Description:FIELD OF INVENTION
[001] The present disclosure relates to nucleic acid amplification and detection that are suitable for rapid pathogen detection. In particular, the present disclosure relates to an assay to detect Mycobacterium tuberculosis and its resistance to rifampicin, as well as primers and kits.
BACKGROUND OF THE INVENTION
[002] Tuberculosis (TB) is an airborne disease caused by the bacterium Mycobacterium tuberculosis (MTB). WHO reported that TB is a persistent problem in developing countries and ranks as the second leading cause of death from an infectious disease worldwide after the human immunodeficiency virus (HIV). The bacterium is slow-growing and needs 1-2 months to grow in a culture; however, a rapid and timely diagnosis of TB is essential to combat the disease. The Ziehl-Neelsen (ZN) stain for direct specimen examination is a conventional diagnostic tool but lacks sensitivity.
[003] The tests based on PCR have shown promise for the detection of mycobacteria in clinical samples, but this amplification process requires additional processing time, reagents, and devices, which affect the cost of the assay. Moreover, PCR analysis needs well-trained personnel. The laboratory demonstration of the presence of M. tuberculosis in the sputum samples requires isolation, microscopy, or PCR-based tests. Since culture and isolation of M. tuberculosis are difficult and take 15-30 days’ time, PCR is also costly and takes more than a day since it is normally carried out after culturing the swabs on suitable media. Additionally, the specimens with the M. tuberculosis species resistant to the antibiotic rifampicin are even more difficult to detect. If the resistant infections are not detected, it is difficult to find a suitable drug of choice to treat the TB patient. Therefore, the currently available diagnostic tests cannot be applied for surveillance and detection at the point of care.
[004] Further, sputum samples are composed of mucus produced by coughing and oral emission from the lower respiratory tract. These samples provide valuable insights into infectious agents present in the respiratory system, especially in the case of tuberculosis. However, sputum samples can be hard to produce in some patients and have a wide spectrum of fluidic properties that make sample preparation complex. Furthermore, their use is generally limited to the diagnosis of respiratory infections.
[005] Tuberculosis is generally treated sequentially with three lines of drugs. Multiple lines of drugs have become necessary as drug resistance has become increasingly problematic. This is due to both patient compliance issues and excessive drug prescription in cases where disease or drug resistance status has not been confirmed beyond a symptomatic diagnosis. Cases of multi-drug-resistant (MDR) TB have become increasingly common, with 2013 global incidences of 3.5% and 20.5% in new cases and previously-treated cases, respectively. The percentage of MDR-TB cases that were also extensively drug-resistant (XDR) was 9.0% in the same year. Patients with extensive pulmonary TB are typically infected with ~1012 bacteria. Drug resistance has been shown to result from spontaneous mutations at rates of 10-7 to 10-10 mutations per bacterium per generation, depending on the specific drug. Patients are thus frequently treated with multiple drugs at once in order to prevent extensive drug resistance. It is thus critical to develop tools to rapidly identify both TB infection and drug resistance profiles for patients presenting with respiratory symptoms in the clinic in order to deliver targeted therapeutics.
[006] CN112126695 discloses a primer composition for loop-mediated isothermal amplification detection of the M. tuberculosis complex and reaction system. The said document also discloses six loop-mediated isothermal amplification primers that were designed with IS6110 as the target gene, namely, an external primer, an internal primer, and a loop primer, and the results of the amplification reaction can be visualized directly under visible light using fluorescent dyes.
[007] CN108531624 discloses materials and processes related to loop-mediated isothermal amplification of the M. tuberculosis DNA Gyrase B subunit (GyrB) target gene for the diagnosis of TB.
[008] CN112011633 discloses materials and processes related to loop-mediated isothermal amplification of M. tuberculosis Beta subunit of RNA polymerase gene (Rpoß) and IS6110 target genes for the diagnosis of tuberculosis/rifampicin resistant tuberculosis.
[009] Despite all prior efforts, there is a need in the art to facilitate a rapid detection assay for M. tuberculosis and its resistance to rifampicin, which is highly sensitive, rapid, reliable, affordable, and easy to perform without highly trained technical manpower.
SUMMARY OF THE INVENTION
[0010] This summary is provided to introduce a selection of concepts in a simplified format that are further described in the detailed description of the invention.
[0011] In a first aspect of the present disclosure, there is provided a set of primers for detecting Mycobacterium tuberculosis and/or rifampicin-resistant M. tuberculosis, the set of primers comprising:
a M. tuberculosis DNA Gyrase B subunit gene-specific primers set;
a M. tuberculosis IS6110 genomic segment-specific primers set;
a M. tuberculosis RNA polymerase ß subunit gene-specific primers set; or
a combination of two or more of these primer sets,
wherein the M. tuberculosis DNA Gyrase B subunit gene-specific primers set comprises primers having oligonucleotide sequences as set forth in SEQ NO: 1 to SEQ NO: 6,
wherein the M. tuberculosis IS6110 genomic segment-specific primers set comprises primers having oligonucleotide sequences as set forth in SEQ NO: 7 to SEQ NO: 12, and
wherein the M. tuberculosis RNA polymerase ß subunit gene-specific primers set comprises primers having oligonucleotide sequences as set forth in SEQ NO: 13 to SEQ NO: 17.
[0012] In another aspect of the present disclosure, there is provided an assay for detecting Mycobacterium tuberculosis and/or rifampicin-resistant M. tuberculosis in a biological sample, the assay comprising:
extracting DNA from the biological sample;
preparing a mixture of the set of primers as claimed;
preparing a reaction mixture by adding the mixture of the set of primers, the extracted DNA, and LAMP reagents;
performing a LAMP reaction using the reaction mixture; and
analyzing results of the LAMP reaction, comparing the results of the LAMP reaction with LAMP reaction results of control samples and detecting the presence and absence of Mycobacterium tuberculosis and/or rifampicin-resistant M. tuberculosis.
[0013] In another aspect of the present disclosure, there is provided a kit for detecting Mycobacterium tuberculosis and/or rifampicin-resistant M. tuberculosis in a biological sample, the kit comprising:
a set of primers as claimed;
reagents; and
an instruction manual for detecting the presence and absence of Mycobacterium tuberculosis and/or rifampicin-resistant M. tuberculosis in the biological sample.
[0014] These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWING
[0015] The following drawing forms part of the present specification and is included to further demonstrate certain aspects of the present disclosure, the invention of which can be better understood in combination with the detailed description of specific embodiments presented herein.
[0016] Figure 1 illustrates the interpretation of a colorimetric isothermal assay for the detection of Mycobacterium tuberculosis with anticipated results.
DETAILED DESCRIPTION OF THE INVENTION
[0017] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps of the process, features of the invention, referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.
[0018] Definitions: For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are collected here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person skilled in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.
[0019] 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 elements or steps but not the exclusion of any other element or step or group of elements or steps.
[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference. The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally equivalent products and methods are clearly within the scope of the disclosure, as described herein.
[0021] As used herein, the terms “method” and “process” have been used interchangeably.
[0022] As used herein, the term “sample” or “biological sample” refers to any known types of samples that are collected in ex-vivo condition from subjects. The subject is mammals. The mammal is preferably Humans (Homo sapiens). The sample is saliva or sputum.
[0023] The term “in-vitro” refers to a medical study or experiment which is done in the laboratory within the control environment at laboratory.
[0024] As used herein, the terms “polynucleotide sequence”, “nucleic acid”, and “gene” mean a chain of two or more nucleotides, such as RNA (ribonucleic acid) and DNA (deoxyribonucleic acid). The term oligonucleotides refer to short nucleic acid polymers.
[0025] As used herein, the term primer refers to a DNA strand that can prime the synthesis of DNA.
[0026] As used herein, the phrase rifampicin-resistant Mycobacterium tuberculosis refers to M. tuberculosis strains that are resistant to rifampicin.
[0027] As used herein, the loop-mediated isothermal amplification (LAMP) assay is a single-tube technique for the amplification of DNA. LAMP is an isothermal nucleic acid amplification technique.
[0028] As used herein, the LAMP reaction refers to a gene amplification method in which DNA is continuously amplified by using primers suitable for the base sequence of the target DNA by a gene isothermal amplification reaction.
[0029] “Specificity” herein indicates the ratio of the amplification of a target gene to the non-specific amplification. So, high specificity means the amplification of a target gene is dominant. “Sensitivity” herein refers to the minimum amount of template required to reliably amplify the target gene.
[0030] As used herein, “Kit” refers to any delivery system for supplying materials or reagents to carry out the method of the present disclosure. In the context of the present disclosure, such delivery systems include systems that allow the storage, transport or delivery of reaction reagents (e.g., primers, enzymes, internal standards, etc. in suitable containers) and/or support materials (e.g. buffers, written instructions for conducting the test, etc.) from one site to another. For example, kits include one or more envelopes (e.g., boxes) containing the relevant reaction reagents and/or support materials. Such content can be supplied to the intended container jointly or separately. For example, the first container may contain an enzyme for use in an assay, and the second container may contain primers.
[0031] As used herein, the term “instruction manual” refers to the instruction manual for the reagent for detecting the presence and absence of M. tuberculosis and/or rifampicin-resistant M. tuberculosis in the biological sample and interpreting the results of the present invention, in which the features, principles, operating procedures, etc. of the method for the present invention are substantially described in text, diagrams, etc. It means a sentence, a pamphlet (leaflet), or the like.
[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.
[0033] Mycobacterium tuberculosis is the causative pathogen in tuberculosis (TB), an infection of the respiratory system that may eventually spread to peripheral organs, leading to acute disease and death if left untreated. One-third of the world’s population suffers from latent TB, with 8.8 million new cases of the disease and 2 million deaths annually. These numbers are dramatically skewed towards the developing world, with TB accounting for the majority of HIV-related deaths. It is thus extremely important to avoid wasting precious resources and medicines on misdiagnosis and retreatment. However, sputum smear microscopy (by far the most common TB test performed in low-income countries at 83 million/year) only achieves 40-60% sensitivity in field conditions, with this value dropping to 20% in cases of co-infection with HIV. Furthermore, the confirmatory diagnostic technique of cell culture is prohibitively slow, yielding results in the time frame of months, during which a treatment regimen has already been pursued regardless of drug susceptibility. Genotyping tests adapted to retron library recombineering (RLR) settings present an attractive option for detection of TB and screening for drug resistance due to their speed and low cost.
[0034] The present disclosure provides a colorimetric LAMP assay for the detection of tuberculosis, and rifampicin-resistant M. tuberculosis. The present disclosure involves designing three sets of LAMP primers, especially those specific to the DNA Gyrase Beta subunit (GyrB) of M. tuberculosis, the Beta (ß) subunit of the RNA polymerase gene (Rpoß) of M. tuberculosis, and the insertion sequence (IS) 6110 genomic segment of M. tuberculosis. The primers are synthesized and dissolved in specific proportions. The genomic DNA is extracted from the known positive sputum samples. The LAMP reaction is set up using the extracted DNA, the specific primer mix, and the commercially available colorimetric LAMP master mix. The reaction was incubated at 65°C for 30 to 40 minutes. The tubes were allowed to cool to room temperature, and the colour change was observed with naked eyes. The positive reaction is indicated by the yellow-coloured reaction mix. On the other hand, the colour of the negative reaction remains pink.
[0035] The present disclosure provides a rapid detection assay for M. tuberculosis and its resistance to rifampicin that is highly sensitive, rapid, reliable, affordable, easy to perform, and whose results can be interpreted visually. The assay of the present disclosure does not require any sophisticated instrument.
[0036] In a first aspect of the present disclosure, there is provided a set of primers for detecting Mycobacterium tuberculosis and/or rifampicin-resistant M. tuberculosis, the set of primers comprising:
A M. tuberculosis DNA Gyrase B subunit gene-specific primers set;
a M. tuberculosis IS6110 genomic segment-specific primers set;
a M. tuberculosis RNA polymerase ß subunit gene-specific primers set; or
a combination of two or more of these primer sets,
wherein the M. tuberculosis DNA Gyrase B subunit gene-specific primers set comprises primers having oligonucleotide sequences as set forth in SEQ NO: 1 to SEQ NO: 6,
wherein the M. tuberculosis IS6110 genomic segment-specific primers set comprises primers having oligonucleotide sequences as set forth in SEQ NO: 7 to SEQ NO: 12, and
wherein the M. tuberculosis RNA polymerase ß subunit gene-specific primers set comprises primers having oligonucleotide sequences as set forth in SEQ NO: 13 to SEQ NO: 17.
[0037] In an embodiment of the present disclosure, there is provided a set of primers for detecting Mycobacterium tuberculosis, the set of primers comprising:
a M. tuberculosis DNA Gyrase B subunit gene-specific primers set; and
a M. tuberculosis IS6110 genomic segment-specific primers set,
wherein the M. tuberculosis DNA Gyrase B subunit gene-specific primers set comprises primers having oligonucleotide sequences as set forth in SEQ NO: 1 to SEQ NO: 6, and
wherein the M. tuberculosis IS6110 genomic segment-specific primers set comprises primers having oligonucleotide sequences as set forth in SEQ NO: 7 to SEQ NO: 12.
[0038] In an embodiment of the present disclosure, there is provided a set of primers for detecting rifampicin-resistant M. tuberculosis, the set of primers comprising:
a M. tuberculosis RNA polymerase ß subunit gene-specific primers set comprising primers having oligonucleotide sequences as set forth in SEQ NO: 13 to SEQ NO: 17.
[0039] In an embodiment of the present disclosure, there is provided a set of primers for detecting Mycobacterium tuberculosis and rifampicin-resistant M. tuberculosis, the set of primers comprising:
a M. tuberculosis DNA Gyrase B subunit gene-specific primers set;
a M. tuberculosis IS6110 genomic segment-specific primers set; and
a M. tuberculosis RNA polymerase ß subunit gene-specific primers set,
wherein the M. tuberculosis DNA Gyrase B subunit gene-specific primers set comprises primers having oligonucleotide sequences as set forth in SEQ NO: 1 to SEQ NO: 6,
wherein the M. tuberculosis IS6110 genomic segment-specific primers set comprises primers having oligonucleotide sequences as set forth in SEQ NO: 7 to SEQ NO: 12, and
wherein the M. tuberculosis RNA polymerase ß subunit gene-specific primers set comprises primers having oligonucleotide sequences as set forth in SEQ NO: 13 to SEQ NO: 17.
[0040] In an embodiment of the present disclosure, wherein the set of primers is designed for loop-mediated isothermal amplification (LAMP) assay.
[0041] In an embodiment of the present disclosure, wherein the M. tuberculosis DNA Gyrase B subunit gene-specific primers set and the M. tuberculosis IS6110 genomic segment-specific primers set identify M. tuberculosis.
[0042] In an embodiment of the present disclosure, wherein the M. tuberculosis RNA polymerase ß subunit gene-specific primers set identifies rifampicin-resistant M. tuberculosis.
[0043] In another aspect of the present disclosure, there is provided an assay for detecting Mycobacterium tuberculosis and/or rifampicin-resistant M. tuberculosis in a biological sample, the assay comprising:
extracting DNA from the biological sample;
preparing a mixture of the set of primers as claimed;
preparing a reaction mixture by adding the mixture of the set of primers, the extracted DNA, and LAMP reagents;
performing a LAMP reaction using the reaction mixture; and
analyzing results of the LAMP reaction, comparing the results of the LAMP reaction with LAMP reaction results of control samples and detecting the presence and absence of Mycobacterium tuberculosis and/or rifampicin-resistant M. tuberculosis.
[0044] In another aspect of the present disclosure, there is provided an assay for detecting Mycobacterium tuberculosis and rifampicin-resistant M. tuberculosis in a biological sample, the assay comprising:
extracting DNA from the biological sample;
preparing a mixture of a set of primers;
preparing a reaction mixture by adding the mixture of the set of primers, the extracted DNA, and LAMP reagents;
performing a LAMP reaction using the reaction mixture; and
analyzing results of the LAMP reaction, comparing the results of the LAMP reaction with LAMP reaction results of control samples and detecting the presence and absence of Mycobacterium tuberculosis and rifampicin-resistant M. tuberculosis,
wherein the set of primers comprises primers having oligonucleotide sequences as set forth in SEQ NO: 1 to SEQ NO: 6, SEQ NO: 7 to SEQ NO: 12, and SEQ NO: 13 to SEQ NO: 17.
[0045] In another aspect of the present disclosure, there is provided an assay for detecting Mycobacterium tuberculosis and rifampicin-resistant M. tuberculosis in a biological sample, the assay comprising:
extracting DNA from the biological sample;
preparing a mixture of a set of primers;
preparing a reaction mixture by adding the mixture of the set of primers, the extracted DNA, and LAMP reagents;
performing a LAMP reaction using the reaction mixture; and
analyzing results of the LAMP reaction, comparing the results of the LAMP reaction with LAMP reaction results of control samples and detecting the presence and absence of Mycobacterium tuberculosis and rifampicin-resistant M. tuberculosis,
wherein the set of primers comprises primers having oligonucleotide sequences as set forth in SEQ NO: 1 to SEQ NO: 6, SEQ NO: 7 to SEQ NO: 12, and SEQ NO: 13 to SEQ NO: 17.
[0046] In another aspect of the present disclosure, there is provided an assay for detecting Mycobacterium tuberculosis in a biological sample, the assay comprising:
extracting DNA from the biological sample;
preparing a mixture of a set of primers;
preparing a reaction mixture by adding the mixture of the set of primers, the extracted DNA, and LAMP reagents;
performing a LAMP reaction using the reaction mixture; and
analyzing results of the LAMP reaction, comparing the results of the LAMP reaction with LAMP reaction results of control samples and detecting the presence and absence of Mycobacterium tuberculosis and rifampicin-resistant M. tuberculosis,
wherein the set of primers comprises primers having oligonucleotide sequences as set forth in SEQ NO: 1 to SEQ NO: 6, and SEQ NO: 7 to SEQ NO: 12.
[0047] In another aspect of the present disclosure, there is provided an assay for detecting rifampicin-resistant M. tuberculosis in a biological sample, the assay comprising:
extracting DNA from the biological sample;
preparing a mixture of a set of primers;
preparing a reaction mixture by adding the mixture of the set of primers, the extracted DNA, and LAMP reagents;
performing a LAMP reaction using the reaction mixture; and
analyzing results of the LAMP reaction, comparing the results of the LAMP reaction with LAMP reaction results of control samples and detecting the presence and absence of Mycobacterium tuberculosis and rifampicin-resistant M. tuberculosis,
wherein the set of primers comprises primers having oligonucleotide sequences as set forth in SEQ NO: 13 to SEQ NO: 17.
[0048] In an embodiment of the present disclosure, wherein the assay is an in vitro assay.
[0049] In an embodiment of the present disclosure, wherein the LAMP reaction is performed by incubating the reaction mixture at a temperature of about 65±1oC for a period of about 30 to 60 minutes.
[0050] In an embodiment of the present disclosure, wherein the LAMP reaction is performed by incubating the reaction mixture at a temperature in a range of 64 to 66oC for a period of about 30 to 60 minutes.
[0051] In an embodiment of the present disclosure, wherein the LAMP reaction is performed by incubating the reaction mixture at a temperature in a range of 64 to 66oC for a period of about 30 to 40 minutes.
[0052] In an embodiment of the present disclosure, wherein the incubation is performed at 65oC using a heating block.
[0053] In an embodiment of the present disclosure, wherein the presence or absence of Mycobacterium tuberculosis and/or rifampicin-resistant M. tuberculosis is detected by a visual color change.
[0054] In an embodiment of the present disclosure, wherein the LAMP reagents comprise modified Bst 2.0 DNA polymerase, nucleoside triphosphates (dNTPs), a visible pH indicator, a thermolabile uracil DNA glycosylase, and enhancer.
[0055] In an embodiment of the present disclosure, wherein the LAMP reagents WarmStart® Colorimetric LAMP 2X Master Mix or any commercially available colorimetric LAMP 2X master mix.
[0056] In an embodiment of the present disclosure, wherein the DNA is extracted by methods well known to those skilled in the art. For example, DNA extraction can be performed using automated DNA extractors and DNA extraction kits.
[0057] In an embodiment of the present disclosure, wherein the biological sample is saliva or sputum.
[0058] In an embodiment of the present disclosure, wherein the assay is performed in 1 hour.
[0059] In another aspect of the present disclosure, there is provided a kit for detecting Mycobacterium tuberculosis and/or rifampicin-resistant M. tuberculosis in a biological sample, the kit comprising:
a set of primers as claimed;
reagents; and
an instruction manual for detecting the presence and absence of Mycobacterium tuberculosis and/or rifampicin-resistant M. tuberculosis in the biological sample.
[0060] In an embodiment of the present disclosure, wherein the kit is designed for loop-mediated isothermal amplification (LAMP) assay.
[0061] In an embodiment of the present disclosure, wherein the reagents comprise modified Bst 2.0 DNA polymerase, nucleoside triphosphates (dNTPs), a visible pH indicator, a thermolabile uracil DNA glycosylase, and enhancer.
[0062] In an exemplary and non-limiting embodiment, the present disclosure provides an indigenous LAMP based assay for the detection of M. tuberculosis and/or rifampicin-resistant M. tuberculosis using an isothermal amplification reaction, which does not require a thermal cycler as in the case of real-time PCR. The present disclosure provides further advantages:
• The assay can be performed using a single-temperature heating device (65 ± 1°C).
• The time required for the assay is only 30 to 60 minutes.
• The results can be interpreted visually; no other sophisticated instruments are required for the interpretation of the results.
• A highly skilled or technical person is not required to perform this assay.
• It is a cost-effective and less time-consuming technology.
• This assay could enable point-of-care testing outside of the diagnostic laboratory.
• The specificity of the assay is 100% and sensitivity is 99%.
EXAMPLES:
[0063] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. Person skilled in the art will be aware of the fact that the present examples will further subject to variations and modifications specifically described herein based on the technical requirement of the experiment and shall not be limiting what specifically mentioned.
EXAMPLE 1: Loop-mediated isothermal amplification (LAMP) based method
[0064] LAMP technology uses multiple forward and reverse (backward) primers and one or two loop primers in a reaction. In addition to this, LAMP requires a modified Bst 2.0 DNA polymerase with strand displacement activity, which is responsible for isothermal amplification of DNA. The template DNA strand along with Bst 2.0 DNA polymerase and primers, is incubated at a constant temperature of 60°C-65°C. The results are interpreted in the form of color change from pink to yellow.
EXAMPLE 2: Primers
[0065] LAMP primers set contains:
a) F3/ B3 (Forward/ Backward outermost primers) that are similar to PCR primers;
b) FIP/ BIP (Forward/ Backward inner primers), FIP and BIP are specialized primers with complementary and non-complementary nucleotide segments; and
c) LF or LB (forward/ backward loop primers) or both.
[0066] FIP and BIP are specialized primers consisting of two parts, F2/F1c and B2/B1c, respectively. F2 and B2 bind with the template strand to initiate the amplification process, while F1c and B1c sequences serve as overhangs, which help loop formation as the LAMP reaction continues. The short distance between the F2 and F1c (and B2/ B1c) helps form a loop structure within the amplicon. The loop primers increase the number of initiation points for DNA synthesis by binding complementarily to the single-stranded loops and increasing the pace of amplification.
[0067] For this assay, three sets of LAMP primers were designed in-house by the inventors of the present disclosure targeting the DNA Gyrase B subunit (GyrB) and IS6110 genomic segments of Mycobacterium tuberculosis, and the rifampicin-resistant Beta subunit of the RNA polymerase gene (Rpoß) of tuberculosis. Each set contains 5 or 6 primers (F3, B3, FIP, BIP, and/or LF/LB) as listed in Table 1.
[0068] Table 1: List of LAMP primers for detection of M. tuberculosis and/or rifampicin-resistant M. tuberculosis
SEQ IDs Name Primers Sequence
Set 1
SEQ NO: 1 GyrB F3 5’-GGCCGGGAAGAAGATCAAC-3’
SEQ NO: 2 GyrB B3 5’-CGGGTGATAAAGCTGCGC-3’
SEQ NO: 3 GyrB FIP 5’-CATGGTGGTCTCCCACAACTCCACGGCATTCAGCGGTACA-3’
SEQ NO: 4 GyrB BIP 5’-TCGGTTCGTGTGTTGCGTCAAGTCCTCGCCCATCAGGAT-3’
SEQ NO: 5 GyrB LF 5’-GCGTCCATTTCACCTAGACCCT-3’
SEQ NO: 6 GyrB LB 5’-GCCGCCGACGAGTTGTTCT-3’
Set 2
SEQ NO: 7 IS6110 F3 5’-TCCCGCCGATCTCGTC-3’
SEQ NO: 8 IS6110 B3 5’-TCGATCGCGTCGAGGAC-3’
SEQ NO: 9 IS6110 FIP 5’-TGCCCAGGTCGACACATAGGTGGCTTCGGACCACCAGCAC-3’
SEQ NO: 10 IS6110 BIP 5’-GGTTCGCCTACGTGGCCTTTGGTGGCCATCGTGGAAG-3’
SEQ NO: 11 IS6110 LF 5’-CTGCTACCCACAGCCGGTTAG-3’
SEQ NO: 12 IS6110 LB 5’-ACGCCTACGCTCGCAGGAT-3’
Set 3
SEQ NO: 13 Rpoß F3 5’-AGCCAATTCATGGACCAGAA-3’
SEQ NO: 14 Rpoß B3 5’-CGCGTACACCGACAGC-3’
SEQ NO: 15 Rpoß FIP 5’-CTCACGTGACAGACCGCCGGGTCGGGGTTGACCCACA-3’
SEQ NO: 16 Rpoß BIP 5’-ACGTGCACCCGTCGCACTAGCCGATCAGACCGATGT-3’
SEQ NO: 17 Rpoß LB 5’-TGCCCGATCGAAACCCCTGA-3’
[0069] Characteristic properties of primers: The four key factors in the LAMP primer design are the melting temperature (Tm), stability at the 3’ and 5’ ends of each primer (Delta G), GC content, and ability to form secondary structures.
[0070] Tm: Tm is calculated by using the Nearest-Neighbor method. This method is presently considered to be the method that predicts the Tm value closest to the actual value. The Tm for each region is designed to be about 65°C (64 - 66°C) for F1c and B1c, about 60°C (59 - 61°C) for F2, B2, F3, and B3, and about 65°C (64 - 66°C) for the loop primers.
[0071] The 3’ end stability of the primers: The 3’ end of the primers acts as the initiating point of the DNA polymerization and therefore must be very stable and complementary with the target sequence. The following criteria are taken into consideration while selecting the primer sets. The 3’ ends of F2/B2, F3/B3 are designed so that the free energy is –4 kcal/ mol or less. LF/LB and the 5’ end of F1c/B1c are designed so that the free energy is –4 kcal/ mol or less.
[0072] GC content: Primers are designed so that their GC content is between about 40% and 65%. But primers with GC content between 50% and 60% are selected as they are considered to give relatively better results.
[0073] Secondary structures: Another important property of primers is their ability to form secondary structures, or primer dimers. The primers are designed so that they do not form secondary structures. To avoid this condition, it is important to make sure that the 3’ end of the primer is non-complementary.
EXAMPLE 3: Methodology
[0074] The DNA isolated from clinical cases of tuberculosis was used for the standardization. A commercially available kit was used for LAMP: WarmStart® Colorimetric LAMP 2X Master Mix (DNA & RNA) (Catalogue number M1800L). A commercial kit for the extraction of DNA: HiPurA™ Mycobacterium tuberculosis DNA Purification Kit Catalogue number MB545, was used to extract DNA from the saliva or sputum samples.
Primer mixture:
[0075] A primer mixture was made prior to the actual LAMP reaction. The primer mixture comprises 1.6 µM of FIP and BIP, 0.2 µM of F3 and B3, and 0.4 µM of LF and LB (Table 2).
[0076] In an embodiment, FIP is selected from GyrB FIP (SEQ NO: 3), IS6110 FIP (SEQ NO: 9) and Rpoß FIP (SEQ NO: 15); BIP is selected from GyrB BIP (SEQ NO: 4), IS6110 BIP (SEQ NO: 10) and Rpoß BIP (SEQ NO: 16); F3 is selected from GyrB F3 (SEQ NO: 1), IS6110 F3 (SEQ NO: 7) and Rpoß F3 (SEQ NO: 13); B3 is selected from GyrB B3 (SEQ NO: 2), IS6110 B3 (SEQ NO: 8) and Rpoß B3 (SEQ NO: 14); LF is selected from GyrB LF (SEQ NO: 5) and IS6110 LF (SEQ NO: 11) and LB is selected from GyrB LB (SEQ NO: 6), IS6110 LB (SEQ NO: 12) and Rpoß LB (SEQ NO: 17).
[0077] Table 2: Concentration each primer to be incorporated in the primer mixture.

LAMP reaction mixture:
[0078] The assay was performed in a 20 µl reaction mixture, wherein the reaction mixture contained 2 µl of 10X primer mixture, i.e., primer mixture prepared by mixing 16 µM of both forward inner primer (FIP) and backward inner primer (BIP), 2 µM of both F3 and B3 primers, and 4 µM of both loop forward (LF) and loop backward (LB). Further, 10 µl of WarmStart Colorimetric LAMP 2X Master Mix (M1804L), 6.5 µl of nuclease-free water, 2.5 µl of 1X LAMP Primer Mix, and 2 µl of DNA template were added to form a reaction mixture. The reaction mixture was thoroughly mixed by vortexing, followed by a quick spin. Later, the reaction mixture was set at 65°C for 30 to 40 minutes on a water bath. The results were observed with the naked eye by looking at the color change. The reaction mixture for LAMP consists of the following components, as mentioned in Table 3.
[0079] Table 3: The composition of LAMP reaction

EXAMPLE 4: Interpretation of results
[0080] The tubes comprising the reaction mixture are removed from the incubator and observed with the naked eye. The positive reaction is indicated by the yellow-colored reaction mix. On the other hand, the color of the negative reaction remains pink (Figure 1). If the color change is not significant, for example, if the color of the reaction is orange, incubate the reaction at 65° C for 10 minutes and re-examine for the complete color change. The color of the reaction gets intensified by cooling it down to room temperature.
[0081] It was observed that the biological samples of the subject infected with Mycobacterium tuberculosis and resistant to rifampicin showed all the tubes with a yellow colour (sample 1), similar to the positive control (PTC). In the biological samples of the subject infected with Mycobacterium tuberculosis and not resistant to tuberculosis remained a pink color in the tube comprising the M. tuberculosis RNA polymerase ß subunit gene-specific primers set, while the color of the other two tubes comprising the M. tuberculosis DNA Gyrase B subunit gene-specific primers set and M. tuberculosis IS6110 genomic segment-specific primers set changed to yellow (sample 2). However, the tube comprising biological samples not infected with Mycobacterium tuberculosis remained pink, like the negative control (NTC) sample. The negative control sample comprises nuclease free water and positive control samples comprises confirmed samples from subjects infected with Mycobacterium tuberculosis or its rifampicin-resistant strain.
[0082] The assay with the specific sets of primers of the present disclosure showed 99% sensitivity and 100% specificity for Mycobacterium tuberculosis and rifampicin-resistant M. tuberculosis.
[0083] It will thus be seen that the objects set forth above, among those made apparent from the preceding description, are efficiently attained, and since certain changes may be made in the constructions set forth without departing from the spirit and scope of the invention, it is intended that all matter contained in the above description shall be interpreted as illustrative and not in a limiting sense. The invention has been described with reference to preferred and alternate embodiments. Modifications and alterations will become apparent to those skilled in the art upon reading and understanding the detailed discussion of the invention provided herein. This invention is intended to include all such modifications and alterations insofar as they come within the scope of the present invention. These and other modifications of the preferred embodiments as well as other embodiments of the invention will be obvious from the disclosure herein, whereby the foregoing descriptive matter is to be interpreted merely as illustrative of the invention and not as a limitation.
[0084] Finally, to the extent necessary to understand or complete the disclosure of the present invention, all publications, patents, and patent applications mentioned herein are expressly incorporated by reference therein to the same extent as though each were individually so incorporated. , Claims:1. A set of primers for detecting Mycobacterium tuberculosis and/or rifampicin-resistant M. tuberculosis, the set of primers comprising:
a M. tuberculosis DNA Gyrase B subunit gene-specific primers set;
a M. tuberculosis IS6110 genomic segment-specific primers set;
a M. tuberculosis RNA polymerase ß subunit gene-specific primers set; or
a combination of two or more of these primer sets,
wherein the M. tuberculosis DNA Gyrase B subunit gene-specific primers set comprises primers having oligonucleotide sequences as set forth in SEQ NO: 1 to SEQ NO: 6,
wherein the M. tuberculosis IS6110 genomic segment-specific primers set comprises primers having oligonucleotide sequences as set forth in SEQ NO: 7 to SEQ NO: 12, and
wherein the M. tuberculosis RNA polymerase ß subunit gene-specific primers set comprises primers having oligonucleotide sequences as set forth in SEQ NO: 13 to SEQ NO: 17.

2. The set of primers as claimed in claim 1, wherein the set of primers is designed for loop-mediated isothermal amplification (LAMP) assay.

3. The set of primers as claimed in claim 1, wherein the M. tuberculosis DNA Gyrase B subunit gene-specific primers set and the M. tuberculosis IS6110 genomic segment-specific primers set identify M. tuberculosis, and the M. tuberculosis RNA polymerase ß subunit gene-specific primers set identifies rifampicin-resistant M. tuberculosis.

4. An assay for detecting Mycobacterium tuberculosis and/or rifampicin-resistant M. tuberculosis in a biological sample, the assay comprising:
extracting DNA from the biological sample;
preparing a mixture of the set of primers as claimed in claim 1;
preparing a reaction mixture by adding the mixture of the set of primers, the extracted DNA, and LAMP reagents;
performing a LAMP reaction using the reaction mixture; and
analyzing results of the LAMP reaction, comparing the results of the LAMP reaction with LAMP reaction results of control samples and detecting the presence and absence of Mycobacterium tuberculosis and/or rifampicin-resistant M. tuberculosis.

5. The assay as claimed in claim 4, wherein the LAMP reaction is performed by incubating the reaction mixture at a temperature of about 65±1oC for a period of about 30 to 60 minutes.

6. The assay as claimed in claim 4, wherein the presence or absence of Mycobacterium tuberculosis and/or rifampicin-resistant M. tuberculosis is detected by a visual color change.

7. The assay as claimed in claim 4, wherein the LAMP reagents comprise modified Bst 2.0 DNA polymerase, nucleoside triphosphates, a visible pH indicator, a thermolabile uracil DNA glycosylase, and enhancer.

8. The assay as claimed in claim 4, wherein the biological sample is saliva or sputum.

9. A kit for detecting Mycobacterium tuberculosis and/or rifampicin-resistant M. tuberculosis in a biological sample, the kit comprising:
a set of primers as claimed in claim 1;
reagents; and
an instruction manual for detecting the presence and absence of Mycobacterium tuberculosis and/or rifampicin-resistant M. tuberculosis in the biological sample,

10. The kit as claimed in claim 9, wherein the kit is designed for loop-mediated isothermal amplification (LAMP) assay.