WO2018065830A1

WO2018065830A1 - Multiplex realtime pcr kit for diagnosing multidrug resistance (mdr) and extensively drug resistance (xdr) tuberculosis

Info

Publication number: WO2018065830A1
Application number: PCT/IB2017/050693
Authority: WO
Inventors: Anitha RAJAGOPAL; Arivan SADHU GURU; Anbhan SAADHU GURU
Original assignee: Rajagopal Anitha; Sadhu Guru Arivan
Priority date: 2016-10-04
Filing date: 2017-02-09
Publication date: 2018-04-12

Abstract

A system and process is provided for detecting M. tuberculosis strains or mutants thereof, particularly those strains resistant to first line anti-tuberculosis drugs, second line anti-tuberculosis drugs or a combination of both for e.g. isoniazid, rifampin, fluoroquinolone / aminoglycoside, kanamycin or pyrazinamide or any combination thereof in a biological sample, said system comprising: isolating and further amplifying nucleic acids under hybridizing conditions with specific sequences of primers and fluorophore-attached-probes that target portions of the katG, inhA, gyrA, rpoB, pncA or rrs or any combinations thereof and real-time detection of the amplified nucleic acids to determine the presence or absence of M. tuberculosis strains or mutants thereof.

Description

TITLE OF INVENTION:

MULTIPLEX REALTIME PCR KIT FOR DIAGNOSING MULTIDRUG RESISTANCE (MDR) AND EXTENSIVELY DRUG RESISTANCE (XDR) TUBERCULOSIS

CROSS-REFERENCE TO RELATED APPLICATIONS AND PRIORITY

The present application claims priority from provisional patent application no. 201641033933 filed on the 4^th day of October, 2016.

TECHNICAL FIELD

The present invention relates to assays, diagnostic kits and methods for the realtime PCR detection of nucleic acids. More particularly, this invention relates to real-time quantitative PCR (qPCR) assays for accurate, rapid and affordable diagnosis of multi-drug resistant (MDR), extensively drug resistant (XDR) and combination of multi-drug resistant and extensively drug resistant Mycobacterium tuberculosis bacteria.

BACKGROUND

The human respiratory-tract infectious disease tuberculosis (TB), affecting nearly l/3rd of the world population, remains a serious global health concern. According to the World Health Organization (WHO), India shoulders the maximum number of TB patients— 2.84 million people. Mycobacterium tuberculosis bacteria are the causative agents of tuberculosis and while vaccines are available, their effect decreases over period of time and the infected patients are commonly treated with antibiotics like isoniazid and rifampicin to control the disease.

In recent years, the development of multidrug-resistant (MDR), extensively drug- resistant (XDR) and totally drug-resistant strains of Mycobacterium tuberculosis have been reported. Multi drug resistance (MDR) connotes resistance of the microbial strain to any first line anti-tuberculosis drug including isoniazid or rifampicin or a combination of the both. Extensively drug resistance connotes resistance of the microbial strain to any first line anti-tuberculosis drug and in addition, resistance of the microbial strain to any second line anti-tuberculosis drug including kanamycin, pyrazinamide, aminoglycoside or fluoroquinolone or any combination of the both. India has been recently described as a 'TB ticking bomb' with scientists warning that the quantum of drug resistant. A report in a leading newspaper on 24^th March 2016 reported that in the state of Karnataka, of the 17,980 culture and drug susceptibility tests done in 2015-16, 1,097 tested positive for MDR TB, a number considered to be conservative since many more cases could have gone missing without detection. Nearly three per cent of the newly diagnosed sputum positive cases in the country are found to develop MDR-TB and considering one patient can directly spread the disease to at least 10 others if it is not checked at the initial stage, this has caused some concern within the national and international community. While most of the multidrug resistant tuberculosis strains are resistant to the two hitherto most effective first line tuberculosis drugs, i.e. isoniazid and rifampicin, even second line drug treatments including kanamycin and fluoroquinolone have also been reported to be ineffective in some cases (second-line Tuberculosis resistance). Inadequate therapy or delayed therapy has a negative effect in that it allows selection of spontaneous mutations in favour of resistant strains which further aggravates the problem. Therefore, the development of fast and accurate detection of multidrug-resistant and / or extensively-drug resistant TB diagnosis, for early detection, blocking the spread of drug-resistant tuberculosis, norms to guide treatment and improve the cure rate of drug-resistant TB has a very important significance.

Accordingly, the present invention provides new assays, kits, systems and methods for the early and accurate diagnosis and detection of resistant M. tuberculosis strains, both MDR and XDR in a single test. The present invention, which addresses this requirement while maintaining a very high specificity, quick and sensitivity, e.g. wherein even only about 10 copies of target nucleic acids can be detected, whereas nucleic acids from other microorganisms shall not be detected and wherein the entire detection takes less time by a factor of at least 3 as compared to conventional state-of-art techniques, may solve long standing pressing need.

SUMMARY

This summary is provided to introduce aspects related to development of a kit or method employing the quantitative real-time polymerase chain reaction (q-PCR) technique to test the sensitivity or resistance of strains of Mycobacterium tuberculosis obtained from a biological sample. This summary is however not intended to disclose essential features of the innovation and nor is intended to determine, limit or restrict the scope of the innovation.

The present invention directs to the intent of finding quicker and accurate methods to test drug resistance of M. tuberculosis. Accordingly, the invention may provide a plurality of methods comprising a plurality of specially designed primers, probes and combinations thereof for detecting multi-drug resistant (MDR) and extensively drug-resistant (XDR) M. tuberculosis bacteria or any combination of them in a biological sample using real-time polymerase chain reaction (q-PCR).

In accordance with one aspect of the present invention, the features of the multiplex q-PCR kit comprising a plurality of specially designed primers, probes and combinations thereof which enable detection of M. tuberculosis strains resistant to first line anti-tuberculosis drugs, including but not limited to, rifampicin or isoniazid or any combination of the same are disclosed.

In accordance with another aspect of the present invention, the features of the multiplex q-PCR kit comprising a plurality of specially designed primers, probes and combinations thereof which may enable detection of M. tuberculosis strains resistant to second line anti-tuberculosis drugs, including but not limited to fluoroquinolone pyrazinamide, aminoglycosides or kanamycin or any combination of the same are disclosed. In accordance with yet another aspect of the invention, the features of the multiplex q-PCR kit comprising a plurality of specially designed primers, probes and combinations thereof which may enable detection of M. tuberculosis strains simultaneously resistant to both rifampicin or isoniazid as well as to fluoroquinolone or aminoglycosides or pyrazinamide or kanamycin or any combination thereof are disclosed.

In one aspect of the invention, the features of the multiplex q-PCR kit comprising a plurality of specially designed primers, probes and combinations thereof which may enable more rapid detection of resistant bacteria, specifically within a time period that is less than time period for detection of resistant bacteria employed by conventional state of art methods by factor between 3 and 10, are disclosed.

In another aspect of the invention, the features of the multiplex q-PCR comprising a plurality of specially designed primers, probes and combinations thereof which may confer extremely high specificity, comprising a specificity not less than 98% for detection of broad range mutations of multi drug resistant strains and extensively drug resistant strains of M. tuberculosis, are disclosed.

In still another aspect of the invention, the features of the multiplex q-PCR kit comprising a plurality of specially designed primers, probes and combinations thereof which may confer extremely high sensitivity, comprising a sensitivity requiring not more than 10 bacilli per millilitre for detection of broad range mutations of multi drug resistant strains and extensively drug resistant strains of M. tuberculosis, are disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

The detailed description is described with reference to the accompanying figure. In the figure, the left-most digit(s) of a reference number identifies the figure in which the reference number first appears. The same numbers are used throughout the drawings to refer like features and components. Figure 1 illustrates an overview of the invention.

Figure 2a illustrates amplification plot for katG assay with 10 random TB samples (FAM channel). The reaction was done using taqman chemistry in 10 random TB samples using katG primers and probes. Water is added as the qPCR negative control. 8 samples consist of wild genotype. Yellow line indicates signal from wild type probe (FAM channel).

Figure 2b illustrates amplification plot for katG assay with 10 random TB samples (HEX channel). The reaction was done using taqman chemistry in 10 random TB samples using katG primers and probes. Water is added as the qPCR negative control. 2 samples consist of mutant genotype. Pink line indicates signal from mutant probe (HEX channel).

Figure 3 illustrates inhA singleplex results and validation of inhA primers and probes that has new dyes with reference strains. Wild type probe of inhA and mutant probe of inhA was used. Bold red line indicates signal from texas red channel of inhA wildtype probe. Dotted red lines show signal from cy5 channel of inhA mutant probe. Water is used as a negative control

Figure 4 illustrates amplification plot for rpoB assay with 10 random TB samples. The reaction was done using taqman chemistry in 10 random TB samples using katG primers and probes. Water is added as the qPCR negative control. 2 samples produced high signal from FAM channel with wild genotype (bold lines) and 7 samples produced high signal from HEX channel with mutant genotype (dotted line).

Figure 5 illustrates amplification plot for gyrA assay with 10 random TB samples. The reaction was done using taqman chemistry in 10 random TB samples using gyrA primers and probes. Water is added as the qPCR negative control. 7 samples produced high signal from FAM channel with wild genotype (orange lines) and 3 samples produced high signal from HEX channel with mutant genotype (blue line).

Figure 6 illustrates pncA singleplex results and validation of pncA primers and probes that has new dyes with reference strains. Wild type probe of pncA and mutant probe of pncA was used. Bold orange line indicates signal from texas red channel of pncA wildtype probe. Dotted orange lines show signal from cy5 channel of pncA mutant probe. Water is used as a negative control.

Figure 7 illustrates an rrs singleplex amplification plot for rrs gene qPCR pilot experiment. The reaction was done using SYBR green chemistry in H37RV gDNA in triplicates using rrs primers. Water is added as the qPCR negative control. The amplification efficiency was checked by viewing the signal in the amplification plot which is drawn with fluorescence in the Y axis contains and number of cycles in the X axis. Figure 8a illustrates processed signals from duplex assays of inhA and katG target region with 20 different TB samples. Four differentially labelled probes were used which produces signal from four different channels. This plot shows signals form FAM and HEX channels of katG probes. Bold green line indicates signals from FAM channel from wild type katG probe. Dotted green line indicates signals from HEX channel form mutant type katG probe. Water is used as a negative control.

Figure 8b illustrates processed signals form duplex assays of inhA and katG target region with 20 different TB samples. Four differentially labelled probes were used which produces signal from four different channels. This plot shows signals from texas red (TxRd) and Cyanin 5 channels of inhA probes. Bold green line indicates signals from texas red channel from wild type inhA probe. Dotted green line indicates signals from Cy5 channel form mutant type inhA probe. Water is used as a negative control.

Figure 9 illustrates testing compatibility of gyrA and pncA primers and probes with reference strains. Wild type probe of gyrA and mutant probe of pncA was used. Bold orange line indicates signal from FAM channel of gyrA wildtype probe. Dotted orange lines show signal from HEX channel of pncA mutant probe. Water is used as a negative control.

Figure 10 illustrates the rpob - rrs duplex assay for detecting resistance to both first line and second - line anti-tuberculosis drugs simultaneously in a single step, namely kanamycin and rifampicin by targeting mutations in the rpoB (solid line) and rrs gene (dotted line).

The figures depict embodiments of the present disclosure for purposes of illustration only. One skilled in the art will readily recognize from the following description that alternative embodiments of the steps illustrated herein may be employed without departing from the principles of the disclosure described herein.

DETAILED DESCRIPTION

The invention relates to kits, methods, systems of detecting Mycobacterium tuberculosis bacteria in a biological sample. Broadly, a number of nucleic acid amplification methods are used for detection of particular microbial / viral nucleic acid and the invention discloses such a nucleic acid amplification method.

In an embodiment, the invention relates to the use of polymerase chain reaction (PCR) for detecting Mycobacterium tuberculosis bacteria in a biological sample. PCR is a technique known to a person of ordinary skill in the art and is used in molecular biology to amplify one or more copies of a piece of deoxyribonucleic acid (DNA) across several orders of magnitude, generating thousands to millions of copies of a particular sequence of DNA.

More specifically, the invention relates to the use of real-time PCR (qPCR) for detecting Mycobacterium tuberculosis bacteria in a biological sample. q-PCR is also a technique known to a person of ordinary skill in the art which monitors the amplification of a specific primer targeted DNA molecule during the PCR, i.e. in real-time, using fluorescent detection probes and the invention also comprises of the primers and probes and the kits and compositions containing such primers and probes for detecting the bacterial DNA.

In the existing art, various inventions relating to the use of real-time PCR exist which can detect multi-drug resistant strains of Mycobacterium tuberculosis. However, a commercial process, kit, assay, system is not available to detect multi- drug resistant strains of Mycobacterium tuberculosis. Also, the commercial kits available purporting to detect MDR and XDR strains suffer from various drawbacks, including but not limited to, lack of specificity, lack of standardization, lack of sensitivity, time consuming processes and necessity of expensive infrastructure like cold storage facilities and biosafety, which the present invention is be capable of resolving.

In one embodiment, the invention comprises of a medical diagnostic kit based on real-time PCR technology for detecting single nucleic acid polymorphisms which confer broad range resistance to Mycobacterium tuberculosis. In some aspects of the invention, the primers and/or probes of the invention can be labelled with a fluorescent moiety. Fluorescent moieties for use in real-time PCR detection are known to persons skilled in the art and are available from various commercial sources.

In one embodiment, the invention comprises; a first set of primers having the sequences shown in SEQ ID No: 3, SEQ ID No 4, SEQ ID No: 11,SEQ ID No 12, SEQ ID No 19 or SEQ ID No 20; a the second set of primers comprises SEQ ID No 5,SEQ ID No 6, SEQ ID No 13, SEQ ID No 14, SEQ ID No 21 or SEQ ID No 22;preferably a first probe has a sequence of SEQ ID No 1, SEQ ID No, 7, SEQ ID No 9, SEQ ID No 15, SEQ ID No 17, or SEQ ID No 23; a second probe has a sequence of SEQ ID No 2, SEQ ID No 8, SEQ ID NolO, SEQ ID No 16, SEQ ID No 18 or SEQ ID No 24. Also, covered by the present invention are complementary sequences or sequences having at least 80%, 85%, 90% or 95% homology or identity with any of the sequences in SEQ ID Nos.: 1 to SEQ ID Nos 24.

In another embodiment, the invention discloses a method of detecting multi-drug resistant Mycobacterium sp. or extensively drug resistant Mycobacterium sp. or a combination of both in a sample, in particular, M. tuberculosis. The said method may comprise contacting a set of primers and fluorescent-tagged probes according to the present invention with a nucleic acid-containing sample, wherein, once a probe is bound to a target M. tuberculosis nucleic acid in the sample resistant to rifampicin and/or isoniazid and/or any first line anti-tuberculosis drug, a detectable signal is provided; and detecting the said detectable signal. For primer and probe design the following method was followed: 1000 bp flanking sequences for these frequently mutated single nucleotide polymorphisms (SNP) that were present in genes were retrieved in FASTA format by using fetch sequence function in Galaxy, in order to design primers and taqman probes. To design specific primers the regions that contain random mutations were masked. Primers and taqman probes were designed using Beacon designer software. Primers were designed around the most frequently mutated SNPs that were found in TB genome database. Followed by the locus tag of the respected SNPs the forward and reverse primers were assigned as A and B respectively. To improve the specificity, the taqman probes were designed in such a way that the SNP is present in the middle of the probe sequence. The wild type taqman probes were labelled with either FAM dye or Texas Red (TxRd) dye while the mutated taqman probes were labelled with either HEX dye or Cyanin 5 dye. BHQ1 quencher was used for wild type probes and BHQ quencher for mutant probes. Followed by the locus tag of the represented SNPs the mutant and wild type probes were assigned as W and M respectively. The designed primers and taqman probes were then ordered form Sigma Aldrich. For primer validation, the following process was followed: Primers were validated by performing a pilot qPCR experiment with SYBR green. Reference strain H37Rv sample was used in triplicates to perform pilot experiment. 20μί qPCR reactions were set up which consist of IX Roche fast SYBR green master mix, 0.3uM forward primer, 0.3uM reverse primer and 5μί of 5ng^L of genomic DNA. Genomic DNA of H37Rv reference strains collected from NIRT, was used in all the qPCR reactions. qPCR reactions were carried out in ABI step one instrument. qPCR cycling conditions are as follows: 95° C for 10 mins; 40X (95° C for 15sec; 60° C for lmin); 95° C for 15sec; 60° C for 1 min; 95° C for 15sec. The primers were validated by looking at the Ct value in the amplification plot and by looking at the peaks in the melting curve. For optimization of primer concentration, the following method was followed: he primer concentrations were optimized by doing qPCR in the following primer concentration: 1000ng^L, 500ng^L, 250ng^L and 125ng^L in all combinations. 1. Prepared and dispensed diluted primers a. 60μΙ. of 8μΜ working solutions of both forward (fwd) and reverse (rev) primers were prepared in the first tubes of 2 separate 8-tube strips. b. 30μΙ. of water was dispensed into tubes 2-4. c. 30μΙ. of the 8μΜ primer solution was transferred from tube 1 into tube 2 and mixed thoroughly by pipetting up and down at least 5 times. d. Transfer and mixing was repeated from tube 2 to 3 and 3 to 4. e. Using a pipette, 5μΙ. from the strip tubes containing diluted fwd primer were transferred into the first 4 wells down columns 1-4 of a 48-well PCR plate. After adding reverse primer, qPCR mix, and template, final concentrations of forward primer will be 1000, 500, 250 and 125nM. f. Similarly, 5μΙ. from the strip-tubes containing diluted rev primer were transferred into the first 4 wells across rows A-D. After adding PCR mix and template, final concentrations of reverse primer will be 1000, 500, 250 and 125nM. 2. qPCR master mix was prepared for 38 x 20 μΐ_^ reactions which contains IX Roche fast SYBR green mastermix and water.

3. 26μΙ. of master mix was aliquoted into all wells in the PCR plate that contain primers (A1-D4).

4. 18μΙ. from each of wells Al through D4 was mixed and transferred to wells A5 through D8.

5. 2μΙ. template-containing 25ng of genomic DNA was added to one set of reaction (columns 1-4) and 2μΙ. water to the other (columns 5-8).

6. qPCR cycling conditions are as follows: 95° C for 10 mins; 40X (95° C for 15sec; 60° C for lmin);95° C for 15sec; 60° C for 1 min; 95° C for 15sec. 7. Fluorescent plot was evaluated for reaction contains target nucleic acid. Primer combination with the lowest Ct and the highest fluorescence were chosen as the optimal primer combination.

8. Melting curves were also analysed to identify the optimal primer combination. Primer combinations with single, sharp peaks in the presence of target nucleic acid

(columns 1-4) and nothing detected in the corresponding no-template control (columns 5-8) was chosen as the optimal primer combination.

For validation of probes, the following method was followed: Probes were validated by performing a pilot qPCR experiment. Reference strain H37Rvsample was used in triplicates to perform pilot experiment. First each probe was validated separately and then wild type and mutant probes were validated together. 20μί qPCR reactions were set up which consist of IX roche probe master mix, forward primer (optimal concentration), reverse primer (optimal concentration), 250nM of probe and 5μί of 5ng^L of genomic DNA. Genomic DNA of H37Rv reference strains collected from NIRT, was used in all the qPCR reactions. qPCR reactions were carried out in ABI step one instrument. qPCR cycling conditions are as follows: 95° C for 10 mins; 40X (95° C for 15sec; 60° C for lmin); 95° C for 15sec; 60° C for 1 min; 95° C for 15sec. The probes were validated by looking at the Ct value in the amplification plot. For optimization of the q-PCR conditions, the following method was followed: The optimal annealing temperature of primers and probes was found by performing a gradient qPCR. Gradient qPCR was performed with the genomic DNA of H37Rv reference strain. Gradient qPCR were set up for a range of 12 different annealing temperatures to find the optimal annealing temperatures of primers and probes. The annealing temperature at which the reaction gave specific and low Ct value was selected and a reproducibility experiment was performed at that temperature. If the primer pair can reproduce the results by giving specific and good yield then that temperature was selected as the optimal annealing temperature. For optimization of the probe concentration, the following method was followed: The optimal probe concentrations were validated by doing a qPCR reaction at 5 different concentrations: 250ng^L, 200ng^L, 150ng^L, lOOng^L and 50ng^L. The concentration at which the fluorescence efficiency is high is taken as the optimal probe concentration.

In one embodiment, the present invention provides a method of detecting multidrug resistant Mycobacterium sp. in a sample, in particular, M. tuberculosis. The said method may comprise contacting a set of primer probes according to the present invention with a nucleic acid-containing sample, wherein, once a fluorescent-tagged probe is bound to a target M. tuberculosis nucleic acid in the sample, resistant to isoniazid or rifampicin or a combination of both or any first line anti-tuberculosis drug, a detectable signal is provided; and detecting the said detectable signal.

In another embodiment, the present invention provides a method of detecting extensively drug resistant Mycobacterium sp. in a sample, in particular, M. tuberculosis. The said method may comprise contacting a set of primer probes according to the present invention with a nucleic acid-containing sample, wherein, once a fluorescent-tagged probe is bound to a target M. tuberculosis nucleic acid in the sample, resistant to fluoroquinolone, aminoglycoside, pyrazinamide or kanamycin or any combination thereof or any second line anti-tuberculosis drug, a detectable signal is provided; and detecting the said detectable signal.

In yet another embodiment, the present invention provides a method of simultaneously detecting extensively drug resistant and multi-drug resistant Mycobacterium sp. in a sample simultaneously in a single step, in particular M. tuberculosis. The said method may comprise contacting a set of primers and fluorescent-tagged probes according to the present invention with a nucleic acid- containing sample, wherein, once a fluorescent-tagged probe is bound to a target M. tuberculosis nucleic acid in the sample resistant to both rifampicin, isoniazid, or any first line anti-tuberculosis drug or any combination thereof and in addition, to pyrazinamide, fluoroquinolone, aminoglycoside or kanamycin or any second line anti-tuberculosis drug or any combination thereof, a detectable signal is provided; and detecting the said detectable signal. The role of the abovementioned drugs and their gene targets have been summed up in Table 1 below:

Table 1: Gene targets for first and second line anti-tuberculosis drugs

In one embodiment of the invention, the multiplex q-PCR kit may comprise of the katG and /or inhA and / or rpoB and / or any other similar gene targeted mutations and / or any combinations of the afore mentioned genes in its assays.

In yet another embodiment of the invention, the multiplex q-PCR kit may comprise of rrs and / or gyrA and / or pncA or any other similar gene targeted mutations and or any combinations of the aforementioned genes in its assays. In still another embodiment of the invention, the multiplex q-PCR kit may comprise of katG and /or inhA and / or pncA and / or gyrA and / or rrs and/or any similarly placed gene which is target of first line anti-tuberculosis drug and / or any similarly placed gene which is the target of any second-line anti-tuberculosis drug and / or any combinations of any of the aforementioned gene targeted mutations in its assays.

Figure 1, illustrates a brief overview of the multiplex q-PCR method comprising the invention. At step 101, the invention-specific primers and probes designed to detect a broad range of MDR and XDR strains of M. tuberculosis are mixed with the sample containing the bacterial DNA obtained from prospective patients in the presence of appropriate enzymes. At step 102, the polymerase action results in polymerization of nucleic acid in the mixture more specifically the polymerization of the region targeted by the invention -specific probes and primers. At step 103, the DNA probes are degraded and fluorescent signals are emanated from the reaction mixture. At step 104, the target DNA of interest from MDR and XDR strains is amplified and the fluorescence is measured for subsequent DNA amount calculations. Referring to Fig. 2, the results of a singleplex assay for detecting resistance to first

- line anti-tuberculosis drugs namely isoniazid by targeting mutations in the katG gene are described. The primers were validated through a pilot q-PCR using SYBR green with the reference strain H37Rv and further the concentrations to be used for the q-PCR reaction were optimized at 250nM (forward primer, Seq. Id No. 3), 500nM (reverse primer, Seq. Id No. 5). Taqman probe Seq. Id No. 2, (250nM) with fluorophore HEX at the 5 and quencher BHQ at the were designed in such a way that the targeted mutation was present in the centre of the sequence. Taqman probe Seq. Id No. 1 (250nM) for wild type katG gene were also designed using 6FAM fluorophore at the 5 end and BHQ1 quencher at the 3 end. An annealing temperature of 62.4 deg C was fixed as optimum annealing temperature after conducting a gradient PCR. Assay performance was tested by employing a Standard Curve technique and the katG assay gave a correlation coefficient (R²) value of 0.999 and efficiency of the assay was 129%. The assay was tested on genomic DNA samples of 10 tuberculosis patients. 8 samples tested showed amplification signals from the FAM channel indicating they are wild-type genotypes (Fig. 2a) while 2 samples tested showed amplification from the HEX channel indicating that they are katG mutant genotypes (Fig. 2b).

Referring to Fig. 3 the results of a singleplex assay for detecting resistance to first

- line anti-tuberculosis drugs namely isoniazid by targeting mutations in the inhA gene are described. The primers were validated through a pilot q-PCR using SYBR green with the reference strain H37Rv and further the concentrations to be used for the q-PCR reaction were optimized at ΙΟΟΟηΜ (forward primer, Seq. Id No. 4), 250nM (reverse primer, Seq. Id No. 6). Taqman probe Seq. Id No. 8, (250nM) with fluorophore Cyanin5 at the 5 and quencher BHQ at the were designed in such a way that the targeted mutation was present in the centre of the sequence. Taqman probe Seq. Id No. 7 (250nM) for wild type inhA gene were also designed using TxRd fluorophore at the 5 end and BHQ1 quencher at the 3 end. An annealing temperature of 57.9 deg C was fixed as optimum annealing temperature after conducting a gradient PCR. Assay performance was tested by employing a Standard Curve technique and the inhA assay gave a correlation coefficient (R²) value of 0.996 and efficiency of the assay was 113%. The assay was tested on genomic DNA samples of 10 tuberculosis patients. 3 samples tested showed amplification signals from the TxRd channel indicating they are wild-type genotypes (Fig. 3, bold lines) while 7 samples tested showed amplification from the Cyanin5 channel indicating that they are inhA mutant genotypes (Fig. 3, dotted lines).

Referring to Fig. 4, the results of a singleplex assay for detecting resistance to first - line anti-tuberculosis drugs, namely rifampicin, by targeting mutations in the rpoB gene are described. The primers were validated through a pilot q-PCR using SYBR green with the reference strain H37Rv and further the concentrations to be used for the q-PCR reaction were optimized at ΙΟΟΟηΜ (forward primer, Seq. Id No. 19), 500nM (reverse primer, Seq. Id No. 21). Taqman probe Seq. Id No. 18, (250nM) with fluorophore HEX at the 5 and quencher BHQ at the were designed in such a way that the targeted mutation was present in the centre of the sequence. Taqman Probe Seq. Id No. 17 (250nM) for wild type rpoB gene were also designed using 6FAM fluorophore at the 5 end and BHQ1 quencher at the 3 end. An annealing temperature of 59.4 deg C was fixed as optimum annealing temperature after conducting a gradient PCR. Assay performance was tested by employing a Standard Curve technique and the rpoB assay gave a correlation coefficient (R²) value of 0.96 and efficiency of the assay was 99.8%. The assay was tested on genomic DNA samples of 10 tuberculosis patients. 2 samples tested showed amplification signals from the FAM channel indicating they are wild-type genotypes (bold lines) while 7 samples tested showed amplification from the HEX channel indicating that they are rpoB mutant genotypes (dotted lines).

Referring to Fig. 5, the results of a singleplex assay for detecting resistance to second - line anti-tuberculosis drugs, namely fluoroquinolones / aminoglycoside, by targeting mutations in the gyrA gene are described. The primers were validated through a pilot q-PCR using SYBR green with the reference strain H37Rv and further the concentrations to be used for the q-PCR reaction were optimized at 250nM (forward primer, Seq. Id No. 11), 125nM (reverse primer, Seq. Id No. 13). Taqman probe Seq. Id No. 10, (250nM) with fluorophore HEX at the 5 and quencher BHQ at the were designed in such a way that the targeted mutation was present in the centre of the sequence. Taqman Probe Seq. Id No. 9 (250nM) for wild type gyrA gene were also designed using 6FAM fluorophore at the 5 end and BHQ1 quencher at the 3 end. An annealing temperature of 59.4 deg C was fixed as optimum annealing temperature after conducting a gradient PCR. Assay performance was tested by employing a Standard Curve technique and the gyrA assay gave a correlation coefficient (R²) value of 0.99 and efficiency of the assay was 112.9%. The assay was tested on genomic DNA samples of 10 tuberculosis patients. 7 samples tested showed amplification signals from the FAM channel indicating they are wild-type genotypes while 3 samples tested showed amplification from the HEX channel indicating that they are gyrA mutant genotypes (Fig. 5).

Referring to Fig. 6, the results of a singleplex assay for detecting resistance to second - line anti-tuberculosis drugs, namely pyrazinamide, by targeting mutations in the pncA gene are described. The primers were validated through a pilot q-PCR using SYBR green with the reference strain H37Rv and further the concentrations to be used for the q-PCR reaction were optimized at 500nM forward primer, Seq. Id No. 12), 250nM (reverse primer, Seq. Id No. 14). Taqman probe Seq. Id No. 16, 250nM with fluorophore Cyanin5 at the 5 and quencher BHQ at the were designed in such a way that the targeted mutation was present in the centre of the sequence. Taqman Probe Seq. Id No. 15 250nM for wild type pncA gene were also designed using TxRd fluorophore at the 5 end and BHQ1 quencher at the 3 end. An annealing temperature of 56.6 deg C was fixed as optimum annealing temperature after conducting a gradient PCR. Assay performance was tested by employing a Standard Curve technique and the pncA assay gave a correlation coefficient (R²) value of 0.98 and efficiency of the assay was 110.6%. The assay was tested on genomic DNA samples of 10 tuberculosis patients. 4 samples tested showed amplification signals from the TxRd channel indicating they are wild-type genotypes while 6 samples tested showed amplification from the Cyanin5 channel indicating that they are pncA mutant genotypes (Fig. 6). Referring to Fig. 7, the results of a singleplex assay for detecting resistance to second - line anti-tuberculosis drugs, namely kanamycin, by targeting mutations in the rrs gene are described. The primers were validated through a pilot q-PCR using SYBR green with the reference strain H37Rv and further the concentrations to be used for the q-PCR reaction were optimized at 250nM (forward primer, Seq. Id No. 12), 125nM (reverse primer, Seq. Id No. 14). Taqman probe Seq. Id No. 16, (250nM) with fluorophore Cyanin5 at the 5 and quencher BHQ at the were designed in such a way that the targeted mutation was present in the centre of the sequence. Taqman Probe Seq. Id No. 15 (250nM) for wild type rrs gene were also designed using TxRd fluorophore at the 5 end and BHQ1 quencher at the 3 end. An annealing temperature of 58.9 deg C was fixed as optimum annealing temperature after conducting a gradient PCR. Assay performance was tested by employing a Standard Curve technique and the rrs assay gave a correlation coefficient (R²) value of 0.99 and efficiency of the assay was 112.6%. The assay was tested on genomic DNA samples of 10 tuberculosis patients. 3 samples tested showed amplification signals from the TxRd channel indicating they are wild-type genotypes while 7 samples tested showed amplification from the Cyanin5 channel indicating that they are rrs mutant genotypes (Fig. 7).

Referring to Fig.8, the results of a duplex assay for detecting resistance to first - line anti-tuberculosis drugs, namely isoniazid, by targeting mutations in the katG and inhA gene are described. The primers were validated through a pilot q-PCR using SYBR green with the reference strain H37Rv and further the concentrations to be used for the q-PCR reaction were optimized. For duplex assay of katG and inhA, a 50 ul reaction that contains two primers of katG, two primers of inhA, two differentially labelled probes of katG target region (fam - wild type, HEX- mutant), two differentially labelled probes of inhA target region (texas red- wild type, Cy5- mutant), probe master mix, water and DNA sample was mixed in a PCR tube. Results with reference strain gave high signal from fam channel and low signal form HEX channel for katG, and high signal from texas red channel and low signal from Cy5 channel for inhA. Subsequently the assay was tested on genomic DNA samples of 20 tuberculosis patients. Samples were evaluated in 2 steps: first by checking the FAM-HEX channel and then by checking the TxRd-Cyanin5 channel. 12 samples tested showed amplification signals from the FAM and 8 samples from HEX channel indicating they are wild-type genotypes and katG mutant genotypes respectively (Fig. 8 a). 10 samples tested showed amplification from the TxRd channel and 10 samples from the Cyanin5 channel indicating that they are wild type genotypes and inhA mutant genotypes respectively (Fig. 8b)

Referring to Fig. 9, the results of a duplex assay for detecting resistance to second - line anti-tuberculosis drugs, namely pyrazinamide and fluoroquinolones and aminoglycosides or any combinations of the three by targeting mutations in the gyrA and pncA gene are described. The primers were validated through a pilot q- PCR using SYBR green with the reference strain H37Rv and further the concentrations to be used for the q-PCR reaction were optimized (Fig. 9). For duplex assay of gyrA and pncA, a 50 uL reaction that contains two primers of gyrA, two primers of pncA, two differentially labelled probes of gyrA target region (FAM - wild type, HEX - mutant), two differentially labelled probes of pncA target region (texas red- wild type, Cy5 -mutant), probe master mix, water and DNA sample was mixed in a PCR tube. Results with reference strain gave high signal from fam channel and low signal form HEX channel for gyrA, and high signal from texas red channel and low signal from Cy5 channel for pncA. Subsequently the assay was tested on genomic DNA samples of 20 tuberculosis patients. Samples were evaluated in 2 steps: first by checking the FAM-HEX channel and then by checking the TxRd-Cyanin5 channel. 12 samples tested showed amplification signals from the FAM and 8 samples from HEX channel indicating they are wild-type genotypes and gyrA mutant genotypes respectively. 11 samples tested showed amplification from the TxRd channel and 9 samples from the Cyanin5 channel indicating that they are wild type genotypes and pncA mutant genotypes respectively.

Referring to Fig. 10, the results of a duplex assay for detecting resistance to both first line and second - line anti-tuberculosis drugs simultaneously in a single step, namely kanamycin and rifampicin by targeting mutations in the rpoB and rrs gene are described. The primers were validated through a pilot q-PCR using S YBR green with the reference strain H37Rv and further the concentrations to be used for the q- PCR reaction were optimized. For duplex assay of rpoB and rrs, a

reaction that contains two primers of rpoB, two primers of rrs, two differentially labelled probes of rpoB target region (FAM - wild type, HEX- mutant), two differentially labelled probes of rrs target region (texas red- wild type, Cy5-mutant), probe master mix, water and DNA sample was mixed in a PCR tube. Results with reference strain gave high signal from fam channel and low signal form HEX channel for rpoB, and high signal from texas red channel and low signal from Cy5 channel for rrs. Subsequently the assay was tested on genomic DNA samples of 20 tuberculosis patients. Samples were evaluated in 2 steps: first by checking the FAM-HEX channel and then by checking the TxRd-Cyanin5 channel. 11 samples tested showed amplification signals from the FAM and 9 samples from HEX channel indicating they are wild-type genotypes and rpoB mutant genotypes respectively. 13 samples tested showed amplification from the TxRd channel and 7 samples from the Cyanin5 channel indicating that they are wild type genotypes and rrs mutant genotypes respectively.

The list of all nucleotide sequences used for the singleplex and duplex experiments have been enlisted in the Table 2 below:

1 ctcgatgccg ctggtgatcg c Probe Wild type - 1

2 ctcgatgccg ttggtgatcg c Probe mutant - 1

Duplex for

3 gggtgttcgt ccatacga Forward Primer - 1

testing resistance

4 cgtggacata ccgatttc Forward Primer - 2

to first line anti-

5 cacactttcg gtaagaccca Reverse Primer - 1

TB drugs

6 gactgaacgg gatacgaa Reverse Primer - 2

(Isoniazid)

7 cgcggcgaga cgataggttg Probe Wild type - 2

8 cgcggcgaga tgataggttg Probe mutant - 2

9 tcgatctacg acagcctggt g Probe Wild type - 1

10 tcgatctacg gcagcctggt g Probe mutant - 1 Duplex for

11 gagaccatgg gcaactac Forward Primer - 1 testing resistance

12 gtcgacgatg atcaacg Forward Primer - 2 to second line

13 gcttcggtgt acctcatc Reverse Primer - 1 anti-TB drugs

14 gtcgctcact acatcac Reverse Primer - 2 (Fluoroquinolone

& Pyrazinamide)

15 acgtccacca tacgttcggg Probe Wild type - 2

16 acgtccacca cacgttcggg Probe mutant - 2

17 cccagcgccg acagtcgg Probe Wild type - 1

18 cccagcgcca acagtcgg Probe mutant - 1 Duplex for testing resistance

19 ccagaacaac ccgctgtc Forward Primer - 1

to first line and

20 cgctagtaat cgcagatc Forward Primer - 2

second line anti-

21 gagccgatca gaccgatg Reverse Primer - 1

TB drugs

22 gtacggctac cttgttac Reverse Primer - 2

(Rifampicin &

23 acacaccgcc cgtcacgtca tgaaag Probe Wild type - 2 Kanamycin)

24 acacaccgcc cgtcgcgtca tgaaag Probe mutant - 2

Table 2: List of nucleotide sequences for primers and probes used for experiments

In one embodiment, the use of multiplex q-PCR kit may enable a rapid detection of first-line and second line anti-tuberculosis drug resistant bacteria simultaneously in a single step, specifically within a time period of less than 6 hours and more specifically within a time period of less than 2 hours, such that the total time period for the detection is at reduced by a factor of at least 3 and maximally by a factor of 10 as compared to conventional state of art methods.

In another embodiment, the sensitivity of the multiplex q-PCR kit is increased in so far as not more than 10 bacilli per millilitre of sample are required for detection of broad range mutations of multi drug resistant strains and extensively drug resistant strains or any combinations thereof of M. tuberculosis.

In another embodiment, the multiplex q-PCR kit has the capacity to rapidly detect first-line and second line anti-tuberculosis drug resistant bacteria simultaneously in a single step even at public health centre level with little / no access to biosafety, cold storage and power supply.

In another embodiment, the specificity of the multiplex q-PCR kit is increased tremendously in so far as to ensure that not less than 98% specificity is achieved for detection of broad range mutations of multi drug resistant strains and extensively drug resistant strains or any combinations thereof of M. tuberculosis. In still another embodiment of the invention, the multiplex q-PCR kit which enables rapid, sensitive and extremely specific detection of multi drug resistant and extensively drug resistant anti-tuberculosis strains of M. tuberculosis, contains a range of primers and fluorescent-tagged probes and sequence information with a potential to add any new mutations of drug resistant tuberculosis in the kit assay. In yet another embodiment of the invention, the multiplex q-PCR kit which enables rapid, sensitive and extremely specific detection of multi drug resistant and / or extensively drug resistant anti-tuberculosis strains or any combinations thereof of M. tuberculosis, can be stored at ambient room temperature and can be made readily available at a commercial scale. The foregoing description shall be interpreted as illustrative and not in any limiting sense. A person of ordinary skill in the art would understand that certain modifications could come within the scope of this disclosure.

Claims

WE CLAIM:

1. A system for detection of multi-drug resistant (MDR) tuberculosis bacterial strains in a biological sample characterizing in that the said system employs a duplex real-time polymerase chain reaction (q-PCR) assay for simultaneous detection of presence of most-frequent mutations in Mycobacterium tuberculosis H37Rv genome which confer resistance to first line anti-TB drugs; said system comprising:

A DNA sequence comprising nucleotides of M. tuberculosis H37Rv genome dataset from base number 2155158 to base number 2155178 (Seq. ID No. 1) characterizing in that said DNA sequence is tagged at the 5 end with a fluorophore and at the 3 end with a quencher and wherein said DNA sequence shares at least 95% sequence similarity with Seq. ID No. 1,

A DNA sequence comprising nucleotides of M. tuberculosis H37Rv genome dataset from base number 2155158 to base number 2155178 wherein the DNA sequence contains a single nucleotide mutation at position 2155168 (Seq. ID No. 2) characterizing in that said DNA sequence is tagged at the 5 end with a fluorophore and at the 3 end with a quencher and wherein said DNA sequence shares at least 95% sequence similarity with Seq. ID No. 2,

A DNA sequence comprising two primer pairs wherein the two forward primers share at least 95% sequence similarity with Seq. ID No. 3 and Seq. ID No. 4 respectively and the two reverse primers share at least 95% sequence similarity with Seq. ID No. 5 and Seq. ID No. 6,

A DNA sequence comprising nucleotides of M. tuberculosis H37Rv genome dataset from base number 1673415 to base number 1673434 (Seq. ID No. 7) characterizing in that said DNA sequence is tagged at the 5 end with a fluorophore and at the 3 end with a quencher and wherein said DNA sequence shares at least 95% sequence similarity with Seq. ID No. 7, and

A DNA sequence comprising nucleotides of M. tuberculosis H37Rv genome dataset from base number 1673415 to base number 1673434 wherein the DNA sequence contains a single nucleotide mutation at position 1673425 (Seq. ID No. 8) characterizing in that said DNA sequence is tagged at the 5 end with a fluorophore and at the 3 end with a quencher and wherein said DNA sequence shares at least 95% sequence similarity with Seq. ID No. 8.

2. The system of Claim 1, characterizing in that the said fluorophores covalently linked to Seq. ID No. 1, Seq. ID No. 2, at the 5 end of DNA sequence comprise preferably of 6FAM, HEX respectively and wherein the said quencher covalently linked to 3 end of DNA sequence Seq. ID No. 1 and Seq. ID No. 2 is preferably BHQ1.

3. The system of Claim 1, characterizing in that the said fluorophores covalently linked to Seq. ID No. 7 and Seq. ID No. 8at the 5 end of DNA sequence comprise preferably of TxRd andCyanin5 respectively and wherein the said quencher covalently linked to 3 end of DNA sequence Seq. ID No. 7 and Seq. ID No. 8 is preferably BHQ.

4. A system for detection of extensively-drug resistant (XDR) tuberculosis bacterial strains in a or biological sample characterizing in that the said system employs a duplex real-time polymerase chain reaction (q-PCR) assay for simultaneous detection of presence of most-frequent mutations in Mycobacterium tuberculosis H37Rv genome which confer resistance to second line anti-TB drugs; said system comprising:

A DNA sequence comprising nucleotides of M. tuberculosis H37Rv genome dataset from base number 7572 to base number 7592 (Seq. ID No. 9) characterizing in that said DNA sequence is tagged at the 5 end with a fluorophore and at the 3 end with a quencher and wherein said DNA sequence shares at least 95% sequence similarity with Seq. ID No. 9,

A DNA sequence comprising nucleotides of M. tuberculosis H37Rv genome dataset from base number 7572 to base number 7592 wherein the DNA sequence contains a single nucleotide mutation at position 7582 (Seq. ID No. 10) characterizing in that said DNA sequence is tagged at the 5 end with a fluorophore and at the 3 end with a quencher and wherein said DNA sequence shares at least 95% sequence similarity with Seq. ID No. 10,

A DNA sequence comprising two primer pairs wherein the two forward primers share at least 95% sequence similarity with Seq. ID No. 11 and Seq. ID No. 12 respectively and the two reverse primers share at least 95% sequence similarity with Seq. ID No. 13 and Seq. ID No. 14,

A DNA sequence comprising nucleotides of M. tuberculosis H37Rv genome dataset from base number 2289242 to base number 2289262 (Seq. ID No. 15) characterizing in that said DNA sequence is tagged at the 5 end with a fluorophore and at the 3 end with a quencher and wherein said DNA sequence shares at least 95% sequence similarity with Seq. ID No. 15, and

A DNA sequence comprising nucleotides of M. tuberculosis H37Rv genome dataset from base number 2289242 to base number 2289262 wherein the DNA sequence contains a single nucleotide mutation at position 2289252 (Seq. ID No. 16) characterizing in that said DNA sequence is tagged at the 5 end with a fluorophore and at the 3 end with a quencher and wherein said DNA sequence shares at least 95% sequence similarity with Seq. ID No. 16.

5. The system of Claim 4, characterizing in that the said fluorophores covalently linked to Seq. ID No. 9, Seq. ID No. 10, at the 5 end of DNA sequence comprise preferably of 6FAM, HEX respectively and wherein the said quencher covalently linked to 3 end of DNA sequence Seq. ID No. 9 and Seq. ID No. 10 is preferably BHQ1.

6. The system of Claim 4, characterizing in that the said fluorophores covalently linked to Seq. ID No. 15 and Seq. ID No. 16at the 5 end of DNA sequence comprise preferably of TxRd andCyanin5 respectively and wherein the said quencher covalently linked to 3 end of DNA sequence Seq. ID No. 15 and Seq. ID No. 16 is preferably BHQ.

7. A system for simultaneous detection of extensively-drug resistant (XDR) tuberculosis bacterial strains and multi-drug resistant (MDR) tuberculosis bacterial strains in a biological sample characterizing in that the said system employs a single- step duplex real-time polymerase chain reaction (q-PCR) assay for simultaneous detection of presence of most-frequent mutations in Mycobacterium tuberculosis H37Rv genome which confer resistance to both first line and second line anti-TB drugs; said system comprising:

A DNA sequence comprising nucleotides of M. tuberculosis H37Rv genome dataset from base number 761147 to base number 761163 (Seq. ID No. 17) characterizing in that said DNA sequence is tagged at the 5 end with a fluorophore and at the 3 end with a quencher and wherein said DNA sequence shares at least 95% sequence similarity with Seq. ID No. 17,

A DNA sequence comprising nucleotides of M. tuberculosis H37Rv genome dataset from base number 761147 to base number 761163 wherein the DNA sequence contains a single nucleotide mutation at position 761155 (Seq. ID No. 18) characterizing in that said DNA sequence is tagged at the 5 end with a fluorophore and at the 3 end with a quencher and wherein said DNA sequence shares at least 95% sequence similarity with Seq. ID No. 18,

A DNA sequence comprising two primer pairs wherein the two forward primers share at least 95% sequence similarity with Seq. ID No. 19 and Seq. ID No. 20 respectively and the two reverse primers share at least 95% sequence similarity with Seq. ID No. 21 and Seq. ID No. 22,

A DNA sequence comprising nucleotides of M. tuberculosis H37Rv genome dataset from base number 1473233 to base number 1473257 (Seq. ID No. 23) characterizing in that said DNA sequence is tagged at the 5 end with a fluorophore and at the 3 end with a quencher and wherein said DNA sequence shares at least 95% sequence similarity with Seq. ID No. 23, and

A DNA sequence comprising nucleotides of M. tuberculosis H37Rv genome dataset from base number 1473233 to base number 1473257 wherein the DNA sequence contains a single nucleotide mutation at position 1473246 (Seq. ID No. 24) characterizing in that said DNA sequence is tagged at the 5 end with a fluorophore and at the 3 end with a quencher and wherein said DNA sequence shares at least 95% sequence similarity with Seq. ID No. 24.

8. The system of Claim 7, characterizing in that the said fluorophores covalently linked to Seq. ID No. 17, Seq. ID No. 18, at the 5 end of DNA sequence comprise preferably of 6FAM, HEX respectively and wherein the said quencher covalently linked to 3 end of DNA sequence Seq. ID No. 17 and Seq. ID No. 18 is preferably BHQ1.

9. The system of Claim 7, characterizing in that the said fluorophores covalently linked to Seq. ID No. 23, Seq. ID No. 24, at the 5 end of DNA sequence comprise preferably of TxRd, Cyanin5 respectively and wherein the said quencher covalently linked to 3 end of DNA sequence Seq. ID No. 23 and Seq. ID No. 24 is preferably BHQ.

10. The system of Claim 7, wherein said system can also detect only multi-drug resistant (MDR) tuberculosis bacterial strains in a biological sample characterizing in that the said system employs a singleplex real-time polymerase chain reaction (q-PCR) assay for detection of presence of most- frequent mutations in Mycobacterium tuberculosis H37Rv genome which confer resistance to a first line anti-TB drug, more specifically to rifampicin; said system comprising:

A DNA sequence comprising nucleotides of M. tuberculosis H37Rv genome dataset from base number 761147 to base number 761163 wherein the DNA sequence contains a single nucleotide mutation at position 761155 (Seq. ID No. 18) characterizing in that said DNA sequence is tagged at the 5 end with a fluorophore and at the 3 end with a quencher and wherein said DNA sequence shares at least 95% sequence similarity with Seq. ID No. 18, and

A DNA sequence comprising a primer pair wherein the forward primers shares at least 95% sequence similarity with Seq. ID No. 19 and reverse primer shares at least 95% sequence similarity with Seq. ID No. 21.

11. The system of Claim 10, characterizing in that the said fluorophores covalently linked to Seq. ID No. 17, Seq. ID No. 18, at the 5 end of DNA sequence comprise preferably of 6FAM, HEX respectively and wherein the said quencher covalently linked to 3 end of DNA sequence Seq. ID No. 17 and Seq. ID No. 18 is preferably BHQ1.

12. The system of Claim 7, wherein said system can also detect only extensively- drug resistant(XDR) tuberculosis bacterial strains in a biological sample characterizing in that the said system employs a singleplex real-time polymerase chain reaction (q-PCR) assay for detection of presence of most- frequent mutations in Mycobacterium tuberculosis H37Rv genome which confer resistance to a second line anti-TB drug, more specifically to kanamycin; said system comprising:

A DNA sequence comprising nucleotides of M. tuberculosis H37Rv genome dataset from base number 1473233 to base number 1473257 (Seq. ID No. 23) characterizing in that said DNA sequence is tagged at the 5 end with a fluorophore and at the 3 end with a quencher and wherein said DNA sequence shares at least 95% sequence similarity with Seq. ID No. 23,

A DNA sequence comprising nucleotides of M. tuberculosis H37Rv genome dataset from base number 1473233 to base number 1473257 wherein the DNA sequence contains a single nucleotide mutation at position 1473246 (Seq. ID No. 24) characterizing in that said DNA sequence is tagged at the 5 end with a fluorophore and at the 3 end with a quencher and wherein said DNA sequence shares at least 95% sequence similarity with Seq. ID No. 24, and A DNA sequence comprising a primer pair wherein the forward primers shares at least 95% sequence similarity with Seq. ID No. 20 and reverse primer shares at least 95% sequence similarity with Seq. ID No. 22.

13. The system of Claim 12, characterizing in that the said fluorophores covalently linked to Seq. ID No. 23, Seq. ID No. 24, at the 5 end of DNA sequence comprise preferably of TxRd, Cyanin5 respectively and wherein the said quencher covalently linked to 3 end of DNA sequence Seq. ID No. 23 and Seq. ID No. 24 is preferably BHQ.

14. The system of Claim 4, wherein said system can also detect only extensively- drug resistant (XDR) tuberculosis bacterial strains in a biological sample characterizing in that the said system employs a singleplex real-time polymerase chain reaction (q-PCR) assay for detection of presence of most- frequent mutations in Mycobacterium tuberculosis H37Rv genome which confer resistance to a second line anti-TB drug, more specifically to pyrazinamide; said system comprising:

A DNA sequence comprising nucleotides of M. tuberculosis H37Rv genome dataset from base number 2289242 to base number 2289262 (Seq. ID No. 15) characterizing in that said DNA sequence is tagged at the 5 end with a fluorophore and at the 3 end with a quencher and wherein said DNA sequence shares at least 95% sequence similarity with Seq. ID No. 15,

A DNA sequence comprising nucleotides of M. tuberculosis H37Rv genome dataset from base number 2289242 to base number 2289262 wherein the DNA sequence contains a single nucleotide mutation at position 2289252 (Seq. ID No. 16) characterizing in that said DNA sequence is tagged at the 5 end with a fluorophore and at the 3 end with a quencher and wherein said DNA sequence shares at least 95% sequence similarity with Seq. ID No. 16, and

A DNA sequence comprising a primer pair wherein the forward primer shares at least 95% sequence similarity with Seq. ID No. 12 and reverse primer shares at least 95% sequence similarity with Seq. ID No. 14.

15. The system of Claim 14 characterizing in that the said fluorophores covalently linked to Seq. ID No. 15, Seq. ID No. 16, at the 5 end of DNA sequence comprise preferably of TxRd, Cyanin5 respectively and wherein the said quencher covalently linked to 3 end of DNA sequence Seq. ID No. 15 and Seq. ID No. 16 is preferably BHQ.

16. The system of Claim 4, wherein said system can also detect only extensively- drug resistant (XDR) tuberculosis bacterial strains in a biological sample characterizing in that the said system employs a singleplex real-time polymerase chain reaction (q-PCR) assay for detection of presence of most- frequent mutations in Mycobacterium tuberculosis H37Rv genome which confer resistance to a second line anti-TB drug, more specifically to fluoroquinolone or aminoglycoside; said system comprising:

A DNA sequence comprising nucleotides of M. tuberculosis H37Rv genome dataset from base number 2289242 to base number 2289262 (Seq. ID No. 09) characterizing in that said DNA sequence is tagged at the 5 end with a fluorophore and at the 3 end with a quencher and wherein said DNA sequence shares at least 95% sequence similarity with Seq. ID No. 09,

A DNA sequence comprising nucleotides of M. tuberculosis H37Rv genome dataset from base number 2289242 to base number 2289262 wherein the DNA sequence contains a single nucleotide mutation at position 2289252 (Seq. ID No. 10) characterizing in that said DNA sequence is tagged at the 5 end with a fluorophore and at the 3 end with a quencher and wherein said DNA sequence shares at least 95% sequence similarity with Seq. ID No. 10, and

A DNA sequence comprising a primer pair wherein the forward primer shares at least 95% sequence similarity with Seq. ID No. 11 and reverse primer shares at least 95% sequence similarity with Seq. ID No. 13.

17. The system of Claim 16 characterizing in that the said fluorophores covalently linked to Seq. ID No. 09, Seq. ID No. 10, at the 5 end of DNA sequence comprise preferably of 6FAM, HEX respectively and wherein the said quencher covalently linked to 3 end of DNA sequence Seq. ID No. 09 and Seq. ID No. 10 is preferably BHQ1.

18. A process for characterizing and detecting multi-drug resistant (MDR), extensively drug resistant (XDR) tuberculosis bacteria or a combination of both in a biological sample comprising:

Exposing nucleic acids from said biological sample to

A pair or multiple pairs of primers from a plurality of primer pairs comprising forward primers and reverse primers (Seq. ID No. 3, Seq. ID No. 4, Seq. ID No. 5, Seq. ID No. 6, Seq. ID No. 11, Seq. ID No. 12, Seq. ID No. 13, Seq. ID No. 14, Seq. ID No. 19, Seq. ID No. 20, Seq. ID No. 21 or Seq. ID No. 22),

An oligonucleotide probe or multiple oligonucleotide probes from a plurality of oligonucleotide probes (Seq. ID No. 1, Seq. ID No. 7, Seq. ID No. 9, Seq. ID No. 15, Seq. ID No. 17 or Seq. ID No. 23) specific for different regions of the wild-type M. tuberculosis coding for kat G, inh A, gyr A, pncA, rpoB or rrs genes respectively, and

An oligonucleotide probe or multiple oligonucleotide probes from a plurality of oligonucleotide probes (Seq. ID No. 2, Seq. ID No. 8, Seq. ID No. lOSeq. ID No. 16, Seq. ID No. 18or Seq. ID No. 24) specific for mutation variant regions of M. tuberculosis coding for kat G, inh A, gyr A, pncA, rpoB and rrs genes respectively; under conditions and ingredients including but not limited to buffers, deoxynucleotides, enzymes conducive to a polymerase chain reaction wherein each of the said oligonucleotide probes is attached at their 5 end to a distinct fluorophore molecule selected from a plurality of fluorophores like 6-FAM, HEX, TxRd and Cyanin and wherein each of the said oligonucleotide probes is additionally attached at its 3 end to a distinct quencher molecule from amongst BHQ or BHQ1 quencher characterizing in that, said hybridization and amplification of target nucleic acid samples enables quantitative detection and fluorophore based distinction of presence of multi-drug resistant tuberculosis strains, extensively-drug resistant tuberculosis strains or a combination of both.

19. The system of Claim 1, wherein said system can also detect only multi-drug resistant (XDR) tuberculosis bacterial strains in a biological sample characterizing in that the said system employs a singleplex real-time polymerase chain reaction (q-PCR) assay for detection of presence of most- frequent mutations in Mycobacterium tuberculosis H37Rv genome which confer resistance to a first line anti-TB drug, more specifically to isoniazid; said system comprising:

A DNA sequence comprising nucleotides of M. tuberculosis H37Rv genome dataset from base number 1673415 to base number 1673434 (Seq. ID No. 7) characterizing in that said DNA sequence is tagged at the 5 end with a fluorophore and at the 3 end with a quencher and wherein said DNA sequence shares at least 95% sequence similarity with Seq. ID No. 7,

A DNA sequence comprising nucleotides of M. tuberculosis H37Rv genome dataset from base number 1673415 to base number 1673434 wherein the DNA sequence contains a single nucleotide mutation at position 1673425 (Seq. ID No. 8) characterizing in that said DNA sequence is tagged at the 5 end with a fluorophore and at the 3 end with a quencher and wherein said DNA sequence shares at least 95% sequence similarity with Seq. ID No. 8, and

A DNA sequence comprising a primer pair wherein the forward primer shares at least 95% sequence similarity with Seq. ID No. 4 and reverse primer shares at least 95% sequence similarity with Seq. ID No. 6.

20. The system of Claim 1, wherein said multi-drug resistant M. tuberculosis bacteria in a biological sample comprise of bacteria resistant to isoniazid.

21. The system of Claim 4, wherein said extensively drug resistant M. tuberculosis bacteria in a biological sample comprise of bacteria resistant to fluoroquinolone / aminoglycoside, pyrazinamide or a combination of both.

22. The system of Claims 7, 10 and 12 wherein said multi-drug resistant M. tuberculosis bacteria and extensively drug resistant M. tuberculosis bacteria in a biological sample comprise of bacteria resistant to rifampicin, kanamycin or a combination of both.

23. The system of Claims 4, 14 and 16 wherein the said extensively-drug resistant M. tuberculosis bacteria in a biological sample comprise of bacteria resistant to fluoroquinolones / aminoglycosides, pyrazinamide or a combination of both.

24. The system of Claim 1, wherein said system is capable of detecting most- frequent mutations, less-frequent mutations as well any combination of most- frequent mutations and less-frequent mutations in kat G gene by employing singleplex real-time polymerase chain reaction (q-PCR) assays characterizing in that, the less frequent kat G mutation occur in the codons comprising but not limited to R463L of Mycobacterium tuberculosis H37Rv genome.

25. The system of Claim 1, wherein said system is capable of detecting most- frequent mutations, less-frequent mutations as well any combination of most- frequent mutations and less-frequent mutations in inh A gene by employing singleplex or duplex real-time polymerase chain reaction (q-PCR) assays characterizing in that, the less frequent inh A mutations occur in the codons comprising but not limited to T8C, G17T and A16G or any combinations thereof in the promoter region of Mycobacterium tuberculosis H37Rv genome.

26. The system of Claim 4, wherein said system is capable of detecting most- frequent mutations, less-frequent mutations as well any combination of most- frequent mutations and less-frequent mutations in gyr A gene by employing singleplex or duplex real-time polymerase chain reaction (q-PCR) assays characterizing in that, the less frequent gyr A mutations occur in the codons comprising but not limited toA90V, S91P, D94H,D94A and G247S or any combinations thereof of Mycobacterium tuberculosis H37Rv genome.

27. The system of Claim 4, wherein said system is capable of detecting most- frequent mutations, less-frequent mutations as well any combination of most- frequent mutations and less-frequent mutations in pncA gene by employing singleplex or duplex real-time polymerase chain reaction (q-PCR) assays characterizing in that, the less frequent pncA mutations occur in the codons comprising but not limited to the promoter region and initial codons (within 12 codons) or any combinations thereof of Mycobacterium tuberculosis H37Rv genome.

28. The system of Claim 7, wherein said system is capable of detecting most- frequent mutations, less-frequent mutations as well any combination of most- frequent mutations and less-frequent mutations in rpoB gene by employing singleplex or duplex real-time polymerase chain reaction (q-PCR) assays characterizing in that, the less frequent rpoB mutations occur in the codons comprising but not limited to A516V, H526A, H526T and L533P or any combinations thereof of Mycobacterium tuberculosis H37Rv genome.

29. The system of Claim 7, wherein said system is capable of detecting most- frequent mutations, less-frequent mutations as well any combination of most- frequent mutations and less-frequent mutations in rrs gene by employing singleplex or duplex real-time polymerase chain reaction (q-PCR) assays characterizing in that, the less frequent rrs mutations occur in the nucleotide regions comprising but not limited to 1402, 1408 or a combination thereof of Mycobacterium tuberculosis H37Rv genome.

30. A system claimed in any of the claims above, wherein said system enables detection of MDR-TB, XDR-TB and MDR-TB plus XDR-TB in a single test by reducing time required for detection of M. tuberculosis by at least a factor in the range of 3 to 10 as compared to existing state-of-art technologies.

31. A system claimed in any of the claims above, wherein said system enables detection of MDR-TB, XDR-TB and MDR-TB plus XDR-TB with high specificity not less than 98% for detection of broad range mutations in a plurality of genes comprising inhA, katG, rpoB, pncA, gyr A and rrs or combinations thereof.

32. A system claimed in any of the claims above, wherein said system enables detection of broad range mutations of multi drug resistant strains and extensively drug resistant strains of M. tuberculosis with extremely high sensitivity requiring not more than 10 bacilli per millilitre for said detection.