Description:Field of the Invention
The present invention relates to simple and rapid diagnosis of mycobacterium tuberculosis via extraction-free, direct polymeric chain reaction (PCR) techniques. More particularly, the present invention relates to the method contains specific reagents for extracting DNA from the sample using tailor made swab and probes for the detection of Mycobacterium tuberculosis.
Background of the Invention
Tuberculosis (TB) is a common infectious disease caused by mycobacterium, mainly M. tuberculosis. It usually attacks the lungs (as pulmonary TB) but can also affect the central nervous system, the lymphatic system, the circulatory system, the genitourinary system, the gastrointestinal system, bones, joints, and even the skin. TB is easily spread by airborne transmission of small droplets. TB remains major global health threat as it is the 13th leading cause of deaths and the second leading infectious killer after COVID-19 (above HIV and AIDS). According to World Health Organisation, an estimated 10.6 million people fell ill with tuberculosis (TB) worldwide and 1.6 million people died in 2021 (including 187 000 people with HIV). TB is present in all countries with all age groups, but it can be curable and preventable. Multidrug-resistant TB (MDR-TB) remains a public health crisis and a health security threat. Only about 1 in 3 people with drug resistant TB accessed treatment in 2021. An estimated 74 million lives were saved through TB diagnosis and treatment between 2000 and 2021. US$ 13 billion is needed annually for TB prevention, diagnosis, treatment, and care to achieve the global target agreed at the 2018 UN high level-meeting on TB.
Currently, the most commonly used screening methods for people with suspicious symptoms of tuberculosis and people at high risk of tuberculosis are symptom screening and chest X-ray examination. Common symptoms of tuberculosis include cough, sputum production, fever, night sweats and weight loss. Symptoms often come from the chief complaints of suspected persons and are highly subjective. Different diseases often show the same or similar symptoms. Although this method of symptom screening is low-cost, it is not enough to identify true tuberculosis suspects, nor can it distinguish between tuberculosis suspects. and patients with other diseases. Chest X-ray examination is currently the most widely used method to examine lung diseases. Normal and abnormal tissues and structures coexist on chest X-ray films. Even if experienced medical personnel spend enough time reading the pictures, the accuracy of the reading results cannot be guaranteed. Therefore, in actual work, those with abnormal symptom screening or chest X-ray examination results are often recommended to designated tuberculosis medical institutions for further examination.
The oldest method, sputum smear microscopy is the standard diagnostic method used widely for diagnosing TB. The benefit of the test is easy to perform, cost effective but fails to detect more than half of all active cases.
Culture remains the gold standard for laboratory confirmation of TB disease, and growing bacteria are required to perform drug-susceptibility testing and genotyping. However, it is time-consuming and requires adapted infrastructures and well-trained laboratory staff, which can delay effective medical interventions.
The TB skin test is performed by injecting a small amount of fluid (called tuberculin) into the skin on the arm. The interpretation of the tuberculin skin test takes 48-72 h which requires trained supervisor. This test therefore tends to be inaccurate under certain biological conditions and is incapable of distinguishing between latent TB and active TB infection. Additionally, it is labour-intensive for the patient as well as the health-care provider.
Several nucleic acid amplification and nucleic acid hybridization techniques have been demonstrated their use for the detection and diagnosis of infectious diseases, however these methods suffer from drawbacks. One of the notable drawbacks is that current approaches is initial step of isolating and purifying nucleic acids from a clinical sample as part of the nucleic acid testing protocol. This extraction steps required in conventional methods prior to PCR, constitutes a major bottleneck in the diagnostic process, as it remains both manually laborious and expensive, and increases the chances of accidental contamination and human error. Furthermore, in a period of high demand, a shortage of nucleic acid extraction supplies can exacerbate the limitations of such detection methods.
Hence, the current diagnostic techniques are either inaccurate and time consuming or are expensive and demand highly sophisticated laboratories which are not available in resource-poor and developing countries. We developed new methods and techniques where rapid detection of TB can be achieved using extraction-free RT-PCR.
Objective of the Invention
The main objective of the present invention is to provide a simple and rapid method for the detection of MTB.
Another objective of the present invention is to provide an efficient reagent for extracting DNA directly without any DNA extraction procedures.
Yet another objective is to provide a facile and efficient process for the preparation of primers and probes.
Yet another objective of the present invention is to the accuracy of HLS’s Quantiplus® MTB FAST Detection Kit (Real-Time Qualitative PCR Kit) was found to be 100% sensitivity and specificity using the quick extraction reagent.
Summary of the Invention
Accordingly, in respect of the present invention provides a method which involves the detection of mycobacterium tuberculosis, using reagents with samples, sputum/BAL fluid/pus/ascitic fluid/synovial fluid. The method includes collecting a sterile swab and carefully rotate by dipping into the collected sample (Sputum/BAL fluid/Pus/Ascitic fluid) in the container for 10-15 sec. Insert the collected swab into the vial containing reagent (0.6 mL) to form a premixture. Twist the swab head against the bottom and sides of the tube for 10-12 times and pinch the swab head to the side wall of the vial. Placing the reagent vial containing the premixture in a dry bath, which is preheated to 75 °C and incubate for 10 minutes and give a short spin to the lysate at 5000 rpm for 30 sec. Then, all the mixture solutions were subjected to perform RT-PCR. The quick extraction reagents reacted with DNA containing SWAB without any DNA extraction procedures leading to the detection of Mycobacterium tuberculosis with 100% sensitivity and specificity; and can process a relatively larger number of samples in a short time. The present invention has found Quantiplus® MTB FAST Detection Kit which is a Real Time PCR based in-vitro diagnostic assay for detection of MTB Complex (MTBC). The kit works with TB Culture, Body fluids (CSF, Pleural Fluid, Ascetic Fluid and Synovial fluid) Sputum, Pus, Menstrual fluid, Urine and Tissue. The kit contains an advanced formulated qPCR mix with UDG/UNG which enables Performance of Fast PCR in shorter run time, UDG/UNG helps in controlling the PCR carryover contamination, primer probe mix, positive control, and internal control.
Brief description of the drawings
Figure 1 shows a flow diagram of the method of testing for Mycobacterium tuberculosis using quick extraction reagent.
Figure 2 shows a flow chart of the method of testing for Mycobacterium tuberculosis using quick extraction reagent in accordance with an embodiment of the invention.
Figure 3 shows the conventional extraction process of RT-PCR and extraction-free of RT-PCR.
Figure 4 shows a sensitivity of detecting the mycobacterium tuberculosis, in accordance with one embodiment of the present invention.
Figure 5 shows the clinical validation of Quantiplus® MTB FAST Detection Kit.
Figure 6 shows the extraction-free MTB testing with various swab types.
Figure 7 shows the extraction-based MTB testing with various swab types.
Figure 8 shows the evaluation of reagents combinations for extraction free MTB testing.
Figure 9 shows the evaluation of reagents combinations for extraction free MTB testing.
Detail Description of the Invention
The following are only preferred embodiments of the present invention, and the scope of protection of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily make changes or changes that are covered by the present invention. Within the scope of protection. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.
The method provided by the present invention will be described in more detail in conjunction with the following specific examples, which should be understood as specific descriptions and elaborations of the present invention, and should not be construed as a limitation of the scope of the present invention. Unless otherwise specified, the reagent components used in the following examples are all components of the kit of the present invention. Modifications or changes made by those skilled in the art on the basis of the present invention, these equivalent modifications also fall within the scope of the claims described in the claims of the present invention. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.
The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”.
Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.
The term “tuberculosis” or “TB”, as used herein, refers to a disease or infection caused by Mycobacterium tuberculosis. It includes pulmonary tuberculosis and extrapulmonary tuberculosis.
The term “detects” or “detection”, as used herein, refers to ex-vivo detection performed using a sample obtained from a subject.
The term “subject”, as used herein, refers to a mammal. It refers to a mammal having or suspected of having Mycobacterium tuberculosis infection. It includes tuberculosis patients and subjects suspected of having Mycobacterium tuberculosis infection. Mammals include humans, primates, dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, mice, rats, hamsters and guinea pigs. Unless otherwise noted, the terms “patient” or “subject” are interchangeably used herein. In an example, the subject is human.
Sequences used in the present disclosure
SEQ.ID Oligo Name Oligo Sequence 5' to 3' Length Remarks

1 rpoB-F2N CAGACCGCATACGTTGAT 18 Rifamicin Resistance
2 CACACAGCAGACGTTGATT 19
3 AACACCGCAGACGTTGATAC 20
4 ACAAACTGCAGAAGCTGACTA 21
5 GACACACCGCAGACGTTGAT 20
6 rpoB-R CTCACGTGACAGACCGCCG 19
7 ACTCACGTGACAGACCGCGCT 21
8 ACTCACGTGACAGACCGCGCT 21
9 GCCTCACGTGACAGACCGCCGC 22
10 CTCACGTGACAGACCGCCGATT 22
11 rpoB Block PB2 516 ATGGTCCAGAACAACCG 17 3' PHOSPHATE
12 ATGTACCAGAACAACCCG 18
13 GATGTACCAGAACAACCCACG 21
14 CTATGTACCAGAACAACCCTAT 22
15 ATGTACCAGAACAACGC 17
16 KatG-F GATGGGCTGGCGCTGGAA 18 Isoniazid resistance
17 AGATGGGCTGGCGCTGGAA 19
18 TCGATGGGCTGGCGCTGGATA 21
19 GATGCGCTGCgGCTGGAATT 20
20 GATGCGCGCTGGCGCTGGAA 20
21 KatG-R CCGTACAGGATCTCGAGGAA 20
22 GCCGTACAGATATCTCGAGGAA 22
23 GCGTACAGGATCTCGAGGA 19
24 CCGTAATGCGATCTCGAGGAA 21
25 TAGCCGTACAGGATCTCGAGGAA 23
26 InhA-F GGAAATCGCAGCCACGTTAC 20
27 GGAAATCGCAGCCACGTTACCGCAG 25
28 GGAAATCGCAGCCACGTTAC 20
29 AATCGGGAAAGCAGCCACGTTAC 23
30 TTCAGGAAATCGCAGCCACGTTAC 24
31 InhA-R TTCAGTGGCTGTGGCAGTCA 20
32 GGAAATCGCAGCCACGTTACCGCA 24
33 TTCAGTGGCTGTGGCAGTCA 20
34 TTCACACCAAGTGTTTCGACCAG 23
35 ATCGATGGCTGTTGGCGCTGGATA 24
36 M18F AACACCATAGTGTTTCGACCAG 22 MTB Complex Target IS1081
37 TACACCAAGTGTACTCGACCAG 22
38 ACACCAAGTGTTTCGACCAG 20
39 ATACACCAAGTGTTTCGAGCAG 22
40 GCACACCAAGTGTTTCGACCA 21
41 M18R GCATGCTACGCTAGCATGGGAGT 23
42 CTACACCAAGTGTACTCGTATC 22
43 CTGCTACCTGCTGGGAGTATC 21
44 ATTCACTACCTGAGTGTTTCGAC 23
45 TCACACCAAGTGGTCGACCAG 21
46 HLSMTBF2(MPT) ATGACCTGAGCAAGCAGACAT 21 MTB Complex Target MPT 64
47 TGAACTGAGCAAGCAGACCG 20
48 TGAACTGAGCAAGCAGACCG 20
49 HLSMTBR2(MPT) CAAGTGGTGATAATTCACCGGTC 23
50 GTTCTGATAATTCACCGGGTC 21
51 GCTCTGATCAATTCACCGGGTC 22
52 BGF GAGCATCCTCCAACAGACACCA 22 Internal Contro Target Human Betaglobulin
53 AGCAACCTCAAACAGACACCA 21
54 ATAATTCCAGACACCAACCA 20
55 BGR CAACTTCATCCACGTTCACC 20
56 CCTCAAATCATCCACGTTCAC 21
57 CAACTTCAACTTCATCCACGTT 22
Note All red color are Locked Nucleic Acid (LNA)

SEQ.ID Oligo Name Oligo Sequence 5' to 3' Length Modification
5 3'
58 rpoB-WT-A (509- 514) ACTTGTCTCAGCTGCCTT 18 HEX MGBEQ
59 AATTGCGGCTCAGCTGCTC 19 HEX MGBEQ
60 CGATGCGGCTCAGCTGGCTGACTA 24 HEX BHQ1
61 TATTGCGGCTCAGCTGGCTGACGCT 25 HEX BHQ1
62 rpoB- WT-C (520- 524) GTCAACCCCGACAGCG 16 FAM MGBEQ
63 CTCAACGCTCGACAGCG 17 FAM MGBEQ
64 GCTCGTCAACCCCGACAGGGCT 22 FAM BHQ1
65 CGCTAACCCCGACAGGGCTTCAG 23 FAM BHQ1
66 B2 WT rpoB-B (514- 518) TTCATGGACCAGAAC 15 CAL RED MGBEQ
67 GGATTCATGGAGAACCC 17 CAL RED MGBEQ
68 AACCCTATCTCAGCTGGCTGACGCT 25 CAL RED BHQ1
69 GCTCGTCAACCCCGACAGGGCT 22 CAL RED BHQ1
70 rpoB- WT-D (524- 528) ACCCACAAGCGCCTGCGG 18 HEX MGBEQ
71 TTGACCCACAAGCGCC 16 HEX MGBEQ
72 ATTAGACCCACACGCCTGCGGAG 23 HEX BHQ1
73 GCATGACCCACAAGCGCCTGCGG 23 HEX BHQ1
74 EWT P4- rpoB (530-533) GACTGTCGGCGCTGG 15 FAM MGBEQ
75 ACGGTCGGCGCTGGACT 17 FAM MGBEQ
76 CCACGCATGACAAGCGTCGGCC 22 FAM BHQ1
77 CGCTGGGCATGACCCGTGCG 20 FAM BHQ1
78 KatG-P (313-318) ATCACCAGCGGCATC 15 FAM MGBEQ
79 ACCATCAGCGGCATCG 16 FAM MGBEQ
80 ATCACAGCCAAGAGTGGAATGCG 23 FAM BHQ1
81 AAGATCACAGCCAGTGGAATGCGG 24 FAM BHQ1
82 InhA-P (8-24) CGGCGAGACGATAGGT 16 HEX MGBEQ
83 GCGCCGAGACGATAGGT 17 HEX MGBEQ
84 CGAGTGGCGCCGAGACGATAGGT 23 HEX BHQ1
85 ATAGCGAGTGGCCGAGACGATAG 23 HEX BHQ1
86 M18P CGCTCCATCCGGCGAGTG 18 FAM BHQ1
87 GCCGCTCCATCCGGCGAGTA 20 FAM BHQ1
88 HLSMTBP2(MPT) GTATCGATAGCGCCGAATGCCGG 23 FAM BHQ1
89 ATATCGATAGCGCCGAATGCCCGA 24 FAM BHQ1
90 BGP GAAGTCTGCCGTTACTGCCCT 21 CAL RED BHQ2
91 CGCAAGTCTGCCGTTACTGCCCT 23 CAL RED BHQ2

The present invention is to provide a quick extraction agent for the detection of Mycobacterium tuberculosis in a biological sample.
In one embodiment, the process for the preparation of quick extraction reagent according to the present invention comprises of various surfactant such as nonyl phenoxypolyethoxylethanol (NP-40), polysorbate 20 (tween 20), phosphate buffer (PBS), sodium lauryl sulphate, sodium dodecyl sulphate (SDS), cetyl trimethylammonium bromide (CTAB), triton X-80, and triton X-100.
In another embodiment, the process for the preparation of reagent according to the present invention comprises of various solvents such as acetonitrile (CAN), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), ethanol (EtOH), methanol (MeOH), isopropyl alcohol (IPA) , glycerol, 2-ethoxy ethanol and water (H2O).
In yet another embodiment, the process for the preparation of reagent according to the present invention comprises of various bases such as lithium hydroxide (LiOH), sodium hydroxide (NaOH), potassium hydroxide (KOH), triethyl amine (Et3N), ammonia (NH3), calcium hydroxide (Ca(OH)2), barium hydroxide (Ba(OH)2), sodium carbonate (Na2CO3), potassium carbonate (K2CO3), sodium bicarbonate (NaHCO3) etc.
In yet another embodiment, the process for the preparation of reagent according to the present invention comprises of various salts such as sodium chloride (NaCl), potassium chloride (KCl), magnesium chloride (MgCl2), calcium chloride (CaCl2), etc.
In a preferred embodiment, the process for the preparation of reagent according to the present invention comprises of various antifoam such as SE-15, SE-30, antifoam L-30 emulsion, C-emulsion, silicon antifoam, antiformal 204, simethicone, octanal.
The embodiment of the present invention is to provide the preparation of the quick extraction reagent comprising the 500-800mL of sterile water was added to the 10-100mL of solvents [acetonitrile (CAN), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), ethanol (EtOH), methanol (MeOH), isopropyl alcohol (IPA), glycerol, 2-ethoxy ethanol and water (H2O)] to form a mixed solution. To this mixed solution was added to the base solution [lithium hydroxide (LiOH), sodium hydroxide (NaOH), potassium hydroxide (KOH), triethyl amine (Et3N), Ammonia (NH3), calcium hydroxide (Ca(OH)2), barium hydroxide (Ba(OH)2), sodium carbonate (Na2CO3), potassium carbonate (K2CO3), sodium bicarbonate (NaHCO3) 1-3M, 25-100 mL], followed by addition salts [sodium chloride (NaCl), potassium chloride (KCl), magnesium chloride (MgCl2), calcium chloride (CaCl2), 1-5 g]. To this solution were added various surfactants such as nonyl phenoxypolyethoxylethanol (NP-40), polysorbate 20 (tween 20), phosphate buffer (PBS), sodium lauryl sulphate, sodium dodecyl sulphate (SDS), cetyl trimethylammonium bromide (CTAB), triton X-80, and triton X-100. To the above solution was added antifoam (SE-15, SE-30, antifoam L-30 emulsion, C-emulsion, silicon antifoam, antiformal 204, simethicone, octanal, 10-40 mL). The reaction mixture was stirred for 15-45 minutes and stored for use.
In some embodiments, the invention is extraction free TB detection kit named Quantiplus® MTB FAST Detection Kit which is nucleic acid amplification test for the specific detection of all members of the MTB complex. The component of the kit comprises of DNA Fast qPCR Mix with UDG/UNG, Quick extraction reagent, MTB Fast Primer Probe Mix, MTB Fast Positive Control, Molecular Biology grade pure water (MBGPW). Composition of the components are described as below (Table 1).
Table 1: Composition of the components
Contents Description
DNA Fast qPCR Mix with UDG/UNG (2X) PCR Amplification mix Contains Taq Enzyme and UDG/UNG Enzyme and dNTP’s
Quick extraction reagent Contains detergent and nucleic acid stabilizer
MTB Fast PPM Contains target specific and IC specific primer and probe reconstituted in TE buffer
MTB Fast PC MTB Synthetic DNA clone
MBGPW Nuclease free PCR grade water

Taq polymerase is a thermostable DNA polymerase I, which it was originally originated from Thermus aquaticus, it is frequently used in the polymerase chain reaction. Adding the DNA polymerase to the reaction led to exponential DNA replication, greatly amplifying discrete segments of DNA between the primers.
However, after each round of replication the mixture needs to be heated above 90 °C to denature the newly formed DNA, allowing the strands to separate and act as templates in the next round of amplification. This heating step also inactivates the DNA polymerase. Taq polymerase is well-suited for this application because it is able to withstand the temperature of 95°C which is required for DNA strand separation without denaturing.
Thermostable Taq polymerase enables running the PCR at high temperature (~60°C and above), which facilitates high specificity of the primers and reduces the production of nonspecific products, such as primer dimer.
Uracil-DNA glycosylases (UDG) are evolutionary, well-preserved DNA-repair enzymes. The term UDG refers to a superfamily of enzymes comprising six sub-families. Family I UDG enzymes called UNG. Both UDG and UNG perform the same function in qPCR namely to prevent carryover contamination by previous PCR products.
UNG allows previous PCR amplification or mis-primed, nonspecific products to degrade, leaving native nucleic acid templates intended for amplification intact. UNG activation occurs as the first step of PCR at 50°C incubation for 2 minutes.
Preferably, the probe and the primer are single-stranded deoxyribonucleotide molecules. Probes or primers used in the present invention may include naturally occurring Deoxyribonucleotide triphosphate (dNTP's). In PCR, deoxynucleotide triphosphates (dNTPs) serve as building blocks for new DNA strands. There are four different types of dNTPS, these include: dATP(deoxyadenosine triphosphate), dCTP(deoxycytidine triphosphate), dGTP(deoxyguanosine triphosphate), dTTP(deoxythymidine triphosphate).
Molecular biology grade water is ideal for applications where highly pure, RNase-, DNase-, and protease-free water is required (e.g., PCR and other molecular biology reactions setup, DNA/RNA/protein extraction, purification, and storage reagents).
PCR primers are short, single-stranded segments of DNA that are designed to be complementary to the beginning and end of the target sequence that will be amplified. In a PCR, it is the primers that dictate exactly what sequence of DNA gets copied.
Two primers are utilized, one for each of the complementary single strands of DNA released during denaturation. The forward primer attaches to the start codon of the template DNA (the anti-sense strand), while the reverse primer attaches to the stop codon of the complementary strand of DNA (the sense strand).
In another preferred embodiment, oligonucleotides (primers and probes) were designed specific for two target gene (IS1081 and MPT64) of M. tuberculosis complex. They are highly conserved and specific primers and dual labelled hydrolysis probes.
The invention provides the oligonucleotides (primers and probes) for detection of Mycobacterium tuberculosis, which is designed based on the IS1081 and MPT 64 of Mycobacterium tuberculosis. The detection of two target genes and sites can effectively reduce the probability of missed detection and false detection, and improve the reagent quality.
The primers and probes detect the mutations in rpoB having a sequence selected from a group consisting of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77 associated with Rifampicin (RIF) resistance and Isoniazid-resistance (INH) associated mutations in the katG having a SEQ ID NO: 80 and inhA promoter region having a sequence selected from a group consisting of SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, and SEQ ID NO: 85.
Another embodiment of the invention relates to oligonucleotide primers (rpoB) capable of acting as reverse primers in a PCR reaction for amplifying MTB complex DNA, Preferably, reverse primer having a sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.
Yet Another embodiment of the invention relates to oligonucleotide primers (rpoB) capable of acting as forward primers in a PCR reaction for amplifying MTB complex DNA, the forward primers having a sequence selected from a group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.
Another embodiment of the invention relates to oligonucleotide primers (InhA) capable of acting as forward primers in a PCR reaction for amplifying MTB complex DNA, preferably, the forward primer having a sequence selected from a group consisting of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30.
Yet another embodiment of the invention relates to oligonucleotide primers (InhA) capable of acting as reverse primers in a PCR reaction for amplifying MTB complex DNA, preferably, the reverse primer having a sequence selected from a group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35.
Another embodiment of the invention relates to oligonucleotide primers capable of acting as reverse primers in a PCR reaction for amplifying MTB complex DNA. Preferably, the reverse primer having a sequence selected from a group consisting of SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51.
Another embodiment of the invention relates to oligonucleotide primers capable of acting as forward primers in a PCR reaction for amplifying MTB complex DNA. Preferably, the forward primer having a sequence selected from a group consisting of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48.
Another embodiment of the invention relates to oligonucleotide primers capable of acting as a probe for MTB complex DNA. Preferably, the probe having a sequence selected from a group consisting of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, and SEQ ID NO: 91.
In addition, kit contains a primer probe set to detected Human endogenous Internal Control (IC) in a single tube.
Internal control target Human beta globulin primer having a sequence selected from a group consisting of SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57.
The various combination of reagents was used for the detection of TB. The reagent volumes can vary from 5 µL to 100 µL for each adsorbed swab. The sample was heated to the temperature 40°C to 90°C for 3-15 minutes.
The treated samples were added to the primer-probe mixture for evaluation by subjecting to RT-PCR. The sample volumes were in the range of 5-20µL.
Fig. 2 represents the main embodiment of the invention is to provides a Method of Testing for Mycobacterium Tuberculosis (200), comprising the following steps:
• collecting a sterile swab (210) and carefully rotate by dipping into the collected sample (Sputum/BAL fluid/Pus/Ascitic fluid) in the container for 10-15 sec;
• Inserting the collected swab (220) into the vial containing reagent (0.6 mL) and ensure that the absorbent tip of the swab is completely immersed into the reagent to form a premix solution. Twist the swab head against the bottom and sides of the tube for 10-12 times and pinch the swab head to the side wall of the vial. Discard the used swab into the biohazard bag as per local regulations and screw the cap tightly;
• Placing the premix solution in a dry bath (230), which is preheated to 75°C and incubate for 10 minutes to give a pro-incubated mixture solution;
• Giving a short spin to the lysate of pro-incubated mixed solution (240) at 5000 rpm for 30 sec to obtain a supernatant; and
• Using 5µL of the supernatant as a template and proceed with RT-PCR assay (250).
In a preferred embodiment, the method involves various swabs made of different materials such as cotton, polyester, rayon, nylon, polyurethane, and calcium alginate.
In conventional PCR, the first step is the extraction of nucleic acids (DNA or RNA) from the sample. DNA extraction that is consistent across all the possible DNA purification chemistries including disruption of the cellular structure to create a lysate, and separation of the soluble DNA from cell debris and other insoluble material, binding the DNA of interest to a purification matrix, washing proteins and other contaminants away from the matrix and elution of the DNA. All these preliminary process takes place at least 60m.
The advantage of extract-free PCR in our study is that the amount required for testing is small amount. Besides, preprocessing the sample, the nucleic acid extraction process can be omitted, eliminating the need for a nucleic acid extraction kit or equipment. If there is a problem with the supply of equipment or kits, the direct PCR without nucleic acid extraction could be a useful replacement test. Additionally, the number of samples when using automatic nucleic acid extraction equipment is limited. The extraction-free PCR does not have this limitation and can process a relatively larger number of samples.
Yet another embodiment of the invention is Figure 3 shows that extraction-free real-time polymerase chain reaction (RT-PCR) is a method of performing PCR without the need for the traditional extraction steps to isolate and purify DNA from a sample. This approach aims to streamline the process, making it faster and simpler while maintaining the accuracy and sensitivity of traditional PCR methods.
The extraction-free polymerase chain reaction can reduce the time required for testing and solve the shortage of reagents or equipment by skipping the nucleic acid extraction process.
The extraction-free method uses raw samples directly, bypassing the extraction steps. This can be done using special reagents or modified protocols that neutralize inhibitors and protect the DNA. The extraction-free RT-PCR could be useful as a screening test that processes many samples in a short time.
Master Mix Preparation: Prepare a reaction mix containing Taq polymerase, UDG/UNG Enzyme, deoxynucleotide triphosphates (dNTPs), primers (M18F, M18R and HLSMTBF2, HLSMTBR2), the quick extraction reagents, magnesium ions, and a fluorescent dye or probe. Common dyes include SYBR Green, which binds to double-stranded DNA, or sequence-specific probes like TaqMan probes. Place the tubes or plate in the real-time PCR machine (thermal cycler with fluorescence detection capability. The thermal cycler goes through cycles of heating and cooling to denature the DNA, anneal the primers, and extend the new DNA strands.
This is the first step for regular cycling event and consists of heating the reaction chamber to 94–98°C (201–208°F) for 20–30 seconds. This causes DNA melting, or denaturation, of the double-stranded DNA template by breaking the hydrogen bonds between complementary bases, yielding two single-stranded DNA molecules.
In the next step, the reaction temperature is lowered to 50–65°C (122–149°F) for 20–40 seconds, allowing annealing of the primers to each of the single-stranded DNA templates. Two different primers are typically included in the reaction mixture: one for each of the two single-stranded complements containing the target region. The primers are single-stranded sequences themselves, but are much shorter than the length of the target region, complementing only very short sequences at the 3' end of each strand.
The temperature at this step depends on the DNA polymerase used; the optimum activity temperature for the thermostable DNA polymerase of Taq polymerase is approximately 75–80°C (167–176°F), though a temperature of 72°C (162°F) is commonly used with this enzyme. In this step, the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding free dNTPs from the reaction mixture that is complementary to the template in the 5'-to-3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxy group at the end of the nascent (elongating) DNA strand.
During each cycle, the fluorescence emitted by the dye or probe is measured. The increase in fluorescence is proportional to the amount of PCR product.
Determine the cycle number at which the fluorescence exceeds a set threshold, which correlates with the initial quantity of the target nucleic acid.

Evaluation of different swabs for extraction-free reagent
The swab head is made of from various materials such as cotton, polyester, rayon, nylon, polyurethane, and calcium alginate. Each material with a different composition will have unique properties to be assessed by the user depending on the intended use. Hence, various swab types with different compositions were investigated to test their suitability for extraction-free protocol using MTB culture sampling. In addition, all varieties of swabs were tested with the extraction kit method as a reference.
Table 2: Testing the extraction-free reagent with MTB culture using different swabs
Swab Samples 1 2 3 4 5 Reference
Ct Values 31.5 30.8 32.8 31.0 32.0 32.1

Table 3: Testing the different swabs with MTB culture using extraction kit
Swab samples 1 2 3 4 5 PCO NTC NTC
Ct Values 31.7 31.1 31.8 31.7 31.7 24.1 Not Amplified Not Amplified

Evaluation of testing
The Quick extraction reagent (>200) with various chemical compositions was prepared and tested. For example: HLS 1, HLS 2, HLS 3, HLS 4, HLS 5, HLS 6, HLS 7, HLS 8, and Reference. MTB samples used are MTB 84, MTB 189, and MTB 283. All HLS buffers prepared were tested against MTB cultures using an MTB Fast RT-PCR kit. The results were compared with the Extraction Kit as a standard column-based extraction method. The reagents HLS 3, HLS 6 and HLS 8 were found to be good as they amplified lower CT values with sample MTB 189 when compared with reference.
Table 4: MTB (FAM) Ct Values
Sample ID HLS1 HLS 2 HLS 3 HLS 4 HLS 5 HLS 6 HLS 7 HLS 8 REFERENCE
MTB 84 23.6 23.1 22.6 23.2 23.3 23.8 23.4 23.4 21.0
MTB 189 40.5 42.18 Not Amplified 40.8 41.6 Not Amplified 38.5 41.0 Not Amplified
MTB 283 20.0 19.4 19.5 20.0 19.9 20.1 19.2 18.9 18.5

Table 5: MTB (Texas Red/ROX) Ct values
Sample ID HLS 1 HLS 2 HLS 3 HLS 4 HLS 5 HLS 6 HLS 7 HLS 8 REFERENCE
MTB 84 24.9 23.4 23.1 23.4 23.5 23.4 23.3 23.9 23.5
MTB 189 23.8 23.7 23.2 23.4 23.2 23.2 23.4 23.3 23.4
MTB 283 26.9 22.8 23.1 23.5 23.2 23.1 23.8 23.0 24.6

In addition, reagent combinations HLS 9, HLS 10, HLS 11 were prepared and tested against MTB samples (MTB 174, MTB 187 and MTB 208) and compared the results with reference. The reagents HLS 1, HLS 9 and HLS 11 were found to be in good whereas HLS 10 is didn’t showed amplification either for MTB or internal control.

Table 6: MTB (FAM) Ct Values
Sample ID HLS 1 HLS 9 HLS 10 HLS 11
MTB 174 21.7 21.0 Not Amplified 21.4
MTB 187 35.7 35.1 Not Amplified 36.6
MTB 208 19.9 20.0 Not Amplified 19.5

Table 7: MTB (Texas Red/ROX) Ct Values
Sample ID HLS 1 HLS 9 HLS 10 HLS 11
MTB 174 23.0 22.4 Not Amplified 23.0
MTB 187 22.9 22.5 Not Amplified 23.1
MTB 208 22.6 23.9 Not Amplified 22.5

Furthermore, the ability of the reagent for extraction free MTB kit was demonstrated by selecting the best performed HLS reagent and testing their reproducibility and stability. HLS reagent HLS 1, HLS 3, HLS 6, HLS 9 and reference were tested against the MTB samples such as MTB 91, MTB 148 and MTB 178. HLS 3 and 9 are found to reproduce the better results as they showed the 2 Ct values early in comparison with reference. Whereas HLS 6 reagent didn’t reproduce the similar results.
Table 8: MTB (FAM) Ct values
Sample ID HLS 1 HLS 3 HLS 6 HLS 9 REFERENCE
MTB 91 Not Amplified Not Amplified Not Amplified Not Amplified 37.1
MTB 148 20.4 20.2 N/A 18.0 37.1
MTB 178 33.7 35.6 N/A 36.6 38.4

Table 9: MTB (Texas Red/ROX) Ct values
Sample ID REF 1 HLS 3 HLS 6 HLS 9 REFERENCE
MTB 91 23.0 27.2 25.4 24.5 23.5
MTB 148 22.9 23.7 Not Amplified Not Amplified 23.5
MTB 178 25.6 23.0 Not Amplified 24.8 23.8

Analytical sensitivity: The analytical sensitivity or the limit of detection (LOD) was determined using QCMD MTBDNA18S challenge panel. The LOD of the assay is 5 copies or bacilli per assay.
Analytical specificity: The specificity of the test Kit is ensured by the selection of the specific primers and probes. The primers and probes were checked for possible homologies to all sequences published in gene bank. Our selected new primer and probes were very specific M. tuberculosis when tested against potentially cross-reactive other pathogens.
Table 10: Testing the specificity of the kit with potentially cross-reactive pathogens
Control Group FAM (M. tuberculosis) Texas Red (IC)
Mycobacterium kansasii -- +
Mycobacterium abscessus -- +
Mycobacterium xenopi -- +
Mycobacterium fortuitum -- +
E. coli -- +
Staphylococcus aureus -- +
Bacillus subtilis -- +
INF-A -- +
INF-B -- +
Candida albicans -- +

Clinical validation of Quantiplus TB fast kit was done with Quality Control for Molecular Diagnostics (QCMD) MTB panel. QCMD panel results indicated 100% concordance with expected results. 50 clinical samples pretested with GeneXpert were tested with extracted DNA and extraction free protocols using Quantiplus TB Fast kit. All the results were found to in accordance with GeneXpert results.
The Quantiplus TB fast kit efficiency was further demonstrated by testing 37 known MTB positive specimens (extracted DNA) and 32 known MTB negative specimens (extracted DNA). In comparison the specimens were also tested using the reference kit (CB-NAAT). The accuracy of HLSS’s Quantiplus® MTB FAST Detection Kit (Real-Time Qualitative PCR Kit) was found to be 100%.

Sample Actual samples tested Reference Kit Quantiplus® MTB Fast Detection Kit Sensitivity Specificity
MTB Positive 37 37 37 100 % - NA -
MTB Negative 32 32 32 - NA - 100 %
Total 69 69 69 100 % 100 %
, Claims:We Claim:

1. A method of testing for Mycobacterium tuberculosis using quick extraction reagent, comprising:
collecting a sterile swab (210);
inserting the collected swab into a vial containing 0.6 mL of quick extraction reagent (220) to prepare a premix solution;
placing the premix solution in a dry bath (230), wherein the dry bath is preheated to 75°C and further incubated for 10 minutes to give a pro-incubated mixed solution;
giving a short spin to the lysate of the pro-incubated mixed solution (240) at 5000 rpm for 30 sec to obtain a supernatant, and performing an RT-PCR assay (250) to detect Mycobacterium tuberculosis.

2. The method as claimed in Claim 1, wherein the quick extraction reagent includes a surfactant (0.2-2.5%), a solvent (10-100mL), a base (1-3M, 25-100mL), a salt (1-5g), an antifoam (10-40mL), and water (500-800mL).

3. The method as claimed in Claim 1, wherein the surfactant is chosen from at least one of nonyl phenoxypolyethoxylethanol (NP 20), Polysorbate 20 (Tween 20), phosphate buffer (PBS), sodium lauryl sulphate, sodium dodecyl sulphate, cetyltrimethylammonium bromide, Triton X-80, and Triton X-100.

4. The method as claimed in Claim 1, wherein the solvent is chosen from at least one of acetonitrile, dimethylsulfoxide, dimethylformamide, ethanol, methanol, isopropyl alcohol, glycerol, 2-ethoxy ethanol, and water.

5. The method as claimed in Claim 1, wherein the base is chosen from at least one of lithium hydroxide, sodium hydroxide, potassium hydroxide, triethylamine, ammonia, calcium hydroxide, barium hydroxide, sodium carbonate, potassium carbonate, and sodium bicarbonate.

6. The method as claimed in Claim 1, wherein the salt is chosen from at least one of salts such as sodium chloride, potassium chloride, magnesium chloride, and calcium chloride.

7. The method as claimed in Claim 1, wherein the antifoam is chosen from one of SE-15, SE-30, antifoam L-30 emulsion, C-emulsion, silicon antifoam, antiformal 204, simethicone, and octanal.

8. The method as claimed in Claim 1, wherein the reagent volume of 5µL-100µL for each adsorbed swab and the swab was heated to the temperature of 40°C to 90°C for 3-15 minutes.

9. The method as claimed in Claim 1, wherein the swab is selected from the group consisting of at least one of TB Culture, bronchoalveolar lavage fluid, cerebrospinal fluid, pleural fluid, ascetic fluid, synovial fluid, sputum, pus, menstrual fluid, urine, and tissue.

10. The method as claimed in Claim 1, wherein, the various swabs are made of different materials such as cotton, polyester, rayon, nylon, polyurethane, and calcium alginate.

11. The method as claimed in Claim 1, wherein the primers and probes were selected specifically for two target gene sequences MPT64, IS1081 of M. tuberculosis complex, wherein the sample volumes were in the range of 5-20 µL, and they are highly conserved and specific primers and dual labeled hydrolysis probes.

12. The method as claimed in Claim 1, wherein the reverse primer of IS1081 and MPT64 of M. tuberculosis complex having a sequence selected from a group consisting of SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51.

13. The method as claimed in Claim 1, wherein the forward primer of IS1081 and MPT64 of M. tuberculosis complex having a sequence selected from a group consisting of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48.

14. The method as claimed in Claim 1, wherein the primer and probes detect the mutations in rpoB, that having a sequence selected from a group consisting of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77 associated with Rifampicin (RIF) resistance and Isoniazid-resistance (INH) associated mutations in the katG having a SEQ ID NO: 80 and inhA promoter region having a sequence selected from a group consisting of SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, and SEQ ID NO: 85.

15. The method as claimed in Claim 1, wherein the oligonucleotide primers rpoB including a forward and a reversed primer, the reverse primer having a sequence selected from a group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, and the forward primers having a sequence selected from a group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

16. The method as claimed in Claim 1, wherein the oligonucleotide primers KatG including a forward and a reversed primer, the forward primer having a sequence selected from a group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, and the reversed primers having a sequence selected from a group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25.

17. The method as claimed in Claim 1, wherein the oligonucleotide primers InhA including a forward and reversed primer, the forward primer having a sequence selected from a group consisting of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, and the reversed primers having a sequence selected from a group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35.

18. The method as claimed in Claim 1, oligonucleotide primers capable of acting as a probe for MTB complex DNA, that the probe having a sequence selected from a group consisting of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, and SEQ ID NO: 91.

19. The method as claimed in Claim 1, wherein quick extraction reagents reacted with DNA containing SWAB without any DNA extraction procedures leading to the detection of Mycobacterium tuberculosis with 100% sensitivity and specificity; and can process a relatively larger number of samples in a short time.

20. A method for preparation of quick extraction reagent/composition, comprising:
adding 500-800 mL of sterile water to 10-100mL of solvents selected from acetonitrile, dimethyl sulfoxide, dimethylformamide, ethanol, methanol, isopropyl alcohol, glycerol, 2-ethoxy ethanol, and water;
adding 25-100 mL of a 1-3M base solution selected from lithium hydroxide, sodium hydroxide, potassium hydroxide, triethylamine, Ammonia, calcium hydroxide, barium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, followed by the addition of salts selected from sodium chloride, potassium chloride, magnesium chloride, calcium chloride;
adding various surfactants to the solution obtained from the above step, such as nonyl phenoxypolyethoxylethanol (NP-40), polysorbate 20 (Tween 20), phosphate buffer (PBS), sodium lauryl sulphate, sodium dodecyl sulphate (SDS), cetyl trimethylammonium bromide (CTAB), triton X-80, and triton X-100;
adding 10-40mL of antifoam, selected from SE-15, SE-30, antifoam L-30 emulsion, C-emulsion, silicon antifoam, Antifoam 204, simethicone, octanal to the above solution; stirring the reaction mixture for 15-45 minutes and stored it for the usage.
21. A kit for the detection of Mycobacterium tuberculosis, comprising:
a qPCR Amplification mix with Taq Enzyme and UDG/UNG Enzyme and dNTP’s, wherein UDG/UNG which enables performance Fast PCR in a shorter run time;
an MTB Fast PPM Contains target-specific and internal control-specific primer and probe reconstituted in TE buffer;
a quick extraction reagent includes detergent, nucleic acid stabilizer, bases, and water;
an MTB Synthetic DNA clone; and
a Nuclease-free PCR grade water