Diagnostic device and related method

Abstract

The present invention relates to a device (10; 30; 50) for analysing liquid samples comprising amplified nucleic acids, said device comprising: a sample pad (16); a first (18) and a second (19) sample analysis strip configured to analyse different aspects of the nucleic acid sample wherein said segments comprise nucleic acid sequences which are complementary to the predetermined sequences of target nucleic acids whose presence or absence is analysed; and a housing (11) enclosing said sample pad and at least two elongated sample analysis strips wherein the analysis result is detectable from the outside of the housing from the combination of aligned segments of said first and second analysis strips. The invention furthermore relates to a method for determining the presence or absence of a target nucleic acid, providing confirmatory results, in combination with a predetermined sequence in a sample from a subject using the device according to the invention.

Claims

1. A device (10; 30; 50) for analysing a liquid sample comprising amplified nucleic acids, said device comprising: a sample pad (16); a first sample analysis strip (18) and a second sample analysis strip (19), wherein the first and second strips are elongated and extend from the sample pad and each of said first and second strips comprises a straight analysis section (20) divided into multiple segments (22), each segment of said first or second analysis strip having immobilized thereto nucleic acids which sequence is different to a nucleic acid sequence of any other segment of said first or second analysis strip and that is complementary to a target nucleic acid sequence, wherein said segments are configured to indicate the presence or absence of a target nucleic acid sequence in said liquid sample, and wherein the analysis sections of said first and second sample analysis strips comprises a backing film covered by a transparent layer, wherein said transparent layer has a thickness of at least 0.1 mm, said analysis sections of the first and second sample analysis strips having the same length and being arranged side by side such that the analysis result is detectable from the combination of three-dimensional marks appearing in the transparent layer of aligned segments of said first and second analysis strips, and a housing (11) enclosing said sample pad and at least two elongated sample analysis strips, said housing comprising a front side (12) in which a sample inlet passage (15) is formed such that the sample pad is accessible from the outside of the housing and at least one opening (17) such that the analysis result is detectable from the outside of the housing, wherein the sample pad and the sample analysis strips are formed from a material that extends from the sample pad through the analysis strips, said material comprising a single continuous piece of backing film and a single continuous transparent layer, wherein said transparent layer is on top of the backing film.

2. The device according to claim 1, wherein the analysis section of the first sample analysis strip comprises at least one segment comprising at least a partial nucleic acid sequence of a wild type gene and the analysis section of the second sample analysis strip comprises at least one segment comprising the corresponding nucleic acid sequence of a mutant of said gene, which corresponding nucleic acid sequence encompasses at least one mutation.

3. The device according to claim 2, wherein said gene comprises at least one antibiotic resistance marker, such as a Mycobacterium tuberculosis antibiotic resistance marker.

4. The device according to claim 3, wherein said at least one antibiotic resistance marker is selected from the group consisting of rpoB 516 TAC, rpoB 516 GTC, katG 315 ACC, rpoB 531 TTG, rpoB 531 TGG, rpoB 526 TAC, rpoB 526 GAC, rpoB 526 CTC, rpoB 526 ACC, inhA-15 T, rrs 1401 G, gyrA 94 GGC, gyrA 90 GTG and rpoB 533 CCG.

5. The device according to claim 1, wherein said analysis sections are lateral flow biosensors.

6. The device according to claim 1, wherein said analysis sections are formed of a transparent cellulose or polymer material.

7. The device according to claim 1, wherein the backing film (31) and the three-dimensional mark appearing in the segments have different colours.

8. The device according to claim 1, wherein the first and second elongated sample strips first extend in a radial direction from the centre of the sample pad before they are angled such that the analysis sections of the first and second elongated sample analysis strips are arranged parallel to each other and the result is detectable from the combination of segments arranged transverse to the elongated sample analysis strips.

9. The device according to claim 1, wherein the corresponding segments of the first and second analysis section are arranged transverse to the analysis sections.

10. The device according to claim 1, further comprising a third sample analysis strip (24), wherein the third sample analysis strip is elongated and extends from the sample pad between said first and second elongated sample analysis strips, and wherein the third sample analysis strip comprises a straight analysis section divided into multiple segments, each segment of said analysis strip having immobilized thereto nucleic acids which sequence is different to a nucleic acid sequence of any other segment of said first, second or third analysis strip and that is complementary to a target nucleic acid sequence, wherein said segments are configured to indicate the presence or absence of a target nucleic acid in the liquid sample, and said analysis section of said third sample analysis strip having the same length and is arranged between said first and second elongated sample analysis strips such that the analysis result is detectable from the combination of the corresponding segments of the three analysis strips.

11. The device according to claim 8, wherein an angle α within the range of 40°-80° is formed between the first and second elongated sample strip, and a notch (23), extending from the periphery towards the centre of the sample pad, is formed in the sample pad between the first and second elongated sample strip such that the sample is directed towards the first and second elongated sample strip.

12. The device according to claim 10, wherein the third elongated sample strip extends in a radial direction from the sample pad from the tip of the notch.

13. The device according to claim 1, further comprising a fourth (25) and fifth (26) sample analysis strips, wherein the fourth and fifth sample analysis strips are elongated and extends from the sample pad, and wherein the fourth and fifth analysis strips comprises a straight analysis section divided into multiple segments, each segment of said analysis strip having immobilized thereto nucleic acids which sequence is different to a nucleic acid sequence of any other segment of said first, second, third, fourth or fifth analysis strip and that is complementary to a target nucleic acid sequence, wherein said segments are configured to indicate the presence or absence of a target nucleic acid sequence in the liquid sample, and said analysis section of said fourth and fifth sample strips having the same length and are arranged on opposite sides of the first and second elongated sample analysis strip such that the analysis result is detectable from the combination of the corresponding segments of all four or five analysis strips.

14. The device according to claim 10, wherein the fourth and fifth elongated sample strip extend in a radial direction from the centre of the sample pad before they are angled such that the analysis sections are arranged parallel to each other as well as the first and second analysis sections and aligned with the analysis sections of the first and second elongated sample strip.

15. The device according to claim 1, wherein a support structure is formed within the housing to support the sample pad and elongated sample strips in the correct position within the housing.

16. A method for determining the presence or absence of a target nucleic acid sequence in a sample from a subject, the method comprising the steps of: a) providing a biological sample, which has previously been obtained from a subject in a non-invasive manner, b) subjecting the sample to selective amplification of at least one target nucleic acid with a predetermined sequence to obtain amplified target nucleic acid, c) applying the amplified target nucleic acid to the sample pad of the device (10; 30; 50) according to claim 1, d) incubating said device for a period sufficient to enable detection of said target nucleic acid by means of a detection agent, and e) detecting the presence or absence of the at least one target nucleic acid with a predetermined sequence.

17. The method according to claim 16, wherein said nucleic acid is DNA or RNA, such as bacterial DNA, such as DNA from Mycobacterium tuberculosis complex.

18. A kit comprising a device according to claim 1 and at least one probe specific for a multi-resistance marker, at least one probe which is specific for the corresponding wild type sequence, and a detection agent.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows transmission electron microscopic (TEM) images at 50,000× and 300,000× magnification (A and B) shows that the gold nanospheres have an average diameter of 15±0.5 nm. A shift light absorbance spectra (C) from 520 nm to 528 nm shows that the prepared gold particles are conjugated with the oligonucleotides. Single smooth spectrum of DLS measurements (D) confirms conjugation and monodispersion of the AuNP-oligonucleotide conjugates.

(2) FIG. 2 shows the results from the investigation of the limit of detection of the PLP-LF method by testing 10-fold dilutions of genomic MTB DNA (A). The densitograms in B, plotted by computing the local color intensities, depicts the semi-quantitative changes in the control and test lines.

(3) FIG. 3 shows the results from testing characterized genomic DNA samples (300 ng) isolated from clinical MTB containing wild type or mutant codons of rpoB 531 and katG 315. The densitograms qualitatively shows the presence and/or absence of wild type and/or mutant genotypes of the clinical isolates.

(4) FIG. 4 is a listing of the nucleic acid sequences of disclosed in Example 1 with SEQ ID NO:1-18.

(5) FIGS. 5a, 6a and 7a illustrates a perspective view of different embodiments of the device 10; 30; 50 according to the invention. The devices 10; 30; 50 are intended for analysing liquid samples comprising amplified nucleic acids.

(6) FIGS. 5b and 8 illustrate schematically a sample pad and a first and a second elongated analysis strip of an embodiment 10 of the device.

(7) FIG. 6b illustrates schematically a sample pad and a first, a second and a third elongated analysis strip of an embodiment 30 of the device.

(8) FIG. 7b illustrates schematically a sample pad and a first, a second, a third, a fourth and a fifth elongated analysis strip of an embodiment 50 of the device.

(9) The device 10 illustrated in FIG. 5a comprises a rectangular protective housing 11 that has a front side 12, back side 13 and four side surfaces 14. The device 10 is intended to be arranged substantially horizontal with the front side 12 facing upwards during use.

(10) In the front side 12 of the housing 11, a substantially circular sample inlet passage 15 is formed such that the sample that needs to be analysed could access a sample pad 16 from the outside of the housing 11. Furthermore, two openings 17 through which the analysis result could be viewed are formed in the front side of the housing. The openings 17 could be covered by a transparent layer to protect the interior of the housing. The size and shape of the sample inlet passage 15 and the openings 17 could be modified in several ways without departing from the scope of the invention. For example, the two openings could be replaced by one larger opening.

(11) The housing is for example made of a plastic material that provides support for the different components of the device and provides the required protection to the device.

(12) The housing encloses a sample pad 16 and a first 18 and second 19 elongated sample analysis strips illustrated schematically in FIG. 5b.

(13) The sample pad 16 is in the illustrated embodiments substantially circular and made of an absorbent fibre material for example made of glass, synthetic fibres or a cellulose material. The liquid sample is supplied to the sample pad 16 and flowing through the sample pad by capillary movement to the first 18 and second 19 elongated sample analysis strips. This embodiment is illustrated in FIG. 5b and FIG. 8.

(14) In order to facilitate the flow of the sample, a notch 23 extending from the periphery of the sample pad towards the centre of the sample pad is formed in the sample pad between the first and second elongated analysis strip. The illustrated sample pad is substantially circular but the shape could be modified in several ways to facilitate the flow of the sample to the elongated sample analysis strips. An alternative shape could be like a droplet with the narrow end facing the elongated sample analysis strips.

(15) The first section of the sample analysis strips, connected to the sample pad 16, is preferably made of the same material as the sample pad 16 to transfer the sample to the respective sample analysis section 20. In order to further improve the transfer of the sample from the sample pad to the sample analyse strips, the sample pad and the part of the elongated sample analysis strips extending from the sample pad to the sample analysis section is formed in one piece of material.

(16) The analysis sections 20 of the first and second elongated sample strips have the same length and are arranged parallel to each other side by side such that the result is readable from the front side of the housing from the combined result from the two sample analysis sections 20 of each strip.

(17) Each analysis section comprises the same number of segments 22. The segments 22 in each sample analyse section are arranged side by side to provide the desired contact between adjacent segments and make it possible for the sample to flow through the sample analysis section. The sample analysis section, i.e. the different segments arranged adjacent to each other, are made of a transparent material arranged above the backing film 31. The transparent material has a thickness of at least 0.1 mm and once one of the segments is detecting the predetermined gene or substance in the sample, a three-dimensional mark appears within the transparent layer of the segment. The transparent layer in combination with the backing film provides a very distinct mark that is easy for the user to read. The different segments are not visible on the analysis strips but when the sample is applied, a mark will appear in the segment corresponding to the result from the analysed sample. The result is detectable by the combination of the two corresponding transverse segments of the first and second sample analysis stripe.

(18) An angle α within the range of 40°-80° is formed between the first and second elongated sample strip to ensure that the sample flows easily into the elongated sample analysis strips.

(19) In FIGS. 6a and 6b an alternative embodiment of the device is illustrated schematically. The housing has substantially the same design as in the previous embodiment but the device comprises an additional third elongated sample analysis strip arranged between the first and second elongated sample analysis strip. The third elongated sample analysis strip is substantially identical to the first and second elongated sample analysis strip and makes it possible to analyse further characteristics in the same device. The third elongated sample strip extend in substantially radial direction from the centre of the sample pad from the tip of the notch formed between the first and second elongated sample analysis strip.

(20) In FIGS. 7a and 7b a further alternative embodiment 50 of the device 30 is illustrated schematically. The housing has substantially the same design as in the previous embodiment but the device comprises a fourth 25 and a fifth 26 elongated sample analysis strip arranged on outside the first and second elongated sample analysis strip, i.e. on opposite sides of the first and second elongated sample analysis strip. The fourth and fifth elongated sample analysis strips are substantially identical to the first and second elongated sample analysis strip and makes it possible to analyse further characteristics in the same device. The fourth and fifth elongated sample strip first extend in substantially radial direction from the centre of the sample pad before they are angled such that the sample sections are arranged parallel and side by side to the sample sections of the first and second elongated sample analysis section.

(21) An angle β within the range of 60°-100° is formed between the fourth and fifth elongated sample strip to ensure that the sample is flowing easily into all elongated sample analysis strips.

(22) In FIG. 7b sample section is illustrated in perspective. The sample pad 16 and elongated sample analysis strips 18, 19, 24, 25, 26 preferably comprises a plastic backing film 31 arranged opposite to the inlet passage 15 and the openings 17 in the housing to prevent that the liquid sample is leaking from the sample pad and elongated sample analysis strips. Each of the first, second, third, fourth and fifth elongated sample analysis strip comprises a analysis section 20 comprising a number of segments prepared to display a mark if a predetermined nucleic acid sequences are detected in the liquid sample flowing through the sample section 20. Each of the first, second, third, fourth and fifth elongated sample analysis strip furthermore comprises an absorbent pad 28 arranged in the end of the sample strip. The absorbent pad facilitates the flow of sample through the analysis section 20.

(23) The different segments 22 of the sample sections 20 are not visible before the sample has been applied and the result is displayed on the sample analysis section by a line or symbol that are appearing on the sample analysis strip if the particular bacteria or nucleic acid is triggering the marking on that segment is detected in the sample.

(24) The number of segments in the sample section 20 could be adapted to the specific analysis that the device is adapted for but mostly comprises at least 5 segments. The analysis result is read from the combination of the result indicated on the respective sample analysis sections arranged side by side, i.e. the two, three or five segments arranged transverse to the sample sections 20.

EXAMPLE 1

Summary

(25) The following Examples disclose proof of concept the preparation and analysis of samples Mycobacterium tuberculosis complex (MTB) underlying the present invention.

(26) Materials and Methods

(27) Chemicals and Oligonucleotides:

(28) Streptavidin from Streptomyces avidinii, gold(III)chloride trihydrate (HAuCl4), sucrose, dithiothreitol (DTT), Triton X-100, trisodium citrate, Tris (hydroxymethyl)aminomethane hydrochloride (Tris-HCl), Tween 20, Ethylenediaminetetraacetic acid (EDTA) and bovine serum albumin (BSA, for oligonucleotide-AuNP conjugates), Sodium chloride-sodium citrate (SSC) buffer (pH 7.0), phosphate buffer saline (PBS, pH 7.4, 0.01 M), and sodium chloride (NaCl, 5 M, pH 7.0), ATP, dNTPs, oligonucleotides (purchased from Integrated DNA Technologies and Sigma-Aldrich). For the LF assay, binder-free borosilicate glass fiber pads (grade A/C), cellulose fiber absorbent pads (grade 113) and nitrocellulose membrane attached to the laminated cards/strips (0.4 and 0.5 cm width).

(29) Bacterial Strains and DNA Extraction:

(30) The reference strain MTB H37Rv (ATCC 25618) and ten clinical MTB isolates (Table 2) were cultured on Lowenstein-Jensen medium with and without 40 mg/L of RIF, respectively. DNA was extracted and 10 μg of genomic DNA was fragmented enzymatically using 10 U each of Nael and HpyCH4V, and 1× CutSmart buffer (New England Biolabs, Ipswich, Mass., USA) at 37° C. for 90 min followed by enzyme inactivation at 65° C. for 20 min. DNA concentration was measured by the dsDNA HS and BR assays using Qubit 2.0 fluorometer (Life Technologies, Carlsbad, Calif., USA).

(31) TABLE-US-00002 TABLE 2  Genotypic information on strains. Strain ID rpoB genotype katG genotype 2 S531L (TCG/TTG) S315T (AGC/ACC) 4 S531L (TCG/TTG) S315T (AGC/ACC) 8 S531L (TCG/TTG) S315T (AGC/ACC) 9 S531L (TCG/TTG) S315T (AGC/ACC) 12 S531L (TCG/TTG) S315T (AGC/ACC) 13 S531L (TCG/TTG) WT 17 S531L (TCG/TTG) S315T (AGC/ACC) 19 WT WT 20 WT WT 21 WT WT

(32) Padlock Probes and Rolling Circle Amplification:

(33) Sequences of oligonucleotides used in this study are given in FIG. 4. Four PLPs were designed to target two codons in the genes katG and rpoB and their corresponding wild type sequences (katG 315 ACC (MUT), katG 315 AGC (WT), rpoB 531 TTG (MUT) and rpoB 531 TCG (WT). After verifying secondary structures using Mfold Web Server, the PLPs were phosphorylated at the 5′ end by incubating a reaction mixture consisting of 1 μM oligonucleotide, 1×PNK buffer A, 1 mM ATP), and 1 U/μl T4 polynucleotide kinase (Thermo Scientific, Waltham, Mass., USA) at 37° C. for 30 min, followed by enzyme inactivation at 65° C. for 20 min. Confirmation of PLP efficacy was done by performing C2CA [Dahl et al. Proc Natl Acad Sci USA 2004, 101, 4548-4553] with modifications. Sensitivity of the assay was evaluated by LF strips with amplicons prepared from 300 pg, 3 ng, 30 ng and 300 ng of genomic DNA.

(34) Preparation and Characterization of Oligonucleotide Conjugated Gold Nanoparticles:

(35) Gold nanoparticles were prepared by a standard citrate reduction method [Frens G. Nature Phys Sci. 1973, 241, 20-22] with modifications. In a dry 500 mL round-bottom borosilicate glass flask, cleaned in aquaregia (nitric acid and hydrochloric acid in 3:1 ratio), 100 mL of 0.01% HAuCl4 in MilliQ water was boiled with vigorous stirring. Four milliliters of 1% trisodium citrate solution was added and after turning wine-red. Fifty micromolar of thiolated oligonucleotide, designed to hybridize to a sequence present in all C2CA monomers, was reduced by 500 mM of DTT in SSC buffer for 30 min. A NAP™-5 column (GE Healthcare Biosciences, Little Chalfont, UK) was used to purify the oligonucleotides and eluted directly into 1 mL of AuNPs. After incubating it for 2 h at 37° C., 1 M NaCl was incrementally added and kept for ‘aging’ at 4° C. The solution was centrifuged at 13,000 g for 25 min, the supernatant discarded and the AuNP-oligonucleotide conjugates re-dispersed in 1 mL of 5% BSA, 0.25% Tween 20 and 20 mM Tris-HCl (pH 8.0).

(36) Conventional transmission electron microscope (TEM) images of the prepared AuNP were obtained at 100 kV to check the quality of the prepared particles. The AuNP-oligonucleotide conjugates were characterized by measuring their light absorption at 520 nm in a Multi-Mode Microplate Reader (SpectraMaxR M5, Molecular Devices) and their surface charge (ζ-potential) was measured by dynamic light scattering (DLS) using Zetasizer Nano ZS90 (Malvern, UK) equipped with a 4.0 mW HeNe laser and an avalanche photodiode detector.

(37) Design, Assembly and Preparation of Lateral Flow Strips:

(38) The 100×5 mm LF strip consists of a sample application pad, nitrocellulose membrane and absorbent pad that are mounted on a thin plastic backing. The dry sample pad (25×5 mm), after saturation with 1% BSA, 1% Triton X-100, 20 mM Tris-HCl, 100 mM NaCl; pH 8.0), fixed on one end of the nitrocellulose membrane (45×5 mm) with an overlap of 2-3 mm and the absorbent pad (30×5 mm) was fixed on the other end of the nitrocellulose membrane. The biotinylated strip oligonucleotides (FIG. 3) were immobilized in test and control zones on the nitrocellulose membrane. The control zone contained one line of immobilized oligonucleotides complementary to the AuNP-oligonucleotide conjugates. The test zone contained 4 lines separated by 3 mm, where each line consisted of unique strip oligonucleotides for detection of the C2CA monomers corresponding to their specific genotypes of katG 315 WT, katG 315 MUT (AGC/ACC), rpoB 531 WT and rpoB 531 MUT (TCG/TTG) by hybridization. Fifty micromolar of the strip oligonucleotide was mixed with an equal volume of 1×PBS containing 15 μM of streptavidin. After incubation at 37° C. for 2 h, the streptavidin-conjugated oligonucleotide was immobilized on the nitrocellulose membrane using a nanoplotter (Nano-Plotter NP2.0, GeSiM, Grosserkmannsdorf, Germany).

(39) Visualization of C2CA Amplicons on Lateral Flow Strips:

(40) Fifty-five microliters of the C2CA monomers (amplified nucleic acid fragments) were hybridized with 13 μL of AuNP-oligonucleotide conjugates and applied to the sample pad of LF strips, drop by drop. The sample was allowed to flow for 5-7 min and washed with 4×SSC buffer for visualization of the red color bands. Color development in the control line indicated the positive assay control, while the signals from each test line specifically referred to presence of WT and/or MUT genotypes of katG 315 and rpoB 531. Intensity graphs were generated for the bands and pixel-densities were measured to quantify the results in densitograms, using the open source tool ImageJ (Version 1.49q) [Schneider et al. Nat. Meth. 2012, 9, 671-675]

(41) Results

(42) As shown herein, the present Example provides a proof-of-principle assay which able to produce rapid visual signals to discriminate between wild type and the mutations in katG and rpoB genes, causing MDR-TB, i.e. resistance to INH and RIF. The method can produce visual signals in approximately 75 min and the results can guide clinicians in taking informed decisions on public health control actions as well as adjusting to an effective antibiotic regimen. This assay provides preliminary alternative information to the time-consuming conventional DST and offers a compatible solution for resource-limited clinical laboratories. Thus, there present inventors have developed a simple, specific and cost-effective diagnostic test for the prompt identification of drug and/or antibiotic resistance. The DNA-based test is suitable for application in resource-limited clinical laboratories and provides information about the drug and/or antibiotic resistance pattern of bacteria infecting a patient, which is valuable for clinicians in order to take appropriate actions for treatment and infection control.

(43) Evaluation of AuNP-Oligonucleotide Conjugates:

(44) The TEM images of the AuNP (FIGS. 1 A and B) confirm the size of sphered particles to be ±3.5 nm. Size distribution curve based on the light absorption of AuNP showed a λ-max at 520 nm before oligonucleotide conjugation and the peak shifted to 527 nm after the conjugation (FIG. 1C). The DLS measurements (FIG. 1D) revealed an average diameter of 90±4 nm with a single peak indicating monodispersed solution without particle aggregation. The ζ-potential measurements of −37.4 mV and −34.5 mV for AuNP and their oligonucleotide-conjugates, respectively, showed that the preparations were stable.

(45) Limit of Detection of Padlock Probe-Lateral Flow Test:

(46) Limit of detection (LOD) of this PLP-LF assay was performed in triplicates by testing various dilutions of genomic DNA from the reference strain MTB H37Rv. Since both signals were clearly observed with 300 ng of DNA (FIG. 2A), in this proof-of-concept study, further experiments with DNA from clinical samples (Table 2) were performed using this amount. Densitograms (FIG. 2B) based on the local pixel density of red color signals developed on lateral flow strips correlated with the visual observation. The signal intensity of rpoB 531 WT was lower compared to katG 315 WT, which could be due to the high GC content in the target region of the rpoB gene, potentially resulting in a lower yield of C2CA monomers. Minor variations were observed among the densitograms of control lines, even though the lateral flow strips from the same printing session were used. The improvements to achieve higher sensitivity would enable direct testing on sputum samples.

(47) Visual Evaluation of the Padlock Probe-Lateral Flow Test on Clinical Isolates of MTB:

(48) A set of 10 clinical isolates MTB (Table 2) that were resistant and/or susceptible to INH and RIF, were tested by the PLP-LF method. As seen in FIG. 3, six strains (ID numbers: 2, 4, 8, 9, and 12) contained a mutant katG and rpoB codons; three strains (19, 20 and 21) possessed only wild type codons and one strain (13) showed the presence of wild type katG codon but mutant rpoB codon. All the visual signals of PLP-LF assay of the tested strains were fully concordant to genotypic characterization of the strains by pyrosequencing and the respective densitograms below each LF strip correspond to their visual signals. The method could be expanded by including a variety of other mutations causing MDR-TB, and the sensitivity could be improved for application directly on sputum samples from TB patients.