RT-PCR DETECTION REAGENT FOR DETECTING NOVEL CORONAVIRUS, KIT AND DETECTION METHOD THEREOF

Abstract

The present application relates to the molecular biological detection technical field, specifically is a RT-PCR detection reagent for detecting coronavirus virus directly without need of RNA extraction, a kit and a detection method thereof. The RT-PCR detection reagent for detecting coronavirus virus comprises primers having nucleotide sequences as set forth in SEQ ID NO. 1-2, 3-4, and 13-14.

Claims

1. A RT-PCR detection reagent for detecting coronavirus, comprising primer sequences of a SARS-COV-2 ORF1lab gene as following: a upstream primer has the nucleotide sequence of SEQ ID NO. 1; and a downstream primer has the nucleotide sequence of SEQ ID NO. 2; and primer sequences of a SARS-COV-2 N gene fragment as following: a upstream primer has the nucleotide sequence of SEQ ID NO. 3; and a downstream primer has the nucleotide sequence of SEQ ID NO. 4.

2. The RT-PCR detection reagent according to claim 1, further comprising: primer sequences of an influenza A (IFV-A) gene as following: a upstream primer has the nucleotide sequence of SEQ ID NO. 5; and a downstream primer has the nucleotide sequence of SEQ ID NO. 6; and primer sequences of an influenza B (IFV-B) gene as following: a upstream primer has the nucleotide sequence of SEQ ID NO. 7; and a downstream primer has the nucleotide sequence of SEQ ID NO. 8.

3. The RT-PCR detection reagent according to claim 1, further comprising a fluorescent probe sequence of an influenza A (IFV-A) gene having the nucleotide sequence of SEQ ID NO. 9; a fluorescent probe sequence of an influenza B (IFV-B) gene having the nucleotide sequence of SEQ ID NO. 10; a fluorescent probe sequence of a SARS-COV-2 ORF1lab gene having the nucleotide sequence of SEQ ID NO. 11; and a fluorescent probe sequence of a SARS-COV-2 N gene fragment having the nucleotide sequence of SEQ ID NO. 12.

4. The RT-PCR detection reagent according to claim 1, further comprising primer and fluorescent probe sequences of an internal reference gene as following: a upstream primer has the nucleotide sequence of SEQ ID NO. 13; and a downstream primer has the nucleotide sequence of SEQ ID NO. 14.

5. The RT-PCR detection reagent according to claim 4, further comprising a fluorescent probe sequence of an internal reference gene having the nucleotide sequence of SEQ ID NO. 15.

6. The RT-PCR detection reagent according to claim 1, wherein the coronavirus SARS-CoV-2.

7. The RT-PCR detection reagent according to claim 1, further comprising following fluorescent probes: IFV A-probe: FAM-SEQ ID NO. 16-BHQ1; IFV B-probe: HEX-SEQ ID NO. 17-BHQ1; SAS-CoV-2-probe: ROX-SEQ ID NO. 18-BHQ2, and ROX- SEQ ID NO. 19-BHQ; and IC-probe: Cy5-SEQ ID NO. 20-BHQ2.

8. A RT-PCR detection kit comprising the RT-PCR detection reagent according to claim 1.

9. The RT-PCR detection kit according to claim 8, wherein the kit further comprises one or more of sterile water, dNTP, PCR buffer, Hot Start Taq enzyme, PCR enhancer, and cell lysis buffer.

10. The RT-PCR detection kit according to claim 9, wherein the PCR enhancer is one or more of DMSO, Betaine, TMAC, and Trehalose, and the cell lysis buffer is the solution of 1% Nonidet P-40, 150mM Tris-HCl and 50 mM NaCl.

11. The RT-PCR detection kit according to claim 8, wherein the kit further comprises one or both of a positive control and a negative control.

12. A multiple fluorescent direct RT-PCR detection method for detecting coronavirus in a patient using the detection kit according to claim 8, comprising: simultaneously amplifying the SARS-CoV-2 ORF1lab gene fragment, the SARS-CoV-2 N gene fragment, and an internal reference gene, and subjecting amplified products to a multiple fluorescence quantitative PCR detection by one-step in vitro amplification of multiple fluorescent probes, and determining if the sample is infected by the SARS-CoV-2 virus.

13. The multiple fluorescent direct RT-PCR detection method according to claim 12, comprising obtaining a PCR detection working solution by mixing a PCR reaction solution with a solution mixture of primers and probes, and enzyme mixture of a Hot Start Taq polymerase and a reverse transcriptase in the detection kit; using a clinical sample containing SARS-CoV-2 virus as a template, to release virus RNA directly, and to transcribe a corresponding DNA by a reverse transcriptase at a temperature of 50° C., using the corresponding DNA as a template, to perform a PCR reaction in the PCR reaction solution with Hot Start Taq polymerase under chain reaction conditions; choosing FAM(IFV-A), HEX(IFV-B), ROX(ORF1lab/N), and Cy5(internal reference housekeeping gene) for a multiple reaction simultaneous testing, setting a threshold, analyzed by a computer to obtain a Ct value, and determining if the patient is infected by the SARS-CoV-2 virus.

14. The multiple fluorescent direct RT-PCR detection method according to claim 13, wherein the final concentrations of a PCR reaction system in the PCR step are following: the concentration of Mg.sup.2+ in the PCR buffer is 1-3.5 mM, the concentration of dNTP is 0.1-0.5mM, the concentration of the upstream primers, the downstream primers and the probes of SARS-CoV-2 specific genes is 0.1-1.0M, the concentration of the probes is 0.1-0.5M, the concentration of Hot Start Taq enzyme is 0.5-5U/reaction, and each test comprises 5-1000 copies of viral genome template.

15. The multiple fluorescent direct RT-PCR detection method according to claim 12, wherein PCR reaction conditions are as follows: 50° C. 10 min; 95° C. 30 sec; 5 cycles: 95° C. 5 sec, 55° C. 30 sec; 40 cycles: 95° C. 3 sec, and 60° C. 10 sec.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0065] FIG. 1 is a graph of PCR amplification curve of positive control by FluA/B and SARS-CoV-2 detection kit in accordance with at least one embodiment. Wherein .diamond-solid. line stands for SARS-CoV-2 gene, .square-solid. line stands for FluA gene, .box-tangle-solidup. line stands for FluB gene, and x line stands for internal control gene.

EXAMPLES

[0066] The following non-limiting examples may enable those of ordinary skill in the art to more fully understand the present application, but do not limit the present application. Any person familiar with the technical field in the technical scope disclosed in the present application, according to the technical solution of the present application and the inventive concept to make equivalent substitutions or changes shall belong to the protection category of the present application.

The materials and Equipment used are as Follows:

[0067] DNAZap™ (Ambion, cat. #AM9890) or equivalent.

[0068] RNAse Away™ (Fisher Scientific; cat. #21-236-21) or equivalent.

[0069] 10% bleach (1:10 dilution of commercial 5.25-6.0% hypochlorite bleach).

[0070] 96-well 0.2 mL PCR reaction plates (Applied Biosystems).

[0071] ABI 7500, Roche LightCycler480, Bio-Rad CFX96 and other real-time quantitative PCR instruments that meet the detection channel settings of the kit of the present application.

[0072] The materials used in the present application are commercially available.

Detection Methods

[0073] 6-FAM, 6-carboxy-fluorescein.

[0074] HEX, 5-hexachloro-fluorescein

[0075] TAMRA, tetramethyl-6-carboxyrhodamine

[0076] ROX, 6-carboxy-x-rhodamine

[0077] Cy5, Indodicarbocyanine, the formula of Cy5 is

##STR00001##

[0078] BHQ-1 (Black Hole Quencher 1)

[0079] BHQ-2 (Black Hole Quencher 2)

[0080] Double-labeled fluorescent probes composed of the quenching group TAMRA, Eclipse or BHQ series dyes are often used as hydrolysis probes, or TaqMan probes, for real-time fluorescent quantitative PCR experiments.

[0081] 1) TAMRA is a fluorescent dye that will fluoresce at a higher wavelength while quenching the reporter group. The Eclipse and BHQ series are non-fluorescent dyes. When the reporter group is quenched, the reporter group does not emit fluorescence. The probe fluorescence background is lower than that of TAMRA, and the detection sensitivity is higher.

[0082] 2) The absorption spectrum of TAMRA covers a narrow range, and there are fewer types of reporter groups that can be matched with TAN/IRA; while Eclipse has a wider absorption range (390 nm-625 nm), and there are many types of reporter groups that can be quenched, such as FAM, HEX, TAN/IRA, ROX, etc. can be used; the combined use of BHQ series dyes has a wider range of absorption spectra, from 430 nm to near infrared, and there are more types of quenchable reporter groups, including Cy3, Cy5, etc. Therefore, a set of dual-labeled fluorescent probes can be composed of Eclipse or BHQ series dyes for multiple PCR.

##STR00002##

[0083] FAM, HEX and TET are added in the form of phosphite by B-cyanoethyl chemical action at the end of the synthesis cycle, so FAM, HEX and TET are added to the sugar at the 5′ end of the primer instead of the base at the end of the primer. They are covalently linked to the last sugar ring at the 5′end through a phosphodiester bond.

[0084] Color in liquid: HEX is pink, FAM is yellow, TET is orange.

[0085] Oligonucleotides that have undergone these modifications of FAM, HEX and TET cannot undergo phosphorothioate modification.

Example 1

Kit Contents

[0086]

TABLE-US-00001 TABLE 1 Component Specification Description 1. Reaction 900 μL/tube Containing primer/probes for virus detection, Mix PCR buffer, and 10 mM dNTP 2. Enzyme 200 μL/tube Mix of Hot Start Taq enzyme and reverse Mix transcription enzyme 3. Negative 200 μL/tube Containing synthesis sequences of human Ctrl (NC) actin gene 4. Positive 200 μL/tube Containing synthesis sequences of virus Ctrl (PC) target gene fragments and IC

[0087] *Composition and concentration of the active ingredient (s) of the reagent for Reaction mix by nature is 10 mM dNTP and 0.01 mM phosphate buffer solution, Enzyme mix is 0.5 U Hot Start Taq and reverse transcription enzyme, negative control is 0.1 μM dNTP and 0.01mM distilled water, and positive control contains 0.2 μM dNTP and 0.01 mM distilled water. The device contains no other ingredients, which might influence the measurement.

[0088] Synthesis sequences of virus target gene fragments are primer sequences of a SARS-COV-2 ORF1lab gene and primer sequences of a SARS-COV-2 N gene.

[0089] Synthesis sequences of virus target gene fragments are sequence ID No. 1-2, 3-4, and IC is Sequence ID No. 13-14.

Example 2

Sample Collecting and Treatment

Sample Requirements

[0090] 1. The test samples are clinical samples of novel coronavirus infections (nasal/pharyngeal swabs, sputum, lung lavage fluid/extracts, feces/anal swabs, etc.).

[0091] 2. The kit of present application has a high tolerance to inhibitors, but excessive samples may cause inhibitors to exceed the tolerance of the reaction system, and excessive proteins and blood may coagulate at high temperature and block signal collection. To control the false negative caused by the inhibition of reaction, dilute the sample according to following “sample process”.

[0092] 3. The kit of present application cannot directly detect the samples collected by the inactivated sampling tube. Because the sample solution contains enzyme inactivator, this type of detection will fail. Such samples must be extracted and purified by nucleic acid before detection. It is suggested that the samples with very low virus content should be tested after the virus is concentrated and the nucleic acid is extracted and purified. The kit of present application can be used for high-quality amplification with nucleic acid extract as its template.

[0093] For different types of samples, the treatment suggestions are as follows:

TABLE-US-00002 TABLE 2 Samples types Samples Collection and Proceed the Treatment Nose/throat swab* If collected by the virus sampling tube, after suspension, take 5 μL for detection Deep expectoration, In case of collection by non-virus sampling lung lavage/aspirate* tube, all samples shall be transferred into 3 ml virus sampling solution NS or PBS, distilled water, and the supernatant shall be taken off for sampling after being fully suspended (avoid floating objects), take 5 μL for detection Anal swab The samples collected by 3 ml virus sampling tube shall be directly processed after full suspension (anus swab samples containing a large amount of feces shall be treated as feces) Feces For the non-virus sampling tube, use a swab to take a proper amount (about the size of the swab head) and transfer the swab into 3 ml virus sampling solution or NS, PBS, distilled water. After fully suspending and centrifuging at 3000 rpm for 30 sec, take the supernatant for detection (avoid sediment) Serum, Plasma After 1:1 dilution and suspension with NS, PBS or distilled water Whole blood Dilute and suspend with NS or PBS solution 1:5, (non heparin centrifuged at 3000 rpm for 30 sec, then take anticoagulation) the supernatant and take 5 μL for detection

[0094] The sample with * demonstrated the best results for the kit of the present application. When the specimen is not used for specific purposes such as culture, the specimen should be inactivated under 56° c for 30 min and then proceed the treatment.

Example 3

System Preparation and Loading Sample

[0095] Melt the reagent thoroughly, and centrifuge the reagent to the bottom of the tube before use. The number of reaction tubes “n” corresponds to the number of samples and controls. Prepare a 25 μ1 reaction system according to the following method.

TABLE-US-00003 TABLE 3 Reaction composition Dosage 1. Reaction Mix 18 μL × number of specimens and controls (N) 2. Enzyme Mix 2 μL × number of specimens and controls (N)

[0096] After system preparation for “n” samples, the total system solution for 20 μL/well should be added into 96 well plate separately, then 5 μL sample or positive control/negative control is added into each well. Finally, the centrifuge can be used to spin down the sample.

Example 4

Amplification

[0097] The kit of the present application employs the high-speed PCR with higher amplification efficiency and sensitivity (instrument support is required) and is compatible with conventional standard procedures for amplification.

TABLE-US-00004 TABLE 4 High-speed Standard amplification amplification Detection channel 50° C. 10 min; 50° C. 10 min; Flu-A: FAM, 95° C. 30 sec; 95° C. 30 sec; 520 nm 5 cycles: 45 cycles: Flu-B: HEX/VIC, 95° C. 5 sec, 95° C. 10 sec, 555 nm 55° C. 30 sec 55° C. 30 sec SAS-CoV-2: 40 cycles: (read) ROX, 602 nm 95° C. 3 sec, IC: CY5, 668 nm 60° C. 10 sec (read)

[0098] Set the corresponding wavelength for the Roche machine. At lease, the reference fluorescence of the ABI machine and the quenching genes should both be set to ‘None.’

Example 5

Quality Control

[0099] NC is not amplified, PC/IC is amplified, and Ct<30 indicates that the experiment is established. If these prerequisites are not met, the results will be invalid. The sample quality is acceptable as long as IC<30. If IC>30, resample and test again is necessary.

[0100] IC takes human housekeeper gene as the target (Cy5 as the detection channel), and effective samples (anal swab, pharyngeal swab, etc.) from human body should be positive. Using IC, investigation of the collection quality of human samples and whether there is excessive PCR inhibitor in the samples is possible. In the event of changes in the analytical performance of the device, when the positive control cannot be detected, the experiment should be retested or a new kit should be employed.

Example 6

Result Judgment

[0101] The typical “s” type amplification curve of the target gene of the sample shows that CT≤38 and CT value of IC (Cy5)<30, the target gene can be reported as positive. If the CT value>40, or if there is no amplification, the result can be reported as negative. However, if the CT value falls between 38 and 40, the results are unclear and should be reexamined. In this case, the process is re-conducted in some instances. At that point, the results can be interpreted as follows: and CT≤38 is positive, and CT>38 is negative.

Example 7

Sensitivity Test

Performance Index of the Present Kit

[0102] LOD: the limitation of detection was 700, 500, 350, 500 copies/ml for FluA, FluB, SAS-CoV-2 and IC respectively.

[0103] The linear detection range is 500-2×10.sup.7 copies/ml.

LoD with Pseudo-Virus

[0104] The LoD of the Real-Time Fluorescent RT-PCR Kit for Detecting SARS-2019-nCoV was estimated by testing the standardized dilutions of pseudo-virus (n=3 each). The lowest target level at which all three replicates produced positive results was 100 copies/mL. This value was then confirmed by testing 20 replicates at five different concentrations above and below the estimated LoD (See Table 5).

TABLE-US-00005 TABLE 5 LoD confirmation with pseudo-virus N gene ORF1ab gene Mean Ct Conc. Detection Conc. Detection (copies/ml) rate (copies/ml) rate N ORF1ab IC 500 20/20 500 20/20 20.6 23.5 22.7 300 20/20 300 20/20 23.7 25.2 23.1 150 20/20 150 20/20 26.4 28.1 22.8 100 20/20 100 20/20 26.5 28.2 23.9 50 18/20 50 16/20 33.6 38.1 22.6
LoD with Clinical Specimens

[0105] The quantity of SARS-CoV-2 in three clinical specimens that were known to be positive was estimated by quantitative digital PCR. The remainder of each specimen was then diluted in SARS-CoV-2 negative clinical matrix to achieve the approximate concentrations shown in Table 6.

TABLE-US-00006 TABLE 6 Dilution of clinical specimens for LoD determination Dilution Factor BALF (broncho Concentration alveolar lavage Estimated by Throat swab fluid) 1 BALF2 Digital PCR (1.35 × 10.sup.4 (1.23 × 10.sup.4 (1.65 × l0.sup.4 (copies/mL) copies/mL) copies/mL) copies/mL) 500 27 24.6 33 300 45 41 55 150 90 82 110 100 135 123 165 50 270 246 330

[0106] The LoD of the Real-Time Fluorescent RT-PCR Kit for Detecting SARS-2019-nCoV was evaluated by testing the dilutions of each clinical specimen described below (n=20 each). The LoD was determined to be the highest dilution at which ≥19/20 results were positive (i.e., ≥95% proportion positive) (See Table 7).

TABLE-US-00007 TABLE 7 LoD confirmation Concentration of SARS-CoV-2 estimated by Digital PCR Number Positive/ Proportion Specimen (copies/mL) Number Tested Positive Throat swab 500 20/20 100% 300 20/20 100% 150 20/20 100% 100 19/20  95% 50 17/20  85% BALF1 500 20/20 100% 300 20/20 100% 150 20/20 100% 100 20/20 100% 50 17/20  85% BALF2 500 20/20 100% 300 20/20 100% 150 20/20 100% 100 20/20  10% 50 16/20  80%

Example 8

Specificity Test

[0107] The LoD (150 Copies/mL) for each clinical matrix was further validated for 3 lots of kits on a PCR system (Applied Biosystems™ Real Time PCR System 7500) in 20 replicates, where at least 19 tests confirmed positive for every matrix/kit.

[0108] a) Reactivity/Inclusivity:

[0109] Currently, different SARS-CoV-2 isolates are not available for the validation of reactivity/inclusivity of the kit. Primer/probe inclusivity was therefore evaluated by BLASTn analysis against 280 publicly available SARS-CoV-2 sequences on Mar. 10, 2020. Two Primes/probe set used in our kit exhibited 100% homology with all the available sequences. One set of primes/probe only exhibited 1 mismatch a single mismatch with one published sequence.

[0110] In addition to in silico analysis, 10 specimens from different regions of China confirmed as SARS-CoV-2 positive based on clinical criteria were used to validate the lower detection limit. The concentration of SARS-CoV-2 in each specimen was estimated with ddPCR. Further, each specimen was diluted to estimated concentrations of 5×10.sup.3 Copies/mL and 100 Copies/mL (LoD concentration) and tested in replicates of 10 to evaluate the reproducibility of the test. The coefficient of Variation (CV) of Ct values at 5×10.sup.3 copies/mL was lower than 5%. Table 8 below summarizes the results.

TABLE-US-00008 TABLE 8 Testing results Reproducibility LoD Diluted Diluted Concentration concentration Detection concentration Detection (Copies/mL) (copies/mL) rate CV (copies/mL) rate BALF1 1.23 × 10.sup.4 1000 100% 0.35% 100 100% BALF2 1.65 × 10.sup.4 1000 100% 0.46% 100 100% BALF3 1.55 × 10.sup.5 1000 100% 0.53% 100 100% BALF4 1.15 × 10.sup.4 1000 100% 0.72% 100 100% BALF5 5.35 × 10.sup.4 1000 100% 0.59% 100 100% BALF6 6.75 × 10.sup.4 1000 100% 0.86% 100 100% Throat 1.35 × 10.sup.4 1000 100% 1.24% 100 100% swab 1 Throat 7.85 × 10.sup.4 1000 100% 0.47% 100 100% swab 2 BALF7 1.75 × 10.sup.4 1000 100% 0.52% 100 100% BALF8 1.52 × 10.sup.4 1000 100% 0.74% 100 100%

Analytical Specificity:

[0111] The SARS-CoV-2 Real-time Reverse Transcriptase (RT)-PCR Kit utilizes identical oligo sequences, master mix, and amplification instruments.

[0112] A panel of more than 50 respiratory pathogens was tested with the SARS-CoV-2 direct-RT-PCR Kit of the present application, at clinically relevant concentrations (generally at 10.sup.6 genome copies/mL). All pathogens were tested in triplicate and none produced any detectable reactivity with the SARS-CoV-2 direct-RT-PCR kit of the present application.

[0113] Specificity: No cross reaction with other coronaviruses or other viruses has been found, such as human coronavirus (hkul, OC43, nl63 and 229E), SARS coronavirus and Mers coronavirus; H1N1 (new H1N1 influenza virus (2009), seasonal H1N1 influenza virus), H3N2, H5N1, H7N9, Victoria, respiratory syncytial virus A, B, parainfluenza virus 1, 2, 3, rhinovirus a, B, C, adenovirus 1, 2, 3, 4, 5, 7, 55, enterovirus a, B, C, D, human lung virus, human partial lung virus, EB virus, measles virus, human cytomegalovirus, rotavirus, norovirus, mumps virus, varicella zoster virus; also no cross reaction with bacterial cells as Mycoplasma pneumoniae and Chlamydia pneumonia.

Performance Index of the Present Kit

[0114]

TABLE-US-00009 TABLE 9 Clinical results of Flu A/B and SARS-CoV-2 Direct RT-PCR kit of the present application compared with RT-PCR assay Direct RT-PCR Accordance RT-PCR Kit Sensitivity Specificity rate Target assay + − (%) (%) (%) Kappa P Flu A + 236 1 99.57 100.00 99.81 0.975 6.524 − 0 293 Flu B + 235 2 99.15 100.00 99.62 0.972 2.531 − 0 293 SARS-CoV-2 + 236 1 99.58 99.44 99.43 0.968 3.451 − 2 291 Conclusion + 707 4 99.72 99.54 99.62 0.975 2.610 − 2 877

[0115] Total 530 specimens were enrolled and tested in the study to evaluate the performance of the Flu A/B and SARS-CoV-2 Direct RT-PCR Kit of the present application in detecting Flu A/B and SARS-CoV-2 from of throat swab specimens, BALF, and extracted RNA obtained from Zhejiang Jiaxing Center for Disease Control of China compared to the clinical diagnosis of COVID-19 and results by National CDC kit of China, RT-PCR of Flu A, Flu B and SARS-CoV-2 showed overall positive and negative percent agreement across all specimens of 98.65%, 99.66% and 99.81% (95% CI: 97.8% to 99.9%) respectively. Wherein, the RT-PCR assay contains other primer for detecting Flu A, Flu B and SARS-CoV-2.