and a microchamber. This, essentially, is an ultra-low-cost replacement of the expensive Peltier based thermocycling unit of the RT-PCR machine towards achieving equivalent functionalities in terms of DNA amplification.

[0121] The second functionality is the detection of the c-DNA after amplification. In the RT-PCR machine, the detection process is very complex optical-based real-time monitoring of the reaction progress for estimation of the amount of DNA by interpreting the increment of the fluorescence signal in each cycle during c-DNA amplification. The detection system includes photodiode, CCD camera with cooling system, LED light for fluorescent excitation, excitation filter, emission filter and fluorescent detector etc. On the other hand, the portable device has colorimetric detection unit for detection of the amplified and labelled c-DNA in a sample using microfluidic paper strip selectively functionalized with surface plasmon resonating nano-materials and antibodies. This is one-step colorimetric analysis and detection for specific pathogen from the final amplified product. The simplicity in the detection protocol is highly advantageous for implementation of the POC device.

[0122] The third functionality is data analysis and result display. RT-PCR machine has in-built software associated with the instrumentation for real-time data acquisition and data analysis to estimate the initial load of the pathogen and amount of amplified c-DNA in each cycle. A skilled technician is required to interpret the result for confirming the presence of pathogen and estimate the severity of the infection. On the other hand, the present portable device is integrated with a smartphone means for performing image analysis and dissemination of test results. The colorimetric reaction is captured by the smartphone camera for image analysis and interprets the data using rule-based analysis by the custom-made android app and display the decision of the presence or absence of the targeted pathogen.

[0123] In a further aspect, the present invention provides device achieving DNA amplification involving a single DNA polymerase Bst3 along with multiple sets of primers.

[0124] In a further aspect, the present invention provides a method and device to achieve a single user-step amalgamation of reverse transcription, DNA amplification and specific DNA probing in a thermal unit that is operatively connected to carry out customized piecewise isothermal close-tube reactions, particularly adapted to arrest any undesired amplification of the specific DNA probe added, thereby obviating the need of any intermediate user intervention, and yet enabling the amplification and specific probing reaction to take place harmonically in the desired sequence.

[0125] Another aspect of the present invention provides a specialized app with a machine learning algorithm based on pathogen-specific training image data sets for analyzing exclusive properties mapped to the upstream experimental significance and eventually the decision making based on the analysis offers with unique features of the integrated device.

[0126] Another unique aspect of the present invention is that it comprises a pre-programmable thermal control unit capable of inducing designed temperature-time characteristics. This module is not specific to a particular thermal protocol for detecting a specific pathogenic DNA/RNA, but generic enough to be adopted to test-specific thermal protocols for detection of other microorganisms. Further, low-cost materials such as polydimethylsiloxane (PDMS), paper and Pyrex or similar materials have been tested to be adequate for constituting the thermally activated reactive micro-chamber. Integration of a decisive key step of genomic probe-based detection on a specially functionalized paper strip adds to improved accuracy of the test results. No prior art methodologies have disclosed any PDMS/paper/Pyrex-based microchamber in a portable device for isothermal amplification of the targeted RNA mixed reaction analytes to achieve the amplified c-DNAs. Seamless integration of the microchamber unit with the paper strip ensures spontaneous dispensing of the sample on to the assay strip, without necessitating manual intervention.

[0127] The present invention has introduced, for the first time, a piecewise isothermal amplification reaction seamlessly coupled with a complementary DNA probe-based detection as a combined single-user step procedure implementable in a generic pre-programmable customized device for highly accurate and unambiguous test results. This test relies upon RT-LAMP mediated amplification and subsequent detection of the amplified products by specific hybridization with a complementary DNA oligonucleotide. The method consists of three distinct and extremely generic conceptual reaction steps from RNA analysis to test result dissemination.

[0128] First, RT-LAMP reaction in presence of biotinylated forward inner primer (FIP-5′Bt) results in generation of 5′ biotinylated RT-LAMP products. Second, a 6-fluorescein amidite (6-FAM) labelled DNA oligonucleotide (probe), complementary to the loop regions of the RT-LAMP products, is hybridized through consecutive heat denaturation and annealing process thereby generating dual labelled (Biotin+FAM) products. Third, the dual labelled products and the single labelled free probes get separated on a lateral flow assay strip and captured by the streptavidin and anti-FAM antibody immobilized on the test line and control lines, respectively. The second step acts as a key step towards improving the test specificity via a route that, in contrast to the RNA-mediated CRISPR-Cas based detection, solely relies on DNA probe hybridization, having obvious benefits of inherent stability even outside the ambit of controlled laboratory ambience.

[0129] Cas is an endonuclease enzyme that cuts the DNA at a specific location directed by a guide RNA. This is a target-specific technique that can introduce gene knock out or knock in depending on the double strand repair pathway. Engineered CRISPR systems contain two components: a guide RNA (gRNA or sgRNA) and a CRISPR-associated endonuclease (Cas protein). The gRNA is a short synthetic RNA composed of a scaffold sequence necessary for Cas-binding and a user-defined—20 nucleotide spacer that defines the genomic target to be modified.

[0130] The present method overcomes the constraints of the CRISPR-Cas based detection as attributed to the low stability and hence shorter shelf life of sgRNA, which act as serious bottlenecks against implementation without stringent laboratory-based control. Further, sgRNAs are well known for their off-target effects, which may result in false-positive or negative results. Reagents with such reported artifacts are not used in the present test.

[0131] Another unique aspect of the invention is to provide a one-step thermal protocol that seamlessly integrates the RT-LAMP mediated amplification of the pathogen associates nucleic acid and hybridization of the 6-FAM labelled DNA probe together without requiring any user intervention in between.

[0132] Unique coupling of the reaction chamber and the detection unit in the portable platform avoids manual intervention for dispensing the amplified sample onto the sample pad of the colorimetric detection strip. This is ensured by designing a seamless fluidic pathway using a microfluidic dispensing technique. This eliminates manual pipetting of the sample onto the detection unit. A unique design of the sample introducing chamber ensures that the amplified samples can be introduced directly by pushing the microchamber forward to a position just above the sample pad for direct immobilization of the sample onto the paper strip.

[0133] Another unique aspect of this invention is a single step RNA isolation protocol that could be coupled with the one step amplification-detection reaction to present a simple “sample-to-result” solution for the detection of pathogenic infection with all the pertinent steps formatted in a customizable piece-wise isothermal testing format in a pre-programmable portable device unit.

[0134] A smartphone integrated analytical platform is coupled with the detection unit for implementing machine-learning enabled decision-making features premised on image analysis and algorithms. This not only results in a seamless dissemination of test results (positive, negative, or indecisive) but also ensures efficient data management in the cloud to act as a pointer to medical decision making for community-level interventions.

[0135] Real-time PCR based detection relies upon highly expensive reagents and consumables which increases the overall cost of the detection. The present invention provides a method relying upon a single DNA polymerase Bst3 along with multiple sets of primers.

[0136] Additionally, the detection is performed on functionalized paper based strips instead of complex instrumentation-based detection, which reduces the overall cost per experiment to a significant extent without sacrificing the accuracy of the test.

[0137] The existing RT-LAMP protocols, employed for detection of pathogen associated nucleic acids, relies either upon indirect pH based colorimetric or fluorescence-based detection techniques which makes the results ambiguous and difficult to interpret. The present invention, on the other hand, provides a device to detect the thermally amplified genomic products through specific hybridization with a complementary DNA probe that makes the detection precise and unambiguous.

[0138] A further aspect of the present invention provides a detailed method for SARS CoV-2 detection involving the portable POC device which is carried out comprising:

(i) providing biotin labelled primer sets, hybridization probes and other reagents in an airtight said reaction microchamber including samples (including either non-specific RNA or viral RNA/COVID-19 specific RNA).
(ii) switching on the said thermal control unit for heating the block from room temperature to the targeted temperature of 62-68° C., preferably about 65° C.;
(iii) once the desired temperature is reached, the microchambers hosting the reaction mixture are placed onto the isothermal heating block.
(iv) continuing said isothermal heating for the next 25 to 35 minutes preferably about 30 minutes whereby the RNA gets converted into cDNA and subsequently gets amplified into millions-billions of copies.
(v) after completion of the amplification cycle at said about 65° C., the device ramps up to reach the temperature of 93-98° C. preferably about 95° C. and termination cycle of 3-8 minutes preferably about 5 minutes at 95° C. continues, said heating cycles being performed automatically without requiring any manual intervention.
(vi) after the termination cycle is over, the heating system automatically ramps down to the temperature of 48-55° C. preferably about 50° C. which is required for the hybridization cycle and this cycle carried out for specific binding of the probe to the amplified target DNA.
(vii) after completion of the reaction procedure, the microchambers are pushed forward to the dispensing position using the cartridge and guided rail mechanism and keep the microchambers to cool down to the room temperature automatic/automatic manner by pushing the microchamber holder through a guided rail synchronized.
(viii) introducing the products from the above step onto the sample pad of the paper platform which is carried out in a semi-automatic manner with respect to the paper platform whereby the microchamber is placed in between the dispensing arrangement and the sample introducing chamber and releases the solution onto the sample pad using a needle valve.
(ix) allowing the sample flow through the paper matrices due to the capillary action by the force of surface tension and reach out to the conjugate section wherein the colloidal surface plasmon nanomaterial conjugated anti-FAM antibody binds with the targeted dual labelled amplified cDNA and the conjugate-DNA complex further flows downwards to the reaction area where streptavidin and anti-FAM secondary antibodies are immobilized at the test line and control lines of the strip, respectively.
(x) while flowing from the sample pad, the amplified conjugate-DNA complex reaches the test line and binds with the streptavidin for producing the color by the concentration of the colloidal nanomaterials attached to the same. The free nanomaterial-antibody conjugates bypass the test line and reach the control line to bind with the immobilized anti-FAM secondary antibodies and produce the color of the nanomaterial to enable the detection involving said imaging and dissipation means including a smartphone device.

[0139] Still further aspect of the present invention provides a method wherein the time duration for the exclusive step of colorimetric detection on the paper platform is within 10 minutes. The smartphone app is activated for capturing images of the reactions visible at the control line and test line of the paper strip, based on programmed camera properties, subsequently analyzing the same and displaying the final results onto the smartphone screen.

[0140] Yet another aspect of the present invention provides a method for COVID 19 detection, wherein said primer modifications were carried out involving sequences as hereunder:

TABLE-US-00003 Target Primer Region Primer Sequence (5′ to 3′) Modification ORF 1b F3 GCCATTAGTGCAAAGAATAGAGC SEQ.ID No. 1 B3 GGCATGGCTCTATCACATTTAGG SEQ.ID No. 2 FIP TAGCTCCTCTAGTGGCGGCTATTGCACCG 5′-[Btn] (F1c + F2) TAGCTGGTGTCTC SEQ.ID No. 3 BIP TGTAGTAATTGGAACAAGCAAATTCTAT (B1c + B2) GGTGGCCAACCCATAAGGTGAGGG SEQ.ID No. 4 Loop F TTTTTGATGAAACTGTCTATTGGTCATAG SEQ.ID TACTACAG No. 5 Loop B GGCACAACATGTTAAAAACTGTTTATAGT SEQ.ID GATGTAG No. 6 BLP Probe TTGGCACAACATGTTAAAAACTGTTTATA 5′-[6FAM] SEQ.ID GTGATG No. 7 BLP 3′- TTGGCACAACATGTTAAAAACTGTTTATA 5′-[6FAM], mod Probe GTGATG 3′-[3d_G] SEQ.ID No. 8 FLP Probe GCGGCTATTGATTTCAATAATTTTTGATG 5′-[6FAM] SEQ.ID AAAC No. 9 N gene F3 ACAATGTAACACAAGCTTTCG SEQ.ID No. 10 B3 TTGGATCTTTGTCATCCAATT SEQ.ID No. 11 FIP GGCCAATGTTTGTAATCAGTTCCTTAGAC 5′-[Btn] (F1c + F2) GTGGTCCAGAACAA SEQ.ID No. 12 BIP GCTTCAGCGTTCTTCGGAATCACCTGTGT (B1c + B2) AGGTCAACC SEQ.ID No. 13 Loop F TGGTCCCCAAAATTTCCTTGG SEQ.ID No. 14 Loop B CGCGCATTGGCATGGAAGT SEQ.ID No. 15 BLP Probe TTGGCATGGAAGTCACACCTTC 5′-[6FAM SEQ.ID No. 16 BLP 3′- TTGGCATGGAAGTCACACCTTC 5′-[6FAM], mod Probe 3′-[3d_C] SEQ.ID No. 17 FLP Probe GATTAGTTCCTGGTCCCCAAAATTTCC 5′-[6FAM] SEQ.ID No. 18 E gene F3 TTGTAAGCACAAGCTGATG SEQ.ID No. 19 B3 AGAGTAAACGTAAAAAGAAGGTT SEQ.ID No. 20 FIP CGAAAGCAAGAAAAAGAAGTACGCTAGT 5′-[Btn] (F1c + F2) ACGAACTTATGTACTCATTCG SEQ.ID No. 21 BIP GGTATTCTTGCTAGTTACACTAGCCAAGA (B1c + B2) CTCACGTTAACAATATTGC SEQ.ID No. 22 Loop F ATTAACGTACCTGTCTCTTCCGAAA SEQ.ID No. 23 Loop B ATCCTTACTGCGCTTCGATTGTGTG SEQ.ID No. 24 BLP Probe ATCCTTACTGCGCTTCGATTGTGTG 5′-[6FAM] SEQ.ID No. 25 BLP 3′- ATCCTTACTGCGCTTCGATTGTGTG 5′-[6FAM], mod Probe 3′-[3d_G] SEQ.ID No. 26 FLP Probe ATTAACTATTAACGTACCTGTCTCTTCC 5′-[6FAM] SEQ.ID No. 27 RNaseP F3 TTGATGAGCTGGAGCCA SEQ.ID No. 28 B3 CACCCTCAATGCAGAGTC SEQ.ID No. 29 FIP GTGTGACCCTGAAGACTCGGTTTTAGCCA 5′-[Btn] (F1c + F2) CTGACTCGGATC SEQ.ID No. 30 BIP CCTCCGTGATATGGCTCTTCGTTTTTTTCT (B1c + B2) TACATGGCTCTGGTC SEQ.ID No. 31 Loop F ATGTGGATGGCTGAGTTGTT SEQ.ID No. 32 Loop B CATGCTGAGTACTGGACCTC SEQ.ID No. 33 BLP Probe CATGCTGAGTACTGGACCTCG 5′-[6FAM] SEQ.ID No. 34 FLP Probe ATGTGGATGGCTGAGTTGTT 5′-[6FAM] SEQ.ID No. 35

[0141] Importantly, in keeping with a further aspect of the present advancement, the primer design of the test reagents is made exclusive to enhance the specificity and sensitivity in tune with DNA-probe-hybridization mediated specific probing step introduced under the present advancement as a seamless one-step procedure integrated with the piecewise-isothermal test format. As an essential measure to ensure that, all the outer primers are designed to be non-AT rich with the melting temperatures ranging in between 55° C. to 65° C. Lengths of the amplicons are kept within 200 base pairs and distance between the F2 to F1 and B2 to B1 are maintained within 40-60 base pairs. Additionally, during primer design, the potential dimer formation ability between the biotinylated FIP and FAM labelled probes is evaluated, which could otherwise lead to false positive signals in lateral flow assay. The cross reactivity between different sets of primers, DNA probes and their gene targets is also tested. All these criteria have been highly selective for the desired performance of the seamlessly coupled RT-LAMP reaction backbone with DNA probe hybridization and LFA based detection of the advancement, to enhance specificity and sensitivity of the test as compared to the naïve RT-LAMP format.

[0142] Following examples illustrate the present invention in further detail.

EXAMPLES

Example 1: Sample-to-Answer Portable POC Device for Detection of Viral RNA

[0143] i)Exemplary Illustration of the Integrated Portable Device:

[0144] The RT-PCR simplified portable device is specially designed for seamless integration of all the functionalities together to perform sequential events for execution of the entire detection process (FIG. 1). According to an exemplary embodiment, the outer cover of the device is fabricated using 1.1 mm thick mild steel sheet to ensure structural robustness without sacrificing economy of manufacturing. The top cover, paper-strip cartridge and microchamber holding cartridges are developed using additive manufacturing technology with a 3D printer (TECH B V30, India). This production is up scalable via injection moulding for rapid and inexpensive fabrication. The device included a simplified isothermal heating unit with pre-programmable temperature control for RT-LAMP based simultaneous reverse transcription followed by c-DNA amplification from the RNA mix solution into the microchamber. The aluminum heating block (2) with a cartridge heater and a temperature sensor is inserted into it and the block is seated on the heat-sink fan assembly fixed on a platform over the part (1). The thermal reaction block (heating unit), made of aluminium, was drilled with the numbers of slots same as the numbers of test reactions that may be run in parallel. This aspect of the design is completely flexible and hallmarked by unrestricted scalability, at the expense of altering the size of the device. The microchamber (4) was fabricated using transparent polymers. Materials like polydimethylsiloxane (PDMA), PMMA and Pyrex are considered for this purpose in different versions of the test instrument, with no perceptible difference in the outcome. The microchamber (4) for amplification of RNA was seated on a cartridge (3) and the assembly inserted through a guided rail for positioning the microchamber on the heating block (2). The microchamber (4) with amplified RNA mix product can then further be inserted through the guided rail assembly and precisely positioned in between the microfluidic dispenser and the paper strip. The fluid dispensing mechanism (5) may be implemented via either automated or manual arrangement for dispensing amplified c-DNA product and buffer solution onto the sample pad of the paper-strip (7). The paper-strip was encapsulated within a transparent cassette and fixed on another cartridge (6) which can be inserted through the guided rail assembly to a desired position for automated dispense of the products from microchamber. The guided rail assembly (8) was designed for seamless integration of the thermal reaction unit with the detection unit. The top cover (9) has a viewing window covered with a transparent Pyrex or glass or other non-brittle materials for monitoring the isothermal heating of the RNA mix samples. A smartphone can be put onto the transparent window within a marked area for image acquisition of the colorimetric change of the reaction strip on the paper platform.

[0145] ii) Exemplary Illustration of the Thermal Control Unit:

[0146] A thermal control unit was generically adapted and developed with capabilities of arbitrary multi-step isothermal processes that can be pre-programmed in a customized manner, and was thus established as a platform technology for implementing improvised RT-LAMP-based method. The complex thermal cycle used in RT-PCR for DNA amplification has been simplified in the said device with a pre-programmable piece-wise isothermal heating cycle. The functional block diagram of the said isothermal heating unit is represented in the FIG. 3 whereas FIG. 2 represents the lower part of the device for accommodating all the hardware of the heating unit. The hardware for thermal control according to the exemplary embodiment was obtained involving of an Arduino microcontroller, a 40 W heating cartridge, a DS18B20 one-wire temperature sensor, a fan with heat sink, optocoupler-relay (DC) unit, and a SMPS power unit. The rectangular or circular shaped aluminum heating block was machined and fixed with an arrangement in the middle part of the device (FIG. 4 and FIG. 5). A normally open relay was utilized for triggering alternately the heating of the block through the cartridge heater or cooling of the block through the fan-heat sink assembly. The heating block was attached to the fan-heat sink assembly with the help of thermal paste for effective thermal contact. Candle wax (locally sourced) was put in the tubule holder for ensuring good thermal contact. The Arduino program includes fetching the temperature data and executing one of the following three functions:

a) Raise the temperature to the targeted temperature—here the heater relay is actuated through an active low signal to an optocoupler. During this phase, the optocoupler to the cooling fan relay is set to high and remains off.
b) Hold the temperature to the desired value. This function consists of a temperature threshold so that the temperature is maintained within a specified+− range. Falling below T-dT actuates the heating relay while raising the temperature above T+dT actuates the cooling relay. The temperature oscillates between T+dT and T-dT; the oscillations are minimized due to the size of the thermal mass of the heating block. The total time of holding a particular desired temperature is also specified via pre-programming.
c) Lower the temperature—this function is similar to (a) but instead of actuating the heating circuit, the cooling circuit is actuated. The temperature is read every 3 seconds and redundancy temperature sensor checks are made to ensure no signal-scrambling through the one-wire interface.

[0147] The advantage of utilizing an Arduino microcontroller is that it allows for LCD display of real-time temperatures, easy integrability, temperature logging, as well as smartphone and web-based applications. The entire assembly is found to be extremely easy to operate and adaptable as per user requirements, obviating the need of specially-trained laboratory technicians.

[0148] iii) Exemplary Illustration of Microchamber:

[0149] The microchamber according to an exemplary embodiment is adapted and developed for isothermal heating of a reactant system to amplify the RNA into c-DNA for subsequent colorimetric detection of the targeted pathogen (FIG. 7). The microchambers are circular or rectangular or polygonal in shape and size may vary as per the volume to be amplified. There could be one or more microchambers in a single detection system. The material for fabrication of the microchamber is primarily polydimethylsiloxane/paper/Pyrex or similar biocompatible polycarbonates. The microchamber is fixed onto a cartridge (FIG. 6) for placing it onto heating block as it is the first stop position for the cartridge. The RNA mix sample is airtight sealed into the reaction chamber during the isothermal heating.

[0150] iv) Exemplary Illustration of the Functionalized Microfluidic Paper Platform:

[0151] For colorimetric detection of the target genomic material in the amplified c-DNA, the present invention provides specially functionalized microfluidic paper platform (FIG. 9). The said microfluidic paper platform is simulated by mimicking its properties in each section via computational fluid dynamics, for obtaining the optimum flow rate and reaction of the species based on the chosen paper grades and membranes constituting different sections of the lateral flow assay. This microfluidic paper platform comprises a circular/polygonal/similar shaped sample introducing chamber (part-a), a rectangular binding section for immobilizing the conjugates (part-b), a detection area includes a test line and a control line (part-c) and a waste absorbent section (part-d). The sample introducing part is made of nitrocellulose filter paper comprises a circular section integrated with a rectangular channel section. The construction of the sample introduction part is unique for seamless integration of dispensing of the amplified c-DNA product onto the sample introducing chamber. The binding or conjugate section of the paper device is made of glass fibers or similar materials. This binding area is immobilized with colloidal surface plasmonic nanomaterials (such as gold nanoparticles, nanoshells) conjugated with primary antibodies. The reaction area of the paper platform is made of a nitrocellulose membrane on which specific antibodies (for example, streptavidin and anti-FAM antibodies for COVID-19 detection) may be immobilized on two separate lines, seamlessly interfacing with a smartphone camera-based image grabbing unit (see later). Finally, the waste absorbing section is made of a blotting paper/material with high absorption capacity.

[0152] V) Exemplary Illustration of Smartphone App-Based Image Processing and Result Display:

[0153] The smartphone-based android app works on a simple image processing algorithm and is very user-friendly. Different edge detection techniques with few object detection techniques are used to detect and identify the test lines and control lines from the paper-based test strip. An algorithm (Table 1) has been developed based upon some rule-based decision for optimization using machine learning approach for providing accurate decision about the presence or absence of the infection in terms of positive or negative result.

TABLE-US-00004 TABLE 1 The algorithm for interpretation of the test results. Attempt-1 Attempt-2 Possible result Possible result Sample N N case RNaseP ORF-1b gene Inference Remarks RNaseP ORF-1b gene Conclusion 1 − − − Test not Repeat + − − Negative valid test + + + Positive − + + Repeat test with freshly collected sample. 2 − + + Positive Conclusive 3 + + + Positive Conclusive NA NA NA NA 4 + − − Negative Conclusive NA NA NA NA 5 + + − Presumptive Repeat + + + Positive positive test + + − Positive + − − Negative 6 + − + Presumptive Repeat + + + Positive positive test + − + Positive + − − Negative

[0154] The android app (named as COVIRAP) was developed based on the interpretation algorithm in the Table 1 on the API level 21 to 30 used in most of the available commercial smartphones. The user-friendly smartphone app performs multiple important functionalities starting from image capturing, machine learning-based analysis and processing, display of the final result and in parallel send the data to the cloud server. The overall workflow of the smartphone app is given in FIG. 11. These features are generic and can be implemented in other smartphone platforms as well. Arresting the possibilities of false reaction signal by using training sets of images of true and falsified test results via dynamic machine learning is another feature of the mobile app that improves the overall efficacy of detection.

[0155] v) Exemplary Illustration of Integration of Multiple Functionalities into a Simplified Portable Device:

[0156] The entire process of pathogen associated nucleic acids detection using the portable device is performed by synchronizing two major functionalities of the RT-LAMP based isothermal amplification with functionalized paper-based colorimetric detection together on a single platform. There are four functional modular units to execute these two separate functionalities represented in the functional block diagram in the FIG. 12. The thermal control cum reaction unit, which includes the isothermal amplification unit, performs the activity of reverse transcription of RNA to c-DNA followed by its amplification in the said device in a single user-step reaction. The thermal control unit controls the temperature of the aluminum heating block on which the RNA mix filled microchamber is situated. The microchamber is placed onto the surface of the heating block with the help of a cartridge inserted through a guided rail. An illustrative representation of the guided rail assembly is shown in the FIG. 10. The RNA mix solution is isothermally heated for performing biochemical reaction within the airtight sealed microchamber to produce c-DNA product by the method of reverse transcription and loop-mediated isothermal amplification. On the other hand, the entire colorimetric detection process is executed by colorimetric detection unit and smartphone result display unit. The synchronization between the thermal control cum reaction unit and the colorimetric detection unit is done by the guided rail assembly for smooth transition of the microchamber from the heating block to the position to dispense the amplified sample onto the sample introducing area of the paper strip. The cartridge with microchamber is pushed forward after completion of the isothermal heating to its second stop in between the fluid dispenser and the paper strip. The needle valve based microfluidic dispenser releases the amplified solution from the microchamber onto the sample introducing area of the paper strip to flow downstream for detection in the test line and control line by colorimetric reaction process. The smartphone is placed in its designated place onto the top window of the device for image capturing after generation of colored lines on the paper strip. The android app processes the captured image and displays the decision on smartphone screen after analyzing the colorimetric reaction with the in-house developed image processing algorithm.

[0157] The complete implementation of the workflow from sample collection to result dissemination using the portable device unit is represented in FIG. 13 and FIG. 14. This generic workflow is applicable to any arbitrary nucleic acid based detection protocols (including the RT-LAMP-CRISPR method) where isothermal amplification products are detected using LFA based detection method. (1) The body fluid sample collected from the patient may either be used to extract RNA using established methods or alternatively subjected to a brief heating step which can be performed in heating slots present in the device. (2) The purified RNA or the heat lysed sample is subsequently mixed with the test reagents generically containing primers, probes and enzymes and dispensed altogether in dedicated reaction-chambers. (3) The reactions are held in the portable device under pre-programmed piecewise isothermal time-steps without intermediate manual intervention (4) The final products are dispensed onto the LFA paper strips and the colorimetric readouts developed therein are captured using a smartphone camera, for subsequent image analytics and algorithmic implementation and rapid dissemination of the test outcome.

Example 2: Method of Detection of RNA Virus by the POC Device

[0158] The experimental procedure has been successfully tested for detection of influenza A and COVID-19 virus. For illustration, SARS-CoV-2 specific experimental details are given below. Notably, differences between the procedures of testing different pathogenic infections merely lie in the use of specific primers, DNA probes and specific hybridization temperature within the same overall framework (FIG. 15A).

[0159] The isothermal amplification reaction mixture for SARS-CoV-2 detection is prepared using NEB WarmStart LAMP kit, #1700 s (for fluorescence, agarose gel electrophoresis and paper strip based detection) or NEB WarmStart Colorimetric LAMP 2x Master mix #1800 s (for visual detection) by adding up biotinylated forward inner primer (FIP-5′Bt) sets (seq.no, 3, 12, 21 and 30 for ORF1B, for N gene, E gene, RNase P respectively), 6-fluorescein amidite (6-FAM) labelled DNA oligonucleotide (probes of Seq.no. 7 and 9;16 and 18, 25 and 27;34 and 35 for ORF1B, for N gene, E gene and RNaseP gene respectively) or dual modified (5′-FAM and 3′ ddNTP) complementary DNA probes (Seq. ID.No. 8,17 and 26 for ORF1B, N gene, E gene respectively) and other reagents as directed by the manufacturer in an airtight reaction microchamber.

[0160] Either non-specific RNA or ‘in vitro synthesized SARS-CoV-2 specific RNA’, or RNA extracted from suspected human patient samples was added to separate microchambers as for comparison of positive and negative results. First, the device is switched on for heating the block from room temperature to the targeted temperature of 65° C. The ramp-up time for reaching the target temperature is 4-5 minutes. Once the desired temperature is reached, the microchambers hosting the reaction mixture are placed onto the isothermal heating block. The isothermal heating of the system continues for the next 25-35 minutes preferably for 30 minutes. During this period, the RNA gets converted into c-DNA and subsequently gets amplified into millions of copies. After completion of the amplification cycle at a temperature of 62-68° C. preferably about 65° C., the device ramps up in ˜4 minutes to reach the temperature of 93-98° C. preferably about 95° C. and termination cycle of 3-8 minutes preferably about 5 minutes at 95° C. continues. All these heating cycles are designed to be performed automatically without requiring any intervention by the user. After the termination cycle is over, the heating system automatically ramps down to 48-55° C. preferably about 50° C. which is required for the hybridization step. This step carries out specific binding of the probe to the amplified target DNA. After completion of the reaction procedure, the microchambers are pushed forwards to the second stop and cooled down to the room temperature. The samples are now ready for introducing onto the sample pad of the paper platform. This step is done in a semi-automatic manner by pushing the microchamber holder through a guided rail synchronized with the paper platform. The microchamber is placed just above the sample introducing chamber of the paper strip and releases the solution onto the sample pad with the aid of a microfluidic dispenser (needle valve). Sample flows through the paper matrices due to the capillary action by the force of surface tension and reaches out to the conjugate section. Here, the colloidal gold conjugated Anti-FAM antibody binds with the targeted DNA and the amplified labeled DNA complex further flows downwards to the reaction area where streptavidin and anti-FAM secondary antibodies are immobilized at the test line and control lines of the strip, respectively. While flowing from the sample pad, the amplified DNA complex reaches the test line and binds with the streptavidin for producing the color by the concentration of colloidal gold nanoparticles/nanoshell attached to the same. The free gold nanoparticles/nanoshell-antibody conjugates bypass the test line and reach the control line to bind with the immobilized anti-FAM secondary antibodies and produce the color of colloidal nanoparticles/nanoshells. The time duration exclusive for colorimetric detection on the paper platform is within 10 minutes. After 5-10 minutes of sample introduction onto the paper strip, the smartphone app is activated for capturing images based on programmed camera properties, subsequently analyzing the same and displaying the final results onto the smartphone screen.

[0161] Results:

[0162] Standardization of the Protocol for the Detection of SARS-CoV-2 Genomic RNA:

[0163] To detect SARS-CoV-2 infection using this method, three highly conserved target regions have been identified in the SARS-CoV-2 genomic RNA that reside within the RNA dependent RNA polymerase (RdRp/ORF1b) gene (ORF1B), the nucleocapsid (N) gene and the envelope (E) gene (FIG. 15A and FIG. 16). Choices of such multiple highly conserved regions remain imperative and critical amidst the dynamically mutating strains of the infecting pathogen. Individual sets of RT-LAMP primers with high specificity against the target regions (FIGS. 16A and B) were designed to detect and amplify each of the target regions.

[0164] Short in vitro-transcribed (IVT) RNA fragments mimicking the gene targets mentioned above were first used for the standardization of the RT-LAMP reactions (FIG. 15B upper panel). Primers specific to human RNaseP gene were used as internal control. For complementary DNA probe-based detection via LFA, 6-FAM labelled forward and backward loop probes (FLP and BLP) for each set of viral genes and RNaseP were designed and tested their ability to specifically detect the corresponding RT-LAMP reaction products. BLPs, against all of the target genes, exhibited highly specific detection in the form of the test line on the paper strips which was absent in the negative control sets. FLPs, in contrast, showed weak to moderate nonspecific signals even in the negative control sets for N, E and RNaseP genes (FIG. 15B, lower panel), which could be attributed to partial complementarity of 6-FAM labelled probes with biotin labelled primers. For subsequent experiments, BLPs were therefore used for specific probing and LFA based detection procedure.

[0165] Next, the cross reactivity of individual set of primers and probes were tested against the non-target RNA sequences. RT-LAMP primers designed for one target gene of SARS-CoV-2 showed no cross reactivity against other gene targets (FIG. 15C, upper panel). Additionally, the products amplified from one gene target could only be detected with the corresponding BLPs (FIG. 15C, lower panel), ensuring the high specificity of our double layered detection method. The method also showed no cross reactivity against the genomic RNAs of other corona viruses including SARS, OC43, NL63, 229E and HKU1 (FIG. 17B) as well as other RNA viruses like Influenza A, Influenza B or Japanese Encephalitis Virus (JEV) genomic RNAs (FIGS. 17A and B) and hence confirmed that it can specifically detect and distinguish SARS-CoV-2 infection from infections caused by other viruses with similar clinical presentations.

[0166] In vitro sensitivity assay was performed to ascertain the limit of detection (LOD) of the present method for individual target genes. Individual reaction sets containing 10.sup.4 to 1 copy per microliter of the contrived RNA samples were then subjected to the test reaction protocol in sextuplets. Primers and probes corresponding to all of the gene targets showed the ability to consistently detect 100 copies of RNA molecules in six out of six replicates as illustrated in FIG. 18A and FIGS. 19A-19C. For the replicates containing 10 copies of RNA, N gene represented by FIG. 19B, RdRp/ORF1b represented by FIGS. 19A and E gene represented by FIG. 19C primer sets showed 5/6, 4/6 and 3/6 positive detections, respectively, suggesting the following order of sensitivity: Target 1(N)> Target 2(RdRp/ORF1b)> Target 3(E). All of the gene targets exhibit inconsistent results for higher dilutions suggesting a limit of detection as 10 copies per microliter. Summarily, the generic method demonstrated the capability of detecting 10 copies of viral RNA reliably for majority of replicates and 100 copies of viral RNA for all of the replicates, which is comparable to or superior to other RT-LAMP based detection technologies reported for similar applications.

[0167] Field Validation Test for the Detection of SARS-CoV-2 Infection in Patients Under Resource Limited Settings:

[0168] The high specificity and sensitivity of the test in the in-vitro experiments prompted to evaluate its efficacy in detecting the presence of SARS-CoV-2 genomic RNA in the nasopharyngeal swab samples isolated from patients. For this purpose, an experimental model was set up which could mimic performing the diagnostic procedure in a remote location in resource limited setting. The ready-to-use reaction-mixes in the forms of packed test kits were pre-aliquoted and were transported from the lab to the patient sample testing center over a road-travel duration of several hours and subsequently stored in a normal refrigerator at 4° C. overnight before performing the test procedure (FIG. 20). Purposefully, no specific attention was laid to perform the test in controlled ambience.

[0169] RNA extracted from 200 double-blinded patient samples were used, among which 115 were positive and 85 were negative for SARS-CoV-2 infection as determined by the ICMR-NIV developed RT-PCR assay, with the Ct values distributed in between the range of 15 and 35. Primers specific to N gene and RdRp/ORF1b(ORF1B) were employed to detect the presence of viral RNA as these two target gene sets showed higher sensitivity than E gene target (FIG. 18A). RNaseP was used as internal control. All the test reactions were performed using the portable instrument and the final results were recorded and analysed with the help of the custom-made app trained with the algorithm mentioned in Table 1. Briefly, a test with both the gene targets showing positive results was considered as infection positive while negative results with both of them confirm negative infection. For samples (observed mostly with high CT values) with one gene target showing positive and other showing negative results, the test was repeated and repetition of the exact same trend confirmed positive infection as illustrated in FIG. 18B Considering all 200 patient samples, our method showed a positive percentage agreement 93.91% and negative percent agreement 97.64% as illustrated in FIG. 18C, 18D and Table 2. However, for the samples of Ct values 30 and below, which acts as a suitable reference for clinical decision making, the positive percent agreement values reached to 98.79%. To overrule the cross reactivity of the assay, SARS-CoV-2 negative panel (n=85) included 10 known influenza A positive nasopharyngeal samples. All the Influenza positive samples showed negative results for the presence of SARS-CoV-2. This data confirms that the test is capable of detecting SARS-CoV-2 infection in patient samples with high, moderate and low viral loads with superior sensitivity and specificity in contrast to other LAMP-based detection technologies. Additionally, the present experimental model confirms that the present test method is suitable to be implemented in the remote areas with limited infrastructure and resources.

TABLE-US-00005 TABLE 2 Present test result for patient-sample based validation of SARS-CoV-2 infection. Ct value <=25 >25 to 30 >30 to 35 Negative True Positive 59 24 25 N/A Negative N/A N/A N/A 83 False Positive N/A N/A N/A 2 Negative  0 1 6 N/A Sensitivity 100% 96.15% 80.65% N/A Specificity N/A N/A N/A 97.65%

[0170] One step sample-to-result integration for detection of RNA-signature infection via single step swab/saliva-to-result protocol was standardized that could be implemented without changing the basic principle of the generic test-bench and work-flow of the present invention. This endeavor obviates the tedious workflow of RNA isolation or any intermediate steps, like intermediate addition of DNA probes in the protocol, facilitating implementation in point-of-care setting.

[0171] For this purpose, an integrated approach for detecting genomic RNA directly from the pathogenic particles or from the infected cells using Influenza A virus as a model system was incorporated in the device. The present protocol, for that purpose, was standardized for detecting the segment seven (open reading frame M2) of influenza A/H1N1/WSN/1933 virus genomes (Table 3) using extracted RNA samples (FIG. 21). Subsequently, experiments were performed where influenza A virus infected human lung epithelia cells (A549) were re-suspended in phosphate buffer saline (PBS) spiked with the saliva collected from an infection negative donor. A saliva-PBS suspension containing 100 such cells was then subjected to a brief pre-heating step (93-98° C. preferably about 95° C. for 2-5 minutes preferably about 3 minutes) in the portable instrument before being used as an input for the subsequent steps of the protocol. This heating step was likely to cater multifarious objectives including sample homogenization, breaking up the viral capsid and inactivation of the inhibitory enzymes present in human saliva. This adapted protocol was successful in consistently detecting both influenza virus RNA as well as the RNaseP mRNA from the heat ruptured cell lysate either in absence or presence of saliva (0.5% final concentration) (FIG. 22A and FIG. 23). Additionally, this protocol could detect as low as 500 influenza virion particles (measured in terms of plaque forming units-PFU) diluted in PBS without any additional RNA extraction process in absence of saliva (FIG. 22B and FIG. 24). In dilution containing saliva (0.5% final concentration), the method shows a sensitivity of 2500 plaque forming units which is much lower than the usual viral load in a patient showing clinical symptoms.

[0172] The limit of detection (LOD) as ascertained for SARS-CoV-2 N gene target in the presence and absence of saliva. Tenfold serial dilutions of contrived N gene fragments were prepared either in PBS or in PBS spiked with saliva and subsequently subjected to the present procedure in sextuplets. Encouragingly, even in presence of saliva, the present method could successfully detect up to 100 and 10 copies of viral RNA in 5/6 and 4/6 replicates which is comparable to the saliva negative controls (FIG. 22C and FIG. 25). This ensures high sensitivity and robustness of the N gene target specific primers in amplifying corresponding gene fragment in stringent experimental conditions.

[0173] List of RT-LAMP Primers and Probe

TABLE-US-00006 Target Primer Region Primer Sequence (5′ to 3′) Modification ORF 1b F3 GCCATTAGTGCAAAGAATAGAGC SEQ.ID No. 1 B3 GGCATGGCTCTATCACATTTAGG SEQ.ID No. 2 FIP TAGCTCCTCTAGTGGCGGCTATTGCACCGTAGCTGGT 5′-[Btn] (F1c + F2) GTCTC SEQ.ID No. 3 BIP TGTAGTAATTGGAACAAGCAAATTCTATGGTGGCCAA (B1c + B2) CCCATAAGGTGAGGG SEQ.ID No. 4 Loop TTTTTGATGAAACTGTCTATTGGTCATAGTACTACAG F SEQ.ID No. 5 Loop GGCACAACATGTTAAAAACTGTTTATAGTGATGTAG B SEQ.ID No. 6 BLP TTGGCACAACATGTTAAAAACTGTTTATAGTGATG 5′- Probe [6FAM] SEQ.ID No. 7 BLP TTGGCACAACATGTTAAAAACTGTTTATAGTGATG 5′- 3′- [6FAM], mod 3′- Probe [3d_G] SEQ.ID No. 8 FLP GCGGCTATTGATTTCAATAATTTTTGATGAAAC 5′- Probe [6FAM] SEQ.ID No. 9 N F3 ACAATGTAACACAAGCTTTCG gene SEQ.ID No. 10 B3 TTGGATCTTTGTCATCCAATT SEQ.ID No. 11 FIP GGCCAATGTTTGTAATCAGTTCCTTAGACGTGGTCCA 5′-[Btn] (F1c + F2) GAACAA SEQ.ID No. 12 BIP GCTTCAGCGTTCTTCGGAATCACCTGTGTAGGTCAACC (B1c + B2) SEQ.ID No. 13 Loop TGGTCCCCAAAATTTCCTTGG F SEQ.ID No. 14 Loop CGCGCATTGGCATGGAAGT B SEQ.ID No. 15 BLP TTGGCATGGAAGTCACACCTTC 5′- Probe [6FAM] SEQ.ID No. 16 BLP TTGGCATGGAAGTCACACCTTC 5′- 3′- [6FAM], mod 3′- Probe [3d_C] SEQ.ID No. 17 FLP GATTAGTTCCTGGTCCCCAAAATTTCC 5′- Probe [6FAM] SEQ.ID No. 18 E gene F3 TTGTAAGCACAAGCTGATG SEQ.ID No. 19 B3 AGAGTAAACGTAAAAAGAAGGTT SEQ.ID No. 20 FIP CGAAAGCAAGAAAAAGAAGTACGCTAGTACGAACTT 5′-[Btn] (F1c + F2) ATGTACTCATTCG SEQ.ID No. 21 BIP GGTATTCTTGCTAGTTACACTAGCCAAGACTCACGTTA (B1c + B2) ACAATATTGC SEQ.ID No. 22 Loop ATTAACGTACCTGTCTCTTCCGAAA F SEQ.ID No. 23 Loop ATCCTTACTGCGCTTCGATTGTGTG B SEQ.ID No. 24 BLP ATCCTTACTGCGCTTCGATTGTGTG 5′- Probe [6FAM] SEQ.ID No. 25 BLP ATCCTTACTGCGCTTCGATTGTGTG 5′- 3′- [6FAM], mod 3′- Probe [3d_G] SEQ.ID No. 26 FLP ATTAACTATTAACGTACCTGTCTCTTCC 5′- Probe [6FAM] SEQ.ID No. 27 RNase F3 TTGATGAGCTGGAGCCA P SEQ.ID No. 28 B3 CACCCTCAATGCAGAGTC SEQ.ID No. 29 FIP GTGTGACCCTGAAGACTCGGTTTTAGCCACTGACTCG 5′-[Btn] (F1c + F2) GATC SEQ.ID No. 30 BIP CCTCCGTGATATGGCTCTTCGTTTTTTTCTTACATGGCT (B1c + B2) CTGGTC SEQ.ID No. 31 Loop ATGTGGATGGCTGAGTTGTT F SEQ.ID No. 32 Loop CATGCTGAGTACTGGACCTC B SEQ.ID No. 33 BLP CATGCTGAGTACTGGACCTCG 5′- Probe [6FAM] SEQ.ID No. 34 FLP ATGTGGATGGCTGAGTTGTT 5′- Probe [6FAM] SEQ.ID No. 35 Table 3: In- F3 GGGCTGTGACCACTGAAG fluenza SEQ.ID A No. 36 (Segment B3 AGCAATATCCATGGCCTCTG 7) SEQ.ID No. 37 FIP TGAGACCGATGCTGGGAGTCATGGCATTTGGCCTGGT 5′-[Btn] (F1c + F2) ATG SEQ.ID No. 38 BIP TGGTTCTAGCCAGCACTACAGCCTGCTTGCTCACTCGA (B1c + B2) TCC SEQ.ID No. 39 Loop GCAATCTGTTCACAGGTTGCG F SEQ.ID No. 40 Loop TAAGGCTATGGAGCAAATGGC B SEQ.ID No. 41 BLP TAAGGCTATGGAGCAAATGGCT 5′- Probe [6FAM] SEQ.ID No. 42 FLP GCAATCTGTTCACAGGTTGCG 5′- Probe [6FAM] SEQ.ID No. 43

[0174] The other exclusive aspect of saliva to result integration is combining the specific DNA probe hybridization step with the initial amplification step, thereby excluding any in-between manual intervention during the test reactions. For this purpose, the 6-FAM labelled complementary DNA probe (BLP) was added from very beginning of the reaction, resulting in a master-mix which contains all reaction components in it (enzyme mix, RT-LAMP primers, and DNA probe). Hence, mere addition of the input RNA to the master mix and subsequent initiation of the thermal protocol could result in dual labelled products which then could be subjected to direct detection. Although an attractive proposition, the initial addition of the DNA probe in the RT-LAMP reaction as an integrated single-step protocol necessitated additional safeguards as this could potentially lead to the concomitant amplification of the probe itself and hence might interfere with the amplification or specificity of the detection process. To avoid such adverse artefacts, double modified DNA probe harbouring 6-FAM and a di-deoxy nucleotide were included at its 5′- and 3′-termini respectively. Test reactions were performed in presence of either regular (5′-6-FAM) or dual modified (5′-6-FAM+3′-ddNTP) DNA probes and subsequently monitored. As evidenced, addition of either the 6-FAM or the 6-FMA-3′ddNTP probes from the very beginning of the reaction showed signal comparable to the conventional two-step detection procedure with no non-specific signals in negative control sets in either of these cases (FIG. 22D, FIG. 26 and FIG. 27). Together, this modification along with the body fluid-to result integration in the test protocol, when implemented with the help of the present device, holds the potential to revolutionize the nucleic acid-based detection of pathogenic infection and essentially bring high-end molecular diagnostics from sophisticated labs to the field where controlled laboratory-standard interventions remain challenging.

[0175] Hence, it is evidenced that the portable POC device of the present invention performs the detection of pathogen associated nucleic acid, producing test results with specificity and sensitivity compatible with acceptable gold standards without involving the conventionally used expensive instruments and skilled human resources. Additionally, integration of smartphone app based analysis and result display further obviates manual interpretation and ensures data dissemination and sharing to benefit public health intervention. Thus, the present invention provides a low cost portable platform to achieve the desired task and is a disruptive replacement to PCR machines that act as traditional benchmark thermal control units in nucleic acid based testing.