WO2009087688A2 - Method for detection of nucleic acid of mycobacterium and uses thereof - Google Patents

Method for detection of nucleic acid of mycobacterium and uses thereof Download PDF

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Publication number
WO2009087688A2
WO2009087688A2 PCT/IN2009/000016 IN2009000016W WO2009087688A2 WO 2009087688 A2 WO2009087688 A2 WO 2009087688A2 IN 2009000016 W IN2009000016 W IN 2009000016W WO 2009087688 A2 WO2009087688 A2 WO 2009087688A2
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Prior art keywords
seq
mycobacterium
nucleic acid
oligonucleotide
sample
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PCT/IN2009/000016
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French (fr)
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WO2009087688A3 (en
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Rajeev Soni
Nupur Mehrotra
Prabuddha Kumar Kundu
Kajal Arora
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Premas Biotech Pvt.Ltd
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Publication of WO2009087688A3 publication Critical patent/WO2009087688A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6862—Ligase chain reaction [LCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to a method for detection, enumeration and/or identification of disease and non disease related conditions inclusive of but not limited to microorganisms, pathogens, mutations, cancer and/or SNP variations in a sample.
  • the present invention particularly relates to a nucleic acid based detection method for diagnosis of infectious diseases, genetically transmitted diseases, drug resistance, mutations, cancer and/or SNP variations.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • RT-PCR reverse transcription polymerase chain reaction
  • NASBA Self-Sustained Synthetic Reaction
  • Q.beta.-Replicase Q.beta.
  • NAATs nucleic acid amplification tests
  • infectious diseases NAATs supersede the routine growth-based culture and microscopy methods in terms of their ease of use and a low turn-around time (Report on the "Evaluation of Diagnostic Tests for Detection of Genitourinary Chlamydia Infections by STD Control Branch, California Department of Health Services, Department of Epidemiology & Biostatistics, University of California, San Francisco; published in March 2001).
  • the NAATs provide substantial time and cost saving over traditional culture methods for determining the presence of a given pathogen in a clinical specimen.
  • NAATs also provide quantification of the pathogen thereby producing more efficient results. These technologies vary among themselves in their sensitivity and specificity to provide an accurate diagnosis. There is an increased demand for tests which maintain very high positive predictive value (PPV) and negative predictive value (NPV) for detection of all microorganisms and give reproducible results.
  • PPV positive predictive value
  • NPV negative predictive value
  • EP patent 0395292 (Barry et al, 1997) describes a method for generating DNA probes specific for an organism useful for distinguishing between genera and species.
  • the document provides the DNA probes obtained from a variable intergenic region intermediate the genes coding for 16S rRNA and 23 S rRNA wherein the probes are specific for a variety of species including Aeromonas hydrophilla, A. salmonicida, Clostridium difficile, Mycobacterium bovis, M. tuberculosis and Salmonella typhimurium.
  • US patent no 5786149 (Leckie et al, 1998) provides a method of detecting a target sequences from M.
  • tuberculosis using the gap filling ligase chain reaction and oligonucleotides useful in detecting Mycobacterium tuberculosis in a sample, wherein the oligonucleotides are with recessed ends.
  • the oligonucleotides provided in the disclosure detect only M, tuberculosis and do not detect M. avium.
  • US 5,830,711 (Barany et al, 1998) describes a method for distinguishing a first nucleotide sequence which differs by at least a single base from a second nucleotide sequence by using ligase chain reaction (LCR) utilizing the thermophilic DNA ligase from Thermus aquaticus to detect a target DNA sequence.
  • LCR ligase chain reaction
  • the detection methods described in the prior art give variable results with respect to non- specificity, poor amplification signal and non reproducibility. It is therefore desirable to provide a method of detection of the target nucleic acid in a sample using ligase mediated amplification reaction that overcomes the above-mentioned disadvantages. It is an object of the present invention to provide a method and oligonucleotides for detection of a target nucleic acid with high sensitivity and efficacy.
  • the present invention relates to a nucleic acid based detection method for the diagnosis of disease and non disease related conditions inclusive of but not limited to microorganisms, pathogens, mutations, cancer and/or SNP variations in a sample.
  • the present invention particularly relates to a nucleic acid based detection method for detection of a target nucleic acid of an infectious agent selected from a group consisting of Mycobacterium tuberculosis, M. bovis and M. avium.
  • the method disclosed in the present invention is also useful for detection of single nucleotide polymorphism (SNP) and mutations such as mutations resulting in drug resistance.
  • SNP single nucleotide polymorphism
  • One aspect of the present invention is to provide a method for detecting a target nucleic acid sequence in a sample for detection of an infectious agent using isothermal nucleic acid amplification reaction (INAR), wherein the infectious agent is selected from a group consisting of Mycobacterium tuberculosis, M. avium and M. bovis.
  • INBR isothermal nucleic acid amplification reaction
  • Another aspect of the present invention is to provide a method for detecting a target nucleic acid sequence in a sample for detection of mutations resulting in drug resistance using isothermal nucleic acid amplification reaction (INAR), wherein drug resistance is caused in Mycobacterium species due to mutations.
  • INAR isothermal nucleic acid amplification reaction
  • Another aspect of the present invention is to provide oligonucleotide sequences for detection of an infectious agent or mutations resulting in drug resistance using isothermal nucleic acid amplification reaction (INAR), wherein the infectious agent is selected from a group consisting of Mycobacterium tuberculosis, M. avium and M. bovis.
  • INBR isothermal nucleic acid amplification reaction
  • Another aspect of the present invention is to provide oligonucleotide sequences for detection of Mycobacterium tuberculosis, M. avium, and/or M. bovis in a sample.
  • Another aspect of the present invention is to provide oligonucleotide sequences for detection of mutations in Kat G gene of Mycobacterium tuberculosis resulting in drug resistance against drugs such as isoniazid (INH).
  • the diagnostic kit provided in the invention is highly sensitive, specific, easy to use, has a low turnaround time in comparison to conventional detection methods and capable of detecting low titers.
  • One aspect of the present invention relates to a process for detecting a target nucleic acid of Mycobacterium in a sample, wherein the process comprises (a) providing a sample; (b) providing a set of 4 oligonucleotides, wherein the set of oligonucleotides is selected from a group consisting of set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 17-20), set 5 (SEQ ID NO: 22-25), set 6 ( SEQ ID NO: 27-30), set 7 ( SEQ ID NO: 32-35), set 8 (SEQ ID NO: 37-40), set 9 (SEQ ID NO: 42-45), set 10 (SEQ ID NO: 47-50), and set 13 (SEQ ID NO: 62-65); (c) providing thermostable ligase; (d) mixing the above to obtain a reaction mixture; (e) subjecting the reaction mixture to series of cycles comprising denaturation,
  • Another aspect of the present invention relates to oligonucleotide set selected from a group consisting of set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12- 15), set 4 (SEQ ID NO: 17-20), set 5 (SEQ ID NO: 22-25), set 6 ( SEQ ID NO: 27-30), set 7 ( SEQ ID NO: 32-35), set 8 (SEQ ID NO: 37-40), set 9 (SEQ ID NO: 42-45), set 10 (SEQ ID NO: 47-50), and set 13 (SEQ ID NO: 62-65); wherein the oligonucleotide set is useful for detection of the target nucleic acid of Mycobacterium tuberculosis and/or Mycobacterium bovis in a sample.
  • Another aspect of the present invention relate to oligonucleotide set having nucleotide sequence as set forth in SEQ ID NO: 42-45, wherein the oligonucleotide set detects the target nucleic acid of Mycobacterium tuberculosis, Mycobacterium bovis and M. avium in a sample.
  • Yet another aspect of the present invention relates to oligonucleotide set having nucleotide sequence as set forth in SEQ ID NO: 47-50, wherein the oligonucleotide set detects the target nucleic acid of M. avium in a sample.
  • kits for detection of a target nucleic acid of Mycobacterium in a sample comprises the oligonucleotide set selected from a group consisting of set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 17-20), set 5 (SEQ ID NO: 22-25), set 6 ( SEQ ID NO: 27-30), set 7 ( SEQ ID NO: 32-35), set 8 (SEQ ID NO: 37-40), set 9 (SEQ ID NO: 42-45), set 10 (SEQ ID NO: 47-50), and set 13 (SEQ ID NO: 62-65).
  • set 1 SEQ ID NO: 2-5
  • set 2 SEQ ID NO: 7-10
  • set 3 SEQ ID NO: 12-15
  • set 4 SEQ ID NO: 17-20
  • set 5 SEQ ID NO: 22-25
  • set 6 SEQ ID NO: 27-30
  • set 7 SEQ ID NO: 32-35
  • set 8 SEQ ID NO: 37-40
  • kits for detection of a target nucleic acid of Mycobacterium tuberculosis, Mycobacterium bovis and M. avium in a sample the kit comprises the oligonucleotide set having nucleotide sequence as set forth in SEQ ID NO: 42-45.
  • kits for detection of a target nucleic acid of Mycobacterium avium in a sample comprises the set having nucleotide sequence as set forth in SEQ ID NO: 47-50.
  • Lane 1 M-Low molecular weight marker
  • Lane 2 Positive control- 3.2 Kb plasmid (pPBPC2) comprising 250 bp from the region of 85252-85301 of Mycobacterium genome having accession number AE000516,
  • Lane 3 HGD (negative control with human genomic DNA)
  • Lane 4 Sl (known Tb sample)
  • Lane 5 S2 (known Tb sample)
  • Lane 6 S3 (unknown sample)
  • Lane 7 S4 (unknown sample)
  • the present invention relates to a method for detection, enumeration and/or identification of disease and non disease related conditions inclusive of but not limited to microorganisms, pathogens, mutations, cancer and/or SNP variations in a biological sample and/or a pure culture of a microorganism.
  • the present invention particularly relates to a nucleic acid based detection method for detection of a target nucleic acid of an infectious agent in a sample, wherein the infectious agent is selected from a group consisting of Mycobacterium tuberculosis, M. bovis and/or M. avium.
  • the method disclosed in the present invention is also useful for detection of single nucleotide polymorphism (SNP) and mutations, such as mutations resulting in drug resistance.
  • SNP single nucleotide polymorphism
  • Genomic DNA can be extracted from a sample by using methods well known in the art and used as a template DNA for ligase mediated amplification reaction.
  • Isothermal Nucleic Acid Amplification Reaction describes a reaction of 18-40 cycles in which both annealing and amplification are carried out at a temperature ranging from 55-74°C while denaturation is carried out at a temperature ranging from 90-99°C in the presence of one or more thermostable ligase enzymes.
  • One embodiment of the present invention provides a method for detection of a target nucleic acid of an infectious agent in a sample using isothermal nucleic acid amplification reaction (INAR), wherein the infectious agent is selected from a group consisting of Mycobacterium tuberculosis, M. avium and M. bovis.
  • INBR isothermal nucleic acid amplification reaction
  • Another embodiment of the present invention provides a method for detection of a target nucleic acid of an infectious agent in a sample using isothermal nucleic acid amplification reaction (INAR), wherein the infectious agent is Mycobacterium tuberculosis and/or M. bovis.
  • INAR isothermal nucleic acid amplification reaction
  • Another embodiment of the present invention provides a method for detection of a target nucleic acid of an infectious agent in a sample using isothermal nucleic acid amplification reaction (INAR), wherein the infectious agent is Mycobacterium tuberculosis.
  • Another embodiment of the present invention provides a method for detection of a target nucleic acid of an infectious agent in a sample using isothermal nucleic acid amplification reaction (INAR), wherein the infectious agent is Mycobacterium bovis.
  • Another embodiment of the present invention provides a method for detection of a target nucleic acid of an infectious agent in a sample using isothermal nucleic acid amplification reaction (INAR), wherein the infectious agent is Mycobacterium avium.
  • Another embodiment of the present invention provides a method for detection of a target nucleic acid of an infectious agent in a sample using isothermal nucleic acid amplification reaction (INAR), wherein the infectious agent is Mycobacterium avium subsp. paratuberculosis KlO.
  • INAR isothermal nucleic acid amplification reaction
  • Another embodiment of the present invention provides a method for detection of a target nucleic acid of an infectious agent in a sample using isothermal nucleic acid amplification reaction (INAR) 5 wherein the infectious agent is Mycobacterium avium 104.
  • INAR isothermal nucleic acid amplification reaction
  • Another embodiment of the present invention is to provide a method for detecting a target nucleic acid sequence in a sample for detection of mutations resulting in drug resistance using isothermal nucleic acid amplification reaction (INAR), wherein drug resistance is caused in Mycobacterium species due to mutations.
  • INAR isothermal nucleic acid amplification reaction
  • One of the preferred embodiments of the present invention is to provide oligonucleotide set for detection of Mycobacterium tuberculosis, M. avium, and/or M. bovis nucleic acid in a sample.
  • Still yet another embodiment of the present invention is to provide oligonucleotide set for detection of M. bovis nucleic acid in a sample.
  • Still yet another embodiment of the present invention is to provide oligonucleotide set for detection of M. avium nucleic acid in a sample.
  • Another embodiment of the present invention provides oligonucleotide set for detection of Mycobacterium avium subsp. paratuberculosis KlO. Another embodiment of the present invention provides oligonucleotide set for detection of Mycobacterium avium 104.
  • the present invention provides the nucleotide sequences having oligonucleotide sequence as forth in SEQ ID NO: 1-65. Size of the oligonucleotide sequences provided in the present invention ranges from 15-50 bases.
  • the oligonucleotide set (Set 1) having nucleotide sequence as set forth in SEQ ID NO: 2-5.
  • the oligonucleotides were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 62716-62765 (SEQ ID NO: 1) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis. Region 1 : CAA CAT GTT TTC GAT GGG ACT GTA AGC GGC ACA CAC AAT TTC GTC GCC AA SEQ ID NO: 1
  • oligonucleotide set (Set 2) having nucleotide sequence as set forth in SEQ ID NO: 7-10.
  • the oligonucleotides were synthesized using the methods well known in the art.
  • oligonucleotides were designed from nucleotide position 85252-85301 (SEQ ID NO: 6) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis. Region: 2 AAA CCC AGA CAG ATT AGT GAA TGC GTG GCT CGG CGT TGT AGG CGG TGG AA SEQ ID NO: 6
  • oligonucleotide set (Set 3) having nucleotide sequence as set forth in SEQ ID NO: 12-15.
  • the oligonucleotides were synthesized using the methods well known in the art.
  • oligonucleotides were designed from nucleotide position 82712-82761 (SEQ ID NO: 11) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis. Region: 3 CGT GCG CGT TTG GAG GTC CCT GAG CGA TGG GCG ATC TGA GCA TTA GCC AG SEQ ID NO: 11
  • oligonucleotide set (Set 4) having nucleotide sequence as set forth in SEQ ID NO: 17-20.
  • the oligonucleotides were synthesized using the methods well known in the art.
  • oligonucleotides were designed from nucleotide position 2041-2088 (SEQ ID NO: 16) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis. Region 4: GAA AGG GCG CAA TGG ACG CGG CTA CGA CAA GAG TTG GCC TCA CCG ACT SEQ ID NO: 16
  • oligonucleotide set (Set 5) having nucleotide sequence as set forth in SEQ ID NO: 22-25 were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 79850 to 79897 (SEQ ID NO: 21) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis. Region 5: GAT CGC TGA GAT TCA CTT GTT CGG CAC CCA GGA GTA TCG CTG GGT GCT SEQ ID NO: 21
  • oligonucleotide set (Set 6) having nucleotide sequence as set forth in SEQ ID NO: 27-30.
  • the oligonucleotides were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 4206202-4206249 (SEQ ID NO: 26) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis.
  • Region 6 TGG CGC CCA CGC TCT CGG TAA TGG CCC AGG AAT CGT TGT CGG TCC GCG SEQ ID NO: 26
  • oligonucleotide set (Set 7) having nucleotide sequence as set forth in SEQ ID NO: 32-35.
  • the oligonucleotides were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 4203720-4203769 (SEQ ID NO: 31) of the DNA sequence of Mycobacterium tuberculosis CDC1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis.
  • Region 7 GCG ACG CAG TAA CGC CCG CAG CCG AAG CAC GAC CTC CTC GAT GCT AAA CG SEQ ID NO: 31
  • oligonucleotide set (Set 8) having nucleotide sequence as set forth in SEQ ID NO: 37-40.
  • the oligonucleotides were synthesized using the methods well known in the art.
  • oligonucleotides were designed from nucleotide position 3002631-3002680 (SEQ ID NO: 36) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis.
  • Region 8 GGC TGG GCA ACG TTA TCT CGG CTT C C TCG GCC TGT TCC GCC AAC TCC CGG SEQ ID NO: 36 8A S 1 GGC TGG GCA ACG TTA TCT CGG CTT C S' SEQ ID NO: 37 8B 5' CTC GGC CTG TTC CGC CAA CTC CCG G 3' SEQ ID NO: 38 8C 5' GAA GCC GAG ATA ACG TTG CCC AGC C 3' SEQ ID NO: 39 8D 5'CCG GGA GTT GGC GGA ACA GGC CGA G 3' SEQ ID NO: 40
  • the oligonucleotide set (Set 9) having nucleotide sequence as set forth in SEQ ID NO: 42-45.
  • the oligonucleotides were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 50070 to 50119 (SEQ ID NO: 41) of the DNA sequence of Mycobacterium tuberculosis CDCl 551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium avium paratuberculosis KlO and Mycobacterium avium 104. Region: 9 GATGCCGCGGTCCTTGGCGATCTTGGCCGCCCGCACCGCGTCGATGATGA
  • oligonucleotide set (Set 10) having nucleotide sequence as set forth in SEQ ID NO: 47-50.
  • the oligonucleotides were synthesized using the methods well known in the art.
  • oligonucleotides were designed from nucleotide position 20679 to 20720 (SEQ ID NO: 46) of the DNA sequence of Mycobacterium avium ⁇ 0A (Accession number CP000479). This region is specific to detect Mycobacterium avium 104 and Mycobacterium avium paratuberculosis KlO. Region 10: CCGGTTGGTCATCGGGTTGTCGGGATCGTCGCGCAGCCGCTG SEQ ID NO: 46
  • the oligonucleotide set 11 and 12 having nucleotide sequence as set forth in SEQ ID NO: 52-55 and SEQ ID NO: 57-60 respectively.
  • the oligonucleotides were synthesized using the methods well known in the art.
  • the oligonucleotides set 11 (SEQ ID NO: 52-55) designed from 2152482 to 2152432 nucleotide (SEQ ID NO: 51) of the DNA sequence of Mycobacterium tuberculosis CDC1551 (Accession number AE000516). This region is prone to mutation resulting in drug resistance against drugs such as Isoniazid.
  • Region 12 AAC CGG TAA GGA CGC GAT CAC CAC CGG CAT CGA GGT CGT ATG GAC GAA C SEQ ID NO: 56
  • oligonucleotide set having nucleotide sequence as set forth in SEQ ID NO: 62-65.
  • the oligonucleotides were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 484256 to 484207 (SEQ ID NO: 61) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). Region 13:
  • oligonucleotide sequences having nucleotide sequence as set forth in SEQ. ID. NO: SEQ ID NO:2-5, SEQ ID NO:7-10, SEQ ID NO:12-15, SEQ ID NO:17-20, SEQ ID NO:22-25, SEQ ID NO:27-30, SEQ ID NO:32-35, SEQ ID NO:37-40, SEQ ID NO:42-45, SEQ ID NO:47-50, SEQ ID NO: 52-55, SEQ ID NO: 57-60 and SEQ ID NO: 62-65 were synthesized using the methods well known in the art.
  • Template DNA was isolated form various samples such as blood, sputum, lung tissue, cotyledons, ascitic fluid, pleural fluid, semen, urine, stool sample, milk, bronchoalveolar lavage, FNAC samples by using the method well known in the art. 100 ng of template
  • DNA from each sample was used for INAR.
  • Mycobacterium tuberculosis CDC1551 (Accession number AE000516) were used as positive control in the method for detection of the target nucleic acid using the oligonucleotides disclosed in the present invention.
  • Genomic DNA isolated from the blood sample of healthy subject was used as a negative control.
  • Genomic DNA from known tuberculosis positive patient and suspected tuberculosis patient were used as test samples.
  • the INAR showed amplification of expected fragment of 50 bp in positive control i.e. plasmid containing 250 bp of Mycobacterium nucleotide sequence in samples S l and S2 known to be positive for tuberculosis and in unknown samples S3 and S4.
  • Example 1-1 Detailed procedure of Isothermal Nucleic acid Amplification Reaction (INAR) using the oligonucleotide set disclosed in the present invention is explained in Example 1-1 1.
  • the above samples S l, S2, S3 and S4 were also tested for their MTb positive status using conventional culture methods and by PCR (Ogusku, Mauricio Analise de diferen tMeso prriismhie,r est ault.ilizados na PCR visando ao diagn ⁇ stico da tuberculose no Estado do Amazonas in Jornal Brasileiro de Pneumologia 30(4) - Jul/Ago de 2004), primers for amplifying 541bp region in IS61 10 gene were used.
  • the samples were found positive basis PCR amplification results analysis by 1.2% agarose gel electrophoresis.
  • oligonucleotide set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 (SEQ ID NO: 12-15), set 4 (SEQ ID NO: 17-20), set 5 (SEQ ID NO: 22-25), set 6 (SEQ ID NO: 27-30), set 7 (SEQ ID NO: 32-35), set 8 (SEQ ID NO: 37- 40), set 9 (SEQ ID NO: 42-45), set 10 (SEQ ID NO: 47-50), set 1 1 (SEQ ID NO: 52-55), oligonucleotide set 12 (SEQ ID NO: 57-60) and oligonucleotide set 13 (SEQ ID NO: 62- 65) used for detection of tuberculosis being part of the map positions mentioned in SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21 , SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO:
  • One embodiment of the present invention relates to a process for detecting a target nucleic acid of Mycobacterium in a sample, wherein the process comprises (a) providing a sample; (b) providing a set of 4 oligonucleotides, wherein the set of oligonucleotides is selected from a group consisting of set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 17-20), set 5 (SEQ ID NO: 22-25), set 6 ( SEQ ID NO: 27-30), set 7 ( SEQ ID NO: 32-35), set 8 (SEQ ID NO: 37-40), set 9 (SEQ ID NO: 42-45), set 10 (SEQ ID NO: 47-50), and set 13 (SEQ ID NO: 62-65); (c) providing thermostable ligase; (d) mixing the above to obtain a reaction mixture; (e) subjecting the reaction mixture to series of cycles comprising denaturation,
  • Another embodiment of the present invention relates to the process for detecting a target nucleic acid of Mycobacterium in a sample, wherein Mycobacterium is selected from a group consisting of Mycobacterium tuberculosis, M. bovis and M. avium.
  • Yet another embodiment of the present invention relates to the process for detecting a target nucleic acid of Mycobacterium using the oligonucleotide set disclosed in the present invention, wherein at least one oligonucleotide of said set of oligonucleotides is optionally attached to a label.
  • Still yet another embodiment of the present invention relates to the process for detecting a target nucleic acid of Mycobacterium using the oligonucleotide set disclosed in the present invention, wherein at least one oligonucleotide of said set of oligonucleotides is optionally attached to a label, wherein said label is selected from a group consisting of chromophores, fluorescent moieties, enzymes, antigens, and chemiluminescent moieties.
  • the present invention relates to the process for detecting a target nucleic acid of Mycobacterium using the oligonucleotide set disclosed in the present invention, wherein second and third oligonucleotides of said set of oligonucleotides are modified at 5' end with a phosphate group. Further, the present invention relates to the process for detecting a target nucleic acid of Mycobacterium using the oligonucleotide set disclosed in the present invention, wherein second and third oligonucleotides of said set of oligonucleotides are optionally modified at 3' end with phosphate, deoxy, alkyl or aryl group.
  • the present invention relates to the process for detecting a target nucleic acid of Mycobacterium using the oligonucleotide set disclosed in the present invention, wherein second and third oligonucleotides of said set of oligonucleotides are optionally modified at 3' end with a phosphate group.
  • the present invention relates to the process for detecting a target nucleic acid of Mycobacterium disclosed in the present invention, wherein the reaction mixture was subjected to 18-40 cycles; preferably 32 cycles.
  • the present invention relates to the process for detecting a target nucleic acid of Mycobacterium disclosed in the present invention, wherein annealing and ligation is carried out at a temperature ranging from about 55°C-74° C for 30seconds-5 minutes preferably at 68°C for 2 minutes.
  • the present invention provides oligonucleotide set selected from a group consisting of set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 17-20), set 5 (SEQ ID NO: 22-25), set 6 ( SEQ ID NO: 27-30), set 7 ( SEQ ID NO: 32-35), set 8 (SEQ ID NO: 37-40), set 9 (SEQ ID NO: 42-45), set 10 (SEQ ID NO: 47-50) and set 13 (SEQ ID NO: 62-65), wherein the oligonucleotide set is useful for detection of the target nucleic acid of Mycobacterium tuberculosis, Mycobacterium avium and/or Mycobacterium bovis in a sample.
  • Another embodiment of the present invention is to provide oligonucleotide set selected from a group consisting of set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 17-20), set 5 (SEQ ID NO: 22-25), set 6 ( SEQ ID NO: 27-30), set 7 ( SEQ ID NO: 32-35), set 8 (SEQ ID NO: 37-40), set 9 (SEQ ID NO: 42-45) and set 13 (SEQ ID NO: 62-65), wherein the oligonucleotide set is useful for detection of the target nucleic acid of Mycobacterium tuberculosis and/or Mycobacterium bovis in a sample.
  • Another embodiment of the present invention is to provide oligonucleotide set selected from a group consisting of set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 17-20), set 5 (SEQ ID NO: 22-25), set 6 ( SEQ ID NO: 27-30), set 7 ( SEQ ID NO: 32-35), set 8 (SEQ ID NO: 37-40), set 9 (SEQ ID NO: 42-45) and set 13 (SEQ ID NO: 62-65), wherein the oligonucleotide set is useful for detection of the target nucleic acid of Mycobacterium tuberculosis in a sample.
  • Another embodiment of the present invention is to provide oligonucleotide set selected from a group consisting of set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 17-20), set 5 (SEQ ID NO: 22-25), set 6 ( SEQ ID NO: 27-30), set 7 ( SEQ ID NO: 32-35), set 8 (SEQ ID NO: 37-40), set 9 (SEQ ID NO: 42-45) and set 13 (SEQ ID NO: 62-65), wherein the oligonucleotide set is useful for detection of the target nucleic acid of Mycobacterium bovis in a sample.
  • Yet another embodiment of the present invention is to provide oligonucleotide set for detection of a target nucleic acid of Mycobacterium avium in a sample, wherein the oligonucleotide set is having nucleotide sequence as set forth in SEQ ID NO: 47-50.
  • Another embodiment of the present invention provides oligonucleotide set for detection of Mycobacterium avium subsp. avium 104 in a sample, wherein the oligonucleotide set is having nucleotide sequence as set forth in SEQ ID NO: 47-50.
  • Another embodiment of the present invention provides oligonucleotide set for detection of Mycobacterium avium subsp. paratuberculosis KlO avium in a sample, wherein the oligonucleotide set is having nucleotide sequence as set forth in SEQ ID NO: 47-50.
  • the present invention further provides oligonucleotide set having nucleotide sequence as set forth in SEQ ID NO: 42-45, wherein the oligonucleotide set detects the target nucleic acid of Mycobacterium tuberculosis, Mycobacterium bovis and M. avium in a sample.
  • the present invention provides oligonucleotide set having nucleotide sequence as set forth in SEQ ID NO: 57-60, wherein said oligonucleotide set detects mutation at position 315 of KAT G gene of Mycobacterium.
  • the present invention provides a kit for detection of a target nucleic acid of Mycobacterium in a sample, said kit comprising the oligonucleotide set selected from a group consisting of set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 17-20), set 5 (SEQ ID NO: 22-25), set 6 ( SEQ ID NO: 27-30), set 7 ( SEQ ID NO: 32-35), set 8 (SEQ ID NO: 37-40), set 9 (SEQ ID NO: 42-45), set 10 (SEQ ID NO: 47-50), and set 13 (SEQ ID NO: 62-65).
  • set 1 SEQ ID NO: 2-5
  • set 2 SEQ ID NO: 7-10
  • set 3 SEQ ID NO: 12-15
  • set 4 SEQ ID NO: 17-20
  • set 5 SEQ ID NO: 22-25
  • set 6 SEQ ID NO: 27-30
  • set 7 SEQ ID NO: 32-35
  • the present invention provides a kit for detection of a target nucleic acid in a sample, said kit comprising the oligonucleotide set selected from a group consisting of set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 17-20), set 5 (SEQ ID NO: 22-25), set 6 ( SEQ ID NO: 27-30), set 7 ( SEQ ID NO: 32-35), set 8 (SEQ ID NO: 37-40), set 9 (SEQ ID NO: 42-45) and set 13 (SEQ ID NO: 62- 65) wherein the said kit is useful for detection of the target nucleic acid of Mycobacterium tuberculosis and/or Mycobacterium bovis in a sample.
  • the present invention provides a kit for detection of a target nucleic acid in a sample, said kit comprising the oligonucleotide set selected from a group consisting of set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 17-20), set 5 (SEQ ID NO: 22-25), set 6 ( SEQ ID NO: 27-30), set 7 ( SEQ ID NO: 32-35), set 8 (SEQ ID NO: 37-40), set 9 (SEQ ID NO: 42-45) and set 13 (SEQ ID NO: 62- 65) wherein the said kit is useful for detection of the target nucleic acid of Mycobacterium tuberculosis in a sample.
  • the present invention provides a kit for detection of a target nucleic acid in a sample, said kit comprising the oligonucleotide set selected from a group consisting of set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 2-5), set
  • kits wherein the said kit is useful for detection of the target nucleic acid of Mycobacterium bovis in a sample.
  • kits for detection of a target nucleic acid of Mycobacterium tuberculosis, Mycobacterium bovis and M. avium in a sample the kit comprises the oligonucleotide set having nucleotide sequence as set forth in SEQ ID NO: 42-45.
  • kit for detection of a target nucleic acid of Mycobacterium avium in a sample the kit comprises the set having nucleotide sequence as set forth in SEQ ID NO: 47-50.
  • kits for detection of Mycobacterium avium subsp. avium 104 in a sample the kit comprises the set having nucleotide sequence as set forth in SEQ ID NO: 47-50.
  • kits for detection of Mycobacterium avium subsp. paratuberculosis KlO avium in a sample the kit comprises the set having nucleotide sequence as set forth in SEQ ID NO: 47-50.
  • the present invention provides a diagnostic kit for detection of mutation resulting in drug resistance, said kit comprising oligonucleotide sequences having nucleotide sequence as set forth in SEQ ID NO: 52-55 and SEQ ID NO: 57-60.
  • Each oligonucleotide set provided in the present invention consists of at least 4 oligonucleotides (A, B, C and D) of the size ranging from 15-50 bases, wherein first oligonucleotide (A) is complementary to third oligonucleotide (C) and second oligonucleotide (B) is complementary to fourth oligonucleotide (D).
  • a phosphate group is attached to the 5' end of second and third oligonucleotide (B and C).
  • the 3' end of the second and third oligonucleotide (B and C) is optionally modified.
  • the second and third oligonucleotide (B and C) of each oligonucleotide set disclosed in the present invention are optionally modified by adding phosphate group at the 3' end.
  • the modification can be carried out by adding phosphate, deoxy, alkyl or aryl group at the 3' end.
  • One embodiment relates to labeling agent or a probe that can be optionally attached to 5' end and/or 3' end of any of the four (A, B, C and D) oligonucleotides in each set wherein the labeling agent or a probe is radioactive, enzymatic, colorimetric, fluorescent, chemiluminiscent and/or photo-active.
  • the amplified product obtained using the method disclosed in the present invention can be detected by various methods known in the art such as agarose gel electrophoresis, solution phase colorimetric detection using enzymes and photoactivity assays, solid phase colorimetric detection using enzymes and photoactivity assays, solution phase fluorimetric detection, solid phase fluorimetric detection, solution phase chemiluminiscent detection, solid phase chemiluminiscent detection, solution phase using nanoparticles, solid phase using nanoparticles, solution phase using radioactive detection and solid phase using radioactive detection method.
  • the kit disclosed in the present invention is based on INAR for the detection of a target nucleic acid of an infectious agent in a sample in one assay wherein the infectious agent may be present individually or in combination with each other.
  • the method of detection disclosed in the present invention is also useful for detecting cancer, mutations, single nucleotide polymorphism (SNP) and mutations resulting in drug resistance.
  • SNP single nucleotide polymorphism
  • kits containing all the necessary reagents to perform the methods of detection disclosed herein may contain specific oligonucleotide sequence sets optionally attached to a label, a suitable buffer and a thermostable ligase.
  • the kit may further contain a set of printed instructions indicating that the kit is useful for detection of the specific disease and/or non disease related conditions as disclosed in the present invention.
  • the oligonucleotide set (Set 1) having nucleotide sequence as set forth in SEQ ID NO: 2-5 were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 62716-62765 (SEQ ID NO: 1) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis. Region 1 : CAA CAT GTT TTC GAT GGG ACT GTA AGC GGC ACA CAC AAT TTC GTC GCC AA SEQ ID NO: 1
  • a phosphate group was attached to the 5' end of the oligonucleotides as set forth in SEQ ID NO: 3 and SEQ ID NO: 4.
  • the oligonucleotides as set forth in SEQ ID NO: 3 and SEQ ID NO: 4 were modified at 3' end by adding phosphate group.
  • the oligonucleotide set (Set 1) as set forth in SEQ ID NO: 2-5 was tested with blood, sputum, lung tissue, cotyledons, ascitic fluid, pleural fluid, semen, urine, stool sample, milk, bronchoalveolar lavage, FNAC samples and pure culture of Mycobacterium species.
  • the amplification reaction was carried out in a total volume of 20 ⁇ l containing 2 ⁇ l buffer (10X), 1 ⁇ l Thermostable ligase (5U/ ⁇ l), l ⁇ l of each oligonucleotide (SEQ ID NO: 2-5) at concentration 2.5ng each, lOOng of template DNA and ultrapure water to bring the reaction volume to 20 ⁇ l.
  • reaction conditions employed was as follows: first cycle- 95°C for 10 min and 65 0 C for 2 min followed by 31 cycles - 95°C for 1 min and 65°C for 2 min.
  • Various samples and controls were used for detection of Mycobacterium DNA.
  • the description of the different reactions (reactions 1-7) used as template DNA is given below.
  • Reaction 1 NC-Negative control- no template DNA in the reaction mixture
  • Reaction 2 HGD-Negative control- template DNA from healthy subject (not infected with
  • Reaction 3 Positive control- 3.2 Kb plasmid (pPBPCl) with 250 bp from the region of 62716-62765 of Mycobacterium genome having accession number AE000516.
  • Reaction 4 Sl -template DNA from sputum sample of known Mycobacterium tuberculosis positive patient
  • Reaction 5 S2-template DNA from sputum sample of known Mycobacterium tuberculosis infected patient
  • Reaction 6 S3- template DNA from sputum sample of suspected Mycobacterium tuberculosis infected patient
  • Reaction 7 S4- template DNA from sputum sample of suspected Mycobacterium tuberculosis infected patient
  • the reactions were electrophoresed on a 2.5% agarose gel at 100V for 45 min and captured on a gel documentation system. Amplification of expected fragment of 50 bp was observed in positive control i.e. plasmid containing 250 bp of Mycobacterium nucleotide sequence, in samples Sl and S2 known to be positive for tuberculosis and in unknown samples S3 and S4. Fragment of 25 bp due to primer annealing was obtained in negative control in the absence of the template nucleic acid.
  • the oligonucleotide set 1 (SEQ ID NO: 2-5) detects Mycobacterium tuberculosis and M bovis in a sample and does not detect Mycobacterium avium.
  • thermostable ligase was varied from 1 - 10 units with consistent and reproducible results.
  • oligonucleotide set (Set 2) having nucleotide sequence as set forth in SEQ ID NO: 7- 10 were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 85252-85301 (SEQ ID NO: 6) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis. Region: 2 AAA CCC AGA CAG ATT AGT GAA TGC GTG GCT CGG CGT TGT AGG CGG TGG AA SEQ ID NO: 6
  • the amplification reaction was carried out in a total volume of 20 ⁇ l containing 2 ⁇ l buffer (10X), 1 ⁇ l Thermostable ligase (5U/ ⁇ l), 1 ⁇ l of each oligonucleotide (SEQ ID NO: 7-10) at concentration 2.5ng each, lOOng of template DNA and ultrapure water to bring the reaction volume to 20 ⁇ l.
  • reaction conditions employed was as follows: first cycle -95 0 C for 10 min and 68°C for
  • reactions 1-7) used as template DNA is given below.
  • Reaction 1 NC-Negative control- no template DNA in the reaction mixture
  • Reaction 2 HGD-Negative control- template DNA from healthy subject (not infected with
  • Reaction 3 Positive control- 3.2 Kb plasmid (pPBPC2) with 250 bp from the region of
  • Reaction 4 Sl -template DNA from sputum sample of known Mycobacterium tuberculosis positive patient
  • Reaction 5 S2-template DNA from sputum sample of known Mycobacterium tuberculosis infected patient
  • Reaction 6 S3- template DNA from sputum sample of suspected Mycobacterium tuberculosis infected patient
  • Reaction 7 S4- template DNA from sputum sample of suspected Mycobacterium tuberculosis infected patient The reactions were electrophoresed on a 2.5% agarose gel at 100V for 45 min and captured on a gel documentation system. Amplification of expected fragment of 50 bp was observed in positive control i.e. plasmid containing 250 bp of Mycobacterium nucleotide sequence, in samples Sl and S2 known to be positive for tuberculosis and in unknown samples S3 and S4. Fragment of 25 bp due to primer annealing was obtained in negative control in the absence of the template nucleic acid ( Figure 1). The oligonucleotide set 2 (SEQ ID NO: 7-10 detects Mycobacterium tuberculosis and M. bovis in a sample and do not detect Mycobacterium avium.
  • oligonucleotide set having nucleotide sequence as set forth in SEQ ID NO: 12- 15 were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 82712-82761 (SEQ ID NO: 11) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis. Region: 3 CGT GCG CGT TTG GAG GTC CCT GAG CGA TGG GCG ATC TGA GCA TTA GCC AG SEQ ID NO : 11
  • a phosphate group was attached to the 5' end of the oligonucleotides as set forth in SEQ ID NO: 13 and SEQ ID NO: 14.
  • the oligonucleotides as set forth in SEQ ID NO: 13 and SEQ ID NO: 14 were modified at 3' end by adding phosphate group.
  • the oligonucleotide set as set forth in SEQ ID NO: 12-15 was tested with blood, sputum, lung tissue, cotyledons, ascitic fluid, pleural fluid, semen, urine, stool sample, milk, bronchoalveolar lavage, FNAC samples and pure culture of Mycobacterium species.
  • the reaction used for detection of the target nucleic acid was performed under similar conditions as described in Example 1.
  • the oligonucleotide set 3 detects Mycobacterium tuberculosis and M. bovis in a sample and does not detect Mycobacterium avium.
  • thermostable ligase was varied from 1-10 units with consistent and reproducible results.
  • Example 4 Isothermal Nucleic acid Amplification Reaction (INAR) using oligonucleotide set as set forth in SEQ ID NO: 17-20
  • the oligonucleotide set (Set 4) having nucleotide sequence as set forth in SEQ ID NO: 17- 20 were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 2041-2088 (SEQ ID NO: 16) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This r ( egion is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis.
  • Region 4 GAA AGG GCG CAA TGG ACG CGG CTA CGA CAA GAG TTG GCC TCA
  • a phosphate group was attached to the 5' end of the oligonucleotides as set forth in SEQ ID NO: 18 and SEQ ID NO: 19.
  • the oligonucleotides as set forth in SEQ ID NO: 18 and SEQ ID NO: 19 were modified at 3' end by adding phosphate group.
  • the oligonucleotide set as set forth in SEQ ID NO: 17-20 was tested with blood, sputum, lung tissue, cotyledons, ascitic fluid, pleural fluid, semen, urine, stool sample, milk, bronchoalveolar lavage, FNAC samples and pure culture of Mycobacterium species.
  • the reaction used for detection of the target nucleic acid was performed under similar conditions as described in Example 1. Similar results as described in the Example 1 were obtained in said samples with the oligonucleotide set as set forth in SEQ ID NO: 17-20.
  • the oligonucleotide set 4 detects Mycobacterium tuberculosis and M. bovis in a sample and does not detect Mycobacterium avium.
  • thermostable ligase was varied from 1-10 units with consistent and reproducible results.
  • oligonucleotide set (Set 5) having nucleotide sequence as set forth in SEQ ID NO: 22- 25 were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 79850 to 79897 (SEQ ID NO: 21) of the DNA sequence of Mycobacterium tuberculosis CDC1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis. Region 5: GAT CGC TGA GAT TCA CTT GTT CGG CAC CCA GGA GTA TCG CTG GGT GCT SEQ ID NO : 21
  • a phosphate group was attached to the 5' end of the oligonucleotides as set forth- in SEQ ID NO: 23 and SEQ ID NO: 24.
  • the oligonucleotides as set forth in SEQ ID NO: 23 and SEQ ID NO: 24 were modified at 3' end by adding phosphate group.
  • the oligonucleotide set as set forth in SEQ ID NO: 22-25 was tested with blood, sputum, lung tissue, cotyledons, ascitic fluid, pleural fluid, semen, urine, stool sample, milk, bronchoalveolar lavage, FNAC samples and pure culture of Mycobacterium species.
  • the reaction used for detection of the target nucleic acid was performed under similar conditions as described in Example 1. Similar results as described in the Example 1 were obtained in said samples with the oligonucleotide set as set forth in SEQ ID NO: 22-25.
  • the oligonucleotide set 5 (SEQ ID NO: 22-25) detects Mycobacterium tuberculosis and M bovis in a sample and does not detect Mycobacterium avium.
  • the annealing and ligation temperatures were varied from 50° to 74° C for 30seconds to 5 min. with similar results.
  • the enzyme concentration for thermostable ligase was varied from 1-10 units with consistent and reproducible results.
  • Example 6 Isothermal Nucleic acid Amplification Reaction (INAR) using oligonucleotide set as set forth in SEQ ID NO: 27-30
  • the oligonucleotide set (Set 6) having nucleotide sequence as set forth in SEQ ID NO: 27- 30 were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 4206202-4206249 (SEQ ID NO: 26) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis.
  • the oligonucleotide set as set forth in SEQ ID NO: 27-30 was tested with blood, sputum, lung tissue, cotyledons, ascitic fluid, pleural fluid, semen, urine, stool sample, milk, bronchoalveolar lavage, FNAC samples and pure culture of Mycobacterium species.
  • the reaction used for detection of the target nucleic acid was performed under similar conditions as described in Example 1. Similar results as described in the Example 1 were obtained in said samples with the oligonucleotide set as set forth in SEQ ID NO: 27-30.
  • the oligonucleotide set 6 (SEQ ID NO: 27-30) detects Mycobacterium tuberculosis and M bovis in a sample and does not detect Mycobacterium avium.
  • the annealing and ligation temperatures were varied from 50° to 74° C for 30seconds to 5 min. with similar results.
  • the enzyme concentration for thermostable ligase was varied from 1-10 units with consistent and reproducible results.
  • Example 7 Isothermal Nucleic acid Amplification Reaction (INAR) using oligonucleotide set as set forth in SEQ ID NO: 32-35
  • the oligonucleotide set (Set 7) having nucleotide sequence as set forth in SEQ ID NO: 32- 35 were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 4203720-4203769 (SEQ ID NO: 31) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis.
  • Region 7 GCG ACG CAG TAA CGC CCG CAG CCG AAG CAC GAC CTC CTC GAT GCT AAA CG SEQ ID NO : 31 7A 5 1 GCG ACG CAG TAA CGC CCG CAG CCG A S' SEQ ID NO: 32
  • a phosphate group was attached to the 5' end of the oligonucleotides as set forth in SEQ ID NO: 33 and SEQ ID NO: 34.
  • the oligonucleotides as set forth in SEQ ID NO: 33 and SEQ ID NO: 34 were modified at 3' end by adding phosphate group.
  • the oligonucleotide set as set forth in SEQ ID NO: 32-35 was tested with blood, sputum, lung tissue, cotyledons, ascitic fluid, pleural fluid, semen, urine, stool sample, milk, bronchoalveolar lavage, FNAC samples and pure culture of Mycobacterium species.
  • the reaction used for detection of the target nucleic acid was performed under similar conditions as described in Example 1. Similar results as described in the Example 1 were obtained in said samples with the oligonucleotide set as set forth in SEQ ID NO: 32-35.
  • the oligonucleotide set 7 detects Mycobacterium tuberculosis and M.
  • Example 8 Isothermal Nucleic acid Amplification Reaction (INAR) using oligonucleotide set as set forth in SEQ ID NO: 37-40
  • the oligonucleotide set (Set 8) having nucleotide sequence as set forth in SEQ ID NO: 37- 40 were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 3002631-3002680 (SEQ ID NO: 36) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis. Region 8: GGC TGG GCA ACG TTA TCT CGG CTT C C TCG GCC TGT TCC GCC AAC TCC CGG SEQ ID NO: 36
  • a phosphate group was attached to the 5' end of the oligonucleotides as set forth in SEQ ID NO: 38 and SEQ ID NO: 39.
  • the oligonucleotides as set forth in SEQ ID NO: 38 and SEQ ID NO: 39 were modified at 3' end by adding phosphate group.
  • the oligonucleotide set as set forth in SEQ ID NO: 37-40 was tested with blood, sputum, lung tissue, cotyledons, ascitic fluid, pleural fluid, semen, urine, stool sample, milk, bronchoalveolar lavage, FNAC samples and pure culture of Mycobacterium species.
  • the reaction used for detection of the target nucleic acid was performed under similar conditions as described in Example 1. Similar results as described in the Example 1 were obtained in said samples with the oligonucleotide set as set forth in SEQ ID NO: 37-40.
  • the oligonucleotide set 8 (SEQ ID NO: 37-40) detects Mycobacterium tuberculosis and M bovis in a sample and does not detect Mycobacterium avium.
  • the annealing and ligation temperatures were varied from 50° to 74° C for 30seconds to 5 min. with similar results.
  • the enzyme concentration for thermostable ligase was varied from 1-10 units with consistent and reproducible results.
  • the oligonucleotide set (Set 9) having nucleotide sequence as set forth in SEQ ID NO: 42- 45 were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 50070 to 50119 (SEQ ID NO: 41) of the DNA sequence of Mycobacterium tuberculosis CDCl 551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium avium paratuberculosis KlO and Mycobacterium avium 104. Region: 9
  • a phosphate group was attached to the 5' end of the oligonucleotides as set forth in SEQ ID NO: 43 and SEQ ID NO: 44.
  • the oligonucleotides as set forth in SEQ ID NO: 43 and SEQ ID NO: 44 were modified at 3' end by adding phosphate group.
  • the oligonucleotide set as set forth in SEQ ID NO: 42-45 was tested with blood, sputum, lung tissue, cotyledons, ascitic fluid, pleural fluid, semen, urine, stool sample, milk, bronchoalveolar lavage, FNAC samples and pure culture of Mycobacterium species.
  • the reaction used for detection of the target nucleic acid was performed under similar conditions as described in Example 1. Similar results as described in the Example 1 were obtained in said samples with the oligonucleotide set as set forth in SEQ ID NO: 42-45
  • the oligonucleotide set 9 (SEQ ID NO: 42-45) detects Mycobacterium tuberculosis M. bovis and/or Mycobacterium avium in a sample.
  • thermostable ligase was varied from 1-10 units with consistent and reproducible results.
  • Example 10 Isothermal Nucleic acid Amplification Reaction (INAR) using oligonucleotide set as set forth in SEQ ID NO: 47-50
  • the oligonucleotide set (Set 10) having nucleotide sequence as set forth in SEQ ID NO: 47- 50 were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 20679 to 20720 (SEQ ID NO: 46) of the DNA sequence of Mycobacterium aviumlQA (Accession number CP000479). This region is specific Io detect Mycobacterium avium 104 and Mycobacterium avium paratuberculosis KlO. Region 10: CCG GTT GGT CAT CGG GTT GTC GGG ATC GTC GCG CAG CCG CTG
  • a phosphate group was attached to the 5' end of the oligonucleotides as set forth in SEQ ID NO: 48 and SEQ ID NO: 49.
  • the oligonucleotides as set forth in SEQ ID NO: 48 and SEQ ID NO: 49 were modified at 3' end by adding phosphate group.
  • the oligonucleotide set as set forth in SEQ ID NO: 47-50 was tested with blood, sputum, lung tissue, cotyledons, ascitic fluid, pleural fluid, semen, urine, stool sample, milk, bronchoalveolar lavage, FNAC samples and pure culture of Mycobacterium species.
  • the reaction used for detection of the target nucleic acid was performed under similar conditions as described in Example 1. Similar results as described in the Example 1 were obtained in said samples with the oligonucleotide set as set forth in SEQ ID NO: 47-50.
  • the oligonucleotide set 10 detects Mycobacterium avium in a sample and does not detect Mycobacterium tuberculosis and M. bovis.
  • the annealing and ligation temperatures were varied from 50° to 74° C for 30seconds to 5 min. with similar results.
  • the enzyme concentration for thermostable ligase was varied from 1-10 units with consistent and reproducible results.
  • Example 11 Isothermal nucleic acid amplification reaction (INAR) for detecting Mycobacterium DNA using oligonucleotide set (Set 13)
  • the oligonucleotide set (Set 13) having nucleotide sequence as set forth in SEQ ID NO: 62- 65 were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 484256 to 484207 (SEQ ID NO: 61) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). Region 13:
  • a phosphate group was attached to the 5' end of the oligonucleotides as set forth in SEQ ID NO: 63 and SEQ ID NO: 64.
  • the oligonucleotide set as set forth in SEQ ID NO: 62-65 was tested with sputum, blood, ascitic fluid, bronchial washing samples, pleural fluid, urine, semen, tissue.
  • the reaction used for detection of the target nucleic acid was performed under similar conditions as described in Example 1. Similar results as described in the Example 1 were obtained in said samples with the oligonucleotide set as set forth in SEQ ID NO: 62-65 , The oligonucleotide set 13 (SEQ ID NO: 62-65) detects Mycobacterium tuberculosis and M. bovis in a sample and does not detect Mycobacterium avium.
  • thermostable ligase was varied from 1-10 units with consistent and reproducible results.

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Abstract

The present invention provides a nucleic acid based detection method for diagnosis of infectious diseases, genetically transmitted diseases, cancer and/or SNP variations. The present invention provides oligonucleotides useful for detection of infectious agent such as Mycobacterium tuberculosis, M. avium, M. bovis and mutation resulting in drug resistance. The invention further provides kits for diagnosis of said diseases and mutations. The kit provided in the invention is highly sensitive, specific, easy to use, has a low turnaround time and capable of detecting low titers of the infectious agent.

Description

METHOD FOR DETECTION OF NUCLEIC ACID OF MYCOBACTERIUM AND USES THEREOF
TECHNICAL FIELD OF INVENTION
The present invention relates to a method for detection, enumeration and/or identification of disease and non disease related conditions inclusive of but not limited to microorganisms, pathogens, mutations, cancer and/or SNP variations in a sample. The present invention particularly relates to a nucleic acid based detection method for diagnosis of infectious diseases, genetically transmitted diseases, drug resistance, mutations, cancer and/or SNP variations. BACKGROUND ART
In the last decade, molecular diagnostics have become mainstay in the field of clinical diagnostics. Nucleic acid amplification technology has opened new avenues of detection and characterization of diseases as they provide a rapid and accurate way of assessing deviations in the physiology and pathophysiology in a given population or during developmental stages. The molecular methods are becoming more popular due to their ease of performance, reproducibility, sensitivity and specificity of results obtained compared to traditional methods.
Various methods of amplifying nucleic acid sequences for disease detection are known in the art. Techniques such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), reverse transcription polymerase chain reaction (RT-PCR), Self-Sustained Synthetic Reaction (3SR/NASBA), and Q.beta.-Replicase (Q.beta.) are finding increasing use in the clinical laboratories.
Presently, the most practical and useful application of nucleic acid amplification tests (NAATs) is in detecting and identifying infectious agents and cancer diseases. With regard to infectious diseases, NAATs supersede the routine growth-based culture and microscopy methods in terms of their ease of use and a low turn-around time (Report on the "Evaluation of Diagnostic Tests for Detection of Genitourinary Chlamydia Infections by STD Control Branch, California Department of Health Services, Department of Epidemiology & Biostatistics, University of California, San Francisco; published in March 2001). Apart from providing comparable and/or better confirmatory results in certain cases, the NAATs provide substantial time and cost saving over traditional culture methods for determining the presence of a given pathogen in a clinical specimen. Certain NAATs also provide quantification of the pathogen thereby producing more efficient results. These technologies vary among themselves in their sensitivity and specificity to provide an accurate diagnosis. There is an increased demand for tests which maintain very high positive predictive value (PPV) and negative predictive value (NPV) for detection of all microorganisms and give reproducible results.
EP patent 0395292 (Barry et al, 1997) describes a method for generating DNA probes specific for an organism useful for distinguishing between genera and species. The document provides the DNA probes obtained from a variable intergenic region intermediate the genes coding for 16S rRNA and 23 S rRNA wherein the probes are specific for a variety of species including Aeromonas hydrophilla, A. salmonicida, Clostridium difficile, Mycobacterium bovis, M. tuberculosis and Salmonella typhimurium. US patent no 5786149 (Leckie et al, 1998) provides a method of detecting a target sequences from M. tuberculosis using the gap filling ligase chain reaction and oligonucleotides useful in detecting Mycobacterium tuberculosis in a sample, wherein the oligonucleotides are with recessed ends. The oligonucleotides provided in the disclosure detect only M, tuberculosis and do not detect M. avium. US 5,830,711 (Barany et al, 1998) describes a method for distinguishing a first nucleotide sequence which differs by at least a single base from a second nucleotide sequence by using ligase chain reaction (LCR) utilizing the thermophilic DNA ligase from Thermus aquaticus to detect a target DNA sequence.
The detection methods described in the prior art give variable results with respect to non- specificity, poor amplification signal and non reproducibility. It is therefore desirable to provide a method of detection of the target nucleic acid in a sample using ligase mediated amplification reaction that overcomes the above-mentioned disadvantages. It is an object of the present invention to provide a method and oligonucleotides for detection of a target nucleic acid with high sensitivity and efficacy. SUMMARY OF THE INVENTION
The present invention relates to a nucleic acid based detection method for the diagnosis of disease and non disease related conditions inclusive of but not limited to microorganisms, pathogens, mutations, cancer and/or SNP variations in a sample. The present invention particularly relates to a nucleic acid based detection method for detection of a target nucleic acid of an infectious agent selected from a group consisting of Mycobacterium tuberculosis, M. bovis and M. avium. The method disclosed in the present invention is also useful for detection of single nucleotide polymorphism (SNP) and mutations such as mutations resulting in drug resistance. One aspect of the present invention is to provide a method for detecting a target nucleic acid sequence in a sample for detection of an infectious agent using isothermal nucleic acid amplification reaction (INAR), wherein the infectious agent is selected from a group consisting of Mycobacterium tuberculosis, M. avium and M. bovis.
Another aspect of the present invention is to provide a method for detecting a target nucleic acid sequence in a sample for detection of mutations resulting in drug resistance using isothermal nucleic acid amplification reaction (INAR), wherein drug resistance is caused in Mycobacterium species due to mutations.
Another aspect of the present invention is to provide oligonucleotide sequences for detection of an infectious agent or mutations resulting in drug resistance using isothermal nucleic acid amplification reaction (INAR), wherein the infectious agent is selected from a group consisting of Mycobacterium tuberculosis, M. avium and M. bovis.
Another aspect of the present invention is to provide oligonucleotide sequences for detection of Mycobacterium tuberculosis, M. avium, and/or M. bovis in a sample.
Another aspect of the present invention is to provide oligonucleotide sequences for detection of mutations in Kat G gene of Mycobacterium tuberculosis resulting in drug resistance against drugs such as isoniazid (INH).
Yet another aspect of the present invention is to provide a kit for detection of a target nucleic acid of Mycobacterium comprising oligonucleotide sequences useful for detection of Mycobacterium tuberculosis, M. avium, and/or M. bovis in a sample. Still yet another aspect of the present invention is to provide a kit for detection of mutations resulting in drug resistance; the kit comprises oligonucleotide sequences that detect mutation at position 315 of KATG gene of Mycobacterium.
The diagnostic kit provided in the invention is highly sensitive, specific, easy to use, has a low turnaround time in comparison to conventional detection methods and capable of detecting low titers.
One aspect of the present invention relates to a process for detecting a target nucleic acid of Mycobacterium in a sample, wherein the process comprises (a) providing a sample; (b) providing a set of 4 oligonucleotides, wherein the set of oligonucleotides is selected from a group consisting of set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 17-20), set 5 (SEQ ID NO: 22-25), set 6 ( SEQ ID NO: 27-30), set 7 ( SEQ ID NO: 32-35), set 8 (SEQ ID NO: 37-40), set 9 (SEQ ID NO: 42-45), set 10 (SEQ ID NO: 47-50), and set 13 (SEQ ID NO: 62-65); (c) providing thermostable ligase; (d) mixing the above to obtain a reaction mixture; (e) subjecting the reaction mixture to series of cycles comprising denaturation, annealing and ligation; and (f) detecting the presence of the amplified nucleic acid of Mycobacterium.
Another aspect of the present invention relates to oligonucleotide set selected from a group consisting of set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12- 15), set 4 (SEQ ID NO: 17-20), set 5 (SEQ ID NO: 22-25), set 6 ( SEQ ID NO: 27-30), set 7 ( SEQ ID NO: 32-35), set 8 (SEQ ID NO: 37-40), set 9 (SEQ ID NO: 42-45), set 10 (SEQ ID NO: 47-50), and set 13 (SEQ ID NO: 62-65); wherein the oligonucleotide set is useful for detection of the target nucleic acid of Mycobacterium tuberculosis and/or Mycobacterium bovis in a sample.
Another aspect of the present invention relate to oligonucleotide set having nucleotide sequence as set forth in SEQ ID NO: 42-45, wherein the oligonucleotide set detects the target nucleic acid of Mycobacterium tuberculosis, Mycobacterium bovis and M. avium in a sample.
Yet another aspect of the present invention relates to oligonucleotide set having nucleotide sequence as set forth in SEQ ID NO: 47-50, wherein the oligonucleotide set detects the target nucleic acid of M. avium in a sample. Yet another aspect of the present invention relates to a kit for detection of a target nucleic acid of Mycobacterium in a sample, the kit comprises the oligonucleotide set selected from a group consisting of set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 17-20), set 5 (SEQ ID NO: 22-25), set 6 ( SEQ ID NO: 27-30), set 7 ( SEQ ID NO: 32-35), set 8 (SEQ ID NO: 37-40), set 9 (SEQ ID NO: 42-45), set 10 (SEQ ID NO: 47-50), and set 13 (SEQ ID NO: 62-65).
Still yet another aspect of the present invention relates to a kit for detection of a target nucleic acid of Mycobacterium tuberculosis, Mycobacterium bovis and M. avium in a sample, the kit comprises the oligonucleotide set having nucleotide sequence as set forth in SEQ ID NO: 42-45.
Another aspect of the a kit for detection of a target nucleic acid of Mycobacterium avium in a sample, the kit comprises the set having nucleotide sequence as set forth in SEQ ID NO: 47-50.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWING Figure 1 shows gel photograph for isothermal nucleic acid amplification reaction (INAR)
Lane 1 : M-Low molecular weight marker, Lane 2: Positive control- 3.2 Kb plasmid (pPBPC2) comprising 250 bp from the region of 85252-85301 of Mycobacterium genome having accession number AE000516, Lane 3: HGD (negative control with human genomic DNA), Lane 4: Sl (known Tb sample), Lane 5: S2 (known Tb sample), Lane 6: S3 (unknown sample), Lane 7: S4 (unknown sample)
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method for detection, enumeration and/or identification of disease and non disease related conditions inclusive of but not limited to microorganisms, pathogens, mutations, cancer and/or SNP variations in a biological sample and/or a pure culture of a microorganism.
The present invention particularly relates to a nucleic acid based detection method for detection of a target nucleic acid of an infectious agent in a sample, wherein the infectious agent is selected from a group consisting of Mycobacterium tuberculosis, M. bovis and/or M. avium. The method disclosed in the present invention is also useful for detection of single nucleotide polymorphism (SNP) and mutations, such as mutations resulting in drug resistance.
Various samples such as blood, sputum, tissue, saliva, cerebro-spinal fluid, ascitic fluid, pleural fluid, lymph, synovial fluid, semen and other body fluids, urine and other body excretions, body secretions such as milk, bronchoalveolar lavage and other washings can be collected from a subject to isolate genomic DNA for detection of the target nucleic acid. Genomic DNA can be extracted from a sample by using methods well known in the art and used as a template DNA for ligase mediated amplification reaction.
The term used herein "Isothermal Nucleic Acid Amplification Reaction (INAR)" describes a reaction of 18-40 cycles in which both annealing and amplification are carried out at a temperature ranging from 55-74°C while denaturation is carried out at a temperature ranging from 90-99°C in the presence of one or more thermostable ligase enzymes.
The present invention comprises of the following embodiments:
One embodiment of the present invention provides a method for detection of a target nucleic acid of an infectious agent in a sample using isothermal nucleic acid amplification reaction (INAR), wherein the infectious agent is selected from a group consisting of Mycobacterium tuberculosis, M. avium and M. bovis.
Another embodiment of the present invention provides a method for detection of a target nucleic acid of an infectious agent in a sample using isothermal nucleic acid amplification reaction (INAR), wherein the infectious agent is Mycobacterium tuberculosis and/or M. bovis.
Another embodiment of the present invention provides a method for detection of a target nucleic acid of an infectious agent in a sample using isothermal nucleic acid amplification reaction (INAR), wherein the infectious agent is Mycobacterium tuberculosis. Another embodiment of the present invention provides a method for detection of a target nucleic acid of an infectious agent in a sample using isothermal nucleic acid amplification reaction (INAR), wherein the infectious agent is Mycobacterium bovis. Another embodiment of the present invention provides a method for detection of a target nucleic acid of an infectious agent in a sample using isothermal nucleic acid amplification reaction (INAR), wherein the infectious agent is Mycobacterium avium.
Another embodiment of the present invention provides a method for detection of a target nucleic acid of an infectious agent in a sample using isothermal nucleic acid amplification reaction (INAR), wherein the infectious agent is Mycobacterium avium subsp. paratuberculosis KlO.
Another embodiment of the present invention provides a method for detection of a target nucleic acid of an infectious agent in a sample using isothermal nucleic acid amplification reaction (INAR)5 wherein the infectious agent is Mycobacterium avium 104.
Another embodiment of the present invention is to provide a method for detecting a target nucleic acid sequence in a sample for detection of mutations resulting in drug resistance using isothermal nucleic acid amplification reaction (INAR), wherein drug resistance is caused in Mycobacterium species due to mutations. One of the preferred embodiments of the present invention is to provide oligonucleotide set for detection of Mycobacterium tuberculosis, M. avium, and/or M. bovis nucleic acid in a sample.
Another preferred embodiment of the present invention is to provide oligonucleotide set for detection of Mycobacterium tuberculosis and/or M. bovis nucleic acid in a sample. Still yet another embodiment of the present invention is to provide oligonucleotide set for detection of Mycobacterium tuberculosis nucleic acid in a sample.
Still yet another embodiment of the present invention is to provide oligonucleotide set for detection of M. bovis nucleic acid in a sample.
Still yet another embodiment of the present invention is to provide oligonucleotide set for detection of M. avium nucleic acid in a sample.
Another embodiment of the present invention provides oligonucleotide set for detection of Mycobacterium avium subsp. paratuberculosis KlO. Another embodiment of the present invention provides oligonucleotide set for detection of Mycobacterium avium 104.
The present invention provides the nucleotide sequences having oligonucleotide sequence as forth in SEQ ID NO: 1-65. Size of the oligonucleotide sequences provided in the present invention ranges from 15-50 bases.
In one embodiment of the present invention there is provided the oligonucleotide set (Set 1) having nucleotide sequence as set forth in SEQ ID NO: 2-5. The oligonucleotides were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 62716-62765 (SEQ ID NO: 1) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis. Region 1 : CAA CAT GTT TTC GAT GGG ACT GTA AGC GGC ACA CAC AAT TTC GTC GCC AA SEQ ID NO: 1
IA: 5' CAA CAT GTT TTC GAT GGG ACT GTA A 3' SEQ ID NO: 2 IB 51 GCG GCA CAC ACA ATT TCG TCG CCA A 3' SEQ ID NO: 3 1C 5' TTA CAG TCC CAT CGA AAA CAT GTT G 3' SEQ ID NO: 4 ID 5' TTG GCG ACG AAA TTG TGT GTG CCG C 3' SEQ ID NO: 5 In another embodiment of the present invention there is provided the oligonucleotide set (Set 2) having nucleotide sequence as set forth in SEQ ID NO: 7-10. The oligonucleotides were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 85252-85301 (SEQ ID NO: 6) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis. Region: 2 AAA CCC AGA CAG ATT AGT GAA TGC GTG GCT CGG CGT TGT AGG CGG TGG AA SEQ ID NO: 6
2A: 5' AAA CCC AGA CAG ATT AGT GAA TGC G 3' SEQ ID NO: 7 2B: 5' TGG CTC GGC GTT GTA GGC GGT GGA A 3' SEQ ID NO: 8 2C: 5' CGC ATT CAC TAA TCT GTC TGG GTT T 3' SEQ ID NO: 9 2D: 5' TTC CAC CGC CTA CAA CGC CGA GCC A 3' SEQ ID NO: 10 In another embodiment of the present invention there is provided the oligonucleotide set (Set 3) having nucleotide sequence as set forth in SEQ ID NO: 12-15. The oligonucleotides were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 82712-82761 (SEQ ID NO: 11) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis. Region: 3 CGT GCG CGT TTG GAG GTC CCT GAG CGA TGG GCG ATC TGA GCA TTA GCC AG SEQ ID NO: 11
3A 5' CGT GCG CGT TTG GAG GTC CCT GAG C 3' SEQ ID NO: 12 3B S5 GAT GGG CGA TCT GAG CAT TAG CCA G S' SEQ ID NO: 13 3C S5 GCT CAG GGA CCT CCA AAC GCG CAC G S' SEQ ID NO: 14 3D 5' CTG GCT AAT GCT CAG ATC GCC CAT C 3' SEQ ID NO: 15 In another embodiment of the present invention there is provided the oligonucleotide set (Set 4) having nucleotide sequence as set forth in SEQ ID NO: 17-20. The oligonucleotides were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 2041-2088 (SEQ ID NO: 16) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis. Region 4: GAA AGG GCG CAA TGG ACG CGG CTA CGA CAA GAG TTG GCC TCA CCG ACT SEQ ID NO: 16
4A 5' GAA AGG GCG CAA TGG ACG CGG CTA 3' SEQ ID NO: 17 4B 5' CGA CAA GAG TTG GCC TCA CCG ACT 3' SEQ ID NO: 18
4C 5' TAG CCG CGT CCA TTG CGC CCT TTC 3' SEQ ID NO: 19
4D 5' AGT CGG TGA GGC CAA CTC TTG TCG 5' SEQ ID NO: 20 In another embodiment of the present invention there is provided the oligonucleotide set (Set 5) having nucleotide sequence as set forth in SEQ ID NO: 22-25 were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 79850 to 79897 (SEQ ID NO: 21) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis. Region 5: GAT CGC TGA GAT TCA CTT GTT CGG CAC CCA GGA GTA TCG CTG GGT GCT SEQ ID NO: 21
5A 5' GAT CGC TGA GAT TCA CTT GTT CGG 3' SEQ ID NO: 22
5B 5' CAC CCA GGA GTA TCG CTG GGT GCT 3' SEQ ID NO: 23 5C 51 CCG AAC AAG TGA ATC TCA GCG ATC 3' SEQ ID NO: 24
5D 5' AGC ACC CAG CGA TAC TCC TGG GTG 3' SEQ ID NO: 25 In another embodiment of the present invention there is provided the oligonucleotide set (Set 6) having nucleotide sequence as set forth in SEQ ID NO: 27-30. The oligonucleotides were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 4206202-4206249 (SEQ ID NO: 26) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis.
Region 6: TGG CGC CCA CGC TCT CGG TAA TGG CCC AGG AAT CGT TGT CGG TCC GCG SEQ ID NO: 26
6A 5' TGG CGC CCA CGC TCT CGG TAA TGG 3' SEQ ID NO: 27
6B 5' CCC AGG AAT CGT TGT CGG TCC GCG 3' SEQ ID NO: 28
6C 5' CCA TTA CCG AGA GCG TGG GCG CCA 3' SEQ ID NO: 29 6D 5' CGC GGA CCG ACA ACG ATT CCT GGG 3' SEQ ID NO: 30 In another embodiment of the present invention there is provided the oligonucleotide set (Set 7) having nucleotide sequence as set forth in SEQ ID NO: 32-35. The oligonucleotides were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 4203720-4203769 (SEQ ID NO: 31) of the DNA sequence of Mycobacterium tuberculosis CDC1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis.
Region 7: GCG ACG CAG TAA CGC CCG CAG CCG AAG CAC GAC CTC CTC GAT GCT AAA CG SEQ ID NO: 31
7A 5' GCG ACG CAG TAA CGC CCG CAG CCG A 31 SEQ ID NO: 32 7B 51 AGC ACG ACC TCC TCG ATG CTA AAC G 3' SEQ ID NO: 33 7C 5' TCG GCT GCG GGC GTT ACT GCG TCG C 3' SEQ ID NO: 34 7D 5' CGT TTA GCA TCG AGG AGG TCG TGC T 3' SEQ ID NO: 35 In another embodiment of the present invention there is provided the oligonucleotide set (Set 8) having nucleotide sequence as set forth in SEQ ID NO: 37-40. The oligonucleotides were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 3002631-3002680 (SEQ ID NO: 36) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis. Region 8: GGC TGG GCA ACG TTA TCT CGG CTT C C TCG GCC TGT TCC GCC AAC TCC CGG SEQ ID NO: 36 8A S1GGC TGG GCA ACG TTA TCT CGG CTT C S' SEQ ID NO: 37 8B 5' CTC GGC CTG TTC CGC CAA CTC CCG G 3' SEQ ID NO: 38 8C 5' GAA GCC GAG ATA ACG TTG CCC AGC C 3' SEQ ID NO: 39 8D 5'CCG GGA GTT GGC GGA ACA GGC CGA G 3' SEQ ID NO: 40 In another embodiment of the present invention there is provided the oligonucleotide set (Set 9) having nucleotide sequence as set forth in SEQ ID NO: 42-45. The oligonucleotides were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 50070 to 50119 (SEQ ID NO: 41) of the DNA sequence of Mycobacterium tuberculosis CDCl 551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium avium paratuberculosis KlO and Mycobacterium avium 104. Region: 9 GATGCCGCGGTCCTTGGCGATCTTGGCCGCCCGCACCGCGTCGATGATGA
SEQ ID NO: 41 9A 5'GAT GCC GCG GTC CTT GGC GAT CTT G 3' SEQ ID NO: 42 9B 5' GCC GCC CGC ACC GCG TCG ATG ATG A 3' SEQ ID NO: 43 9C 5' CAA GAT CGC CAA GGA CCG CGG CAT C 3' SEQ ID NO: 44 9D 5' TCA TCA TCG ACG CGG TGC GGG CGG C 3' SEQ ID NO: 45 In another embodiment of the present invention there is provided the oligonucleotide set (Set 10) having nucleotide sequence as set forth in SEQ ID NO: 47-50. The oligonucleotides were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 20679 to 20720 (SEQ ID NO: 46) of the DNA sequence of Mycobacterium avium\0A (Accession number CP000479). This region is specific to detect Mycobacterium avium 104 and Mycobacterium avium paratuberculosis KlO. Region 10: CCGGTTGGTCATCGGGTTGTCGGGATCGTCGCGCAGCCGCTG SEQ ID NO: 46
1 OA 5 ' CCG GTT GGT CAT CGG GTT GTC 3 ' SEQ ID NO : 47
1OB 5' GGG ATC GTC GCG CAG CCG CTG 3' SEQ ID NO: 48
1OC 5' GAC AAC CCG ATG ACC AAC CGG 3' SEQ ID NO: 49 1OD 5' CAG CGG CTG CGC GAC GAT CCC 3' SEQ ID NO: 50
In still another embodiment of the present invention there is provided the oligonucleotide set 11 and 12 having nucleotide sequence as set forth in SEQ ID NO: 52-55 and SEQ ID NO: 57-60 respectively. The oligonucleotides were synthesized using the methods well known in the art. The oligonucleotides set 11 (SEQ ID NO: 52-55) designed from 2152482 to 2152432 nucleotide (SEQ ID NO: 51) of the DNA sequence of Mycobacterium tuberculosis CDC1551 (Accession number AE000516). This region is prone to mutation resulting in drug resistance against drugs such as Isoniazid. In case of absence of drug resistance the wild type KAT G gene has Serine (S) at position 315. Region 11 : AAC CGG TAA GGA CGC GAT CAC CAG CGG CAT CGA GGT CGT ATG GAC GAA C SEQ ID NO: 51
1 IA 5' AAC CGG TAA GGA CGC GAT CAC CAG 3' SEQ ID NO: 52 HB 5' CGG CAT CGA GGT CGT ATG GAC GAA C 3' SEQ ID NO: 53 11C 5' CTG GTG ATC GCG TCC TTA CCG GTT 3' SEQ ID NO: 54 HD 5' GTT CGT CCA TAC GAC CTC GAT GCC G 3' SEQ ID NO: 55 In this case of drug resistance, there is a mutation in KAT G gene at position 315 where Serine is changed to Threonine (S-> T). The nucleotide sequence set forth in the SEQ ID NO: 56 shows the mutation at position 315 (S-> T) in the Kat G gene. Thus this mutation can be detected using the oligonucleotide set 12 (SEQ ID NO: 57-60). Region 12: AAC CGG TAA GGA CGC GAT CAC CAC CGG CAT CGA GGT CGT ATG GAC GAA C SEQ ID NO: 56
REGION: 2152482 to 2152433 in CDC 1551 Accession no. AE000516 12A 5' AAC CGG TAA GGA CGC GAT CAC CAC 3' SEQ ID NO: 57
12B 5' CGG CAT CGA GGT CGT ATG GAC GAA C 3' SEQ ID NO: 58
12C 5' GTG GTG ATC GCG TCC TTA CCG GTT 3' SEQ ID NO: 59
12D 5' GTT CGT CCA TAC GAC CTC GAT GCC G 3' SEQ ID NO: 60 In still yet another embodiment of the present invention there is provided the oligonucleotide set (Set 13) having nucleotide sequence as set forth in SEQ ID NO: 62-65. The oligonucleotides were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 484256 to 484207 (SEQ ID NO: 61) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). Region 13:
CCA TCG ACC TAC TAC GAC CAC ATC AAC CGG GAG CCC AGC CGC CGC GAG CT SEQ ID NO:61
13A 5' CCA TCG ACC TAC TAC GAC CAC ATC A 3' SEQ ID NO:62 BB S' ACC GGG AGC CCA GCC GCC GCG AGC T S' SEQ ID NO:63
13C S1 TGA TGT GGT CGT AGT AGG TCG ATG G S' SEQ ID NO:64
13D 5' AGC TCG CGG CGG CTG GGC TCC CGG T 3' SEQ ID NO:65
Isothermal Nucleic acid Amplification Reaction (INAR)
The oligonucleotide sequences having nucleotide sequence as set forth in SEQ. ID. NO: SEQ ID NO:2-5, SEQ ID NO:7-10, SEQ ID NO:12-15, SEQ ID NO:17-20, SEQ ID NO:22-25, SEQ ID NO:27-30, SEQ ID NO:32-35, SEQ ID NO:37-40, SEQ ID NO:42-45, SEQ ID NO:47-50, SEQ ID NO: 52-55, SEQ ID NO: 57-60 and SEQ ID NO: 62-65 were synthesized using the methods well known in the art.
Template DNA was isolated form various samples such as blood, sputum, lung tissue, cotyledons, ascitic fluid, pleural fluid, semen, urine, stool sample, milk, bronchoalveolar lavage, FNAC samples by using the method well known in the art. 100 ng of template
DNA from each sample was used for INAR. Recombinant plasmids for example pPBPC2 containing 250 bp fragment obtained from different regions of DNA sequence of
Mycobacterium tuberculosis CDC1551 (Accession number AE000516) were used as positive control in the method for detection of the target nucleic acid using the oligonucleotides disclosed in the present invention. Genomic DNA isolated from the blood sample of healthy subject (non-infected with Mycobacterium) was used as a negative control. Genomic DNA from known tuberculosis positive patient and suspected tuberculosis patient were used as test samples. The INAR showed amplification of expected fragment of 50 bp in positive control i.e. plasmid containing 250 bp of Mycobacterium nucleotide sequence in samples S l and S2 known to be positive for tuberculosis and in unknown samples S3 and S4. Fragment of 25 bp due to primer annealing was obtained in negative control in the absence of the template nucleic acid. As expected a fragment of 50 bp was obtained in known Mycobacterium tuberculosis positive samples Sl and S2. A fragment of 50 bp was also seen in unknown samples S3 and S4 suggesting that they are positive for Mycobacterium tuberculosis (Figure 1).
Detailed procedure of Isothermal Nucleic acid Amplification Reaction (INAR) using the oligonucleotide set disclosed in the present invention is explained in Example 1-1 1. The above samples S l, S2, S3 and S4 were also tested for their MTb positive status using conventional culture methods and by PCR (Ogusku, Mauricio Analise de diferen tMeso prriismhie,r est ault.ilizados na PCR visando ao diagnόstico da tuberculose no Estado do Amazonas in Jornal Brasileiro de Pneumologia 30(4) - Jul/Ago de 2004), primers for amplifying 541bp region in IS61 10 gene were used. The samples were found positive basis PCR amplification results analysis by 1.2% agarose gel electrophoresis.
It should be noted and understood that the oligonucleotide set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 (SEQ ID NO: 12-15), set 4 (SEQ ID NO: 17-20), set 5 (SEQ ID NO: 22-25), set 6 (SEQ ID NO: 27-30), set 7 (SEQ ID NO: 32-35), set 8 (SEQ ID NO: 37- 40), set 9 (SEQ ID NO: 42-45), set 10 (SEQ ID NO: 47-50), set 1 1 (SEQ ID NO: 52-55), oligonucleotide set 12 (SEQ ID NO: 57-60) and oligonucleotide set 13 (SEQ ID NO: 62- 65) used for detection of tuberculosis being part of the map positions mentioned in SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21 , SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41, and SEQ ID NO: 46; SEQ ID NO: 51, SEQ ID NO: 56 and SEQ ID NO: 61 respectively may have slightly fewer or greater number of bases but should be considered equivalents of these sequences to fall within the scope of the present invention, provided they will hybridize to the same positions on the target as the listed sequences.
One embodiment of the present invention relates to a process for detecting a target nucleic acid of Mycobacterium in a sample, wherein the process comprises (a) providing a sample; (b) providing a set of 4 oligonucleotides, wherein the set of oligonucleotides is selected from a group consisting of set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 17-20), set 5 (SEQ ID NO: 22-25), set 6 ( SEQ ID NO: 27-30), set 7 ( SEQ ID NO: 32-35), set 8 (SEQ ID NO: 37-40), set 9 (SEQ ID NO: 42-45), set 10 (SEQ ID NO: 47-50), and set 13 (SEQ ID NO: 62-65); (c) providing thermostable ligase; (d) mixing the above to obtain a reaction mixture; (e) subjecting the reaction mixture to series of cycles comprising denaturation, annealing and ligation; and (f) detecting the presence of the amplified nucleic acid of Mycobacterium.
Another embodiment of the present invention relates to the process for detecting a target nucleic acid of Mycobacterium in a sample, wherein Mycobacterium is selected from a group consisting of Mycobacterium tuberculosis, M. bovis and M. avium.
Yet another embodiment of the present invention relates to the process for detecting a target nucleic acid of Mycobacterium using the oligonucleotide set disclosed in the present invention, wherein at least one oligonucleotide of said set of oligonucleotides is optionally attached to a label. Still yet another embodiment of the present invention relates to the process for detecting a target nucleic acid of Mycobacterium using the oligonucleotide set disclosed in the present invention, wherein at least one oligonucleotide of said set of oligonucleotides is optionally attached to a label, wherein said label is selected from a group consisting of chromophores, fluorescent moieties, enzymes, antigens, and chemiluminescent moieties. Further, the present invention relates to the process for detecting a target nucleic acid of Mycobacterium using the oligonucleotide set disclosed in the present invention, wherein second and third oligonucleotides of said set of oligonucleotides are modified at 5' end with a phosphate group. Further, the present invention relates to the process for detecting a target nucleic acid of Mycobacterium using the oligonucleotide set disclosed in the present invention, wherein second and third oligonucleotides of said set of oligonucleotides are optionally modified at 3' end with phosphate, deoxy, alkyl or aryl group. The present invention relates to the process for detecting a target nucleic acid of Mycobacterium using the oligonucleotide set disclosed in the present invention, wherein second and third oligonucleotides of said set of oligonucleotides are optionally modified at 3' end with a phosphate group.
The present invention relates to the process for detecting a target nucleic acid of Mycobacterium disclosed in the present invention, wherein the reaction mixture was subjected to 18-40 cycles; preferably 32 cycles.
The present invention relates to the process for detecting a target nucleic acid of Mycobacterium disclosed in the present invention, wherein annealing and ligation is carried out at a temperature ranging from about 55°C-74° C for 30seconds-5 minutes preferably at 68°C for 2 minutes.
The present invention provides oligonucleotide set selected from a group consisting of set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 17-20), set 5 (SEQ ID NO: 22-25), set 6 ( SEQ ID NO: 27-30), set 7 ( SEQ ID NO: 32-35), set 8 (SEQ ID NO: 37-40), set 9 (SEQ ID NO: 42-45), set 10 (SEQ ID NO: 47-50) and set 13 (SEQ ID NO: 62-65), wherein the oligonucleotide set is useful for detection of the target nucleic acid of Mycobacterium tuberculosis, Mycobacterium avium and/or Mycobacterium bovis in a sample.
Another embodiment of the present invention is to provide oligonucleotide set selected from a group consisting of set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 17-20), set 5 (SEQ ID NO: 22-25), set 6 ( SEQ ID NO: 27-30), set 7 ( SEQ ID NO: 32-35), set 8 (SEQ ID NO: 37-40), set 9 (SEQ ID NO: 42-45) and set 13 (SEQ ID NO: 62-65), wherein the oligonucleotide set is useful for detection of the target nucleic acid of Mycobacterium tuberculosis and/or Mycobacterium bovis in a sample. Another embodiment of the present invention is to provide oligonucleotide set selected from a group consisting of set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 17-20), set 5 (SEQ ID NO: 22-25), set 6 ( SEQ ID NO: 27-30), set 7 ( SEQ ID NO: 32-35), set 8 (SEQ ID NO: 37-40), set 9 (SEQ ID NO: 42-45) and set 13 (SEQ ID NO: 62-65), wherein the oligonucleotide set is useful for detection of the target nucleic acid of Mycobacterium tuberculosis in a sample.
Another embodiment of the present invention is to provide oligonucleotide set selected from a group consisting of set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 17-20), set 5 (SEQ ID NO: 22-25), set 6 ( SEQ ID NO: 27-30), set 7 ( SEQ ID NO: 32-35), set 8 (SEQ ID NO: 37-40), set 9 (SEQ ID NO: 42-45) and set 13 (SEQ ID NO: 62-65), wherein the oligonucleotide set is useful for detection of the target nucleic acid of Mycobacterium bovis in a sample.
Yet another embodiment of the present invention is to provide oligonucleotide set for detection of a target nucleic acid of Mycobacterium avium in a sample, wherein the oligonucleotide set is having nucleotide sequence as set forth in SEQ ID NO: 47-50.
Another embodiment of the present invention provides oligonucleotide set for detection of Mycobacterium avium subsp. avium 104 in a sample, wherein the oligonucleotide set is having nucleotide sequence as set forth in SEQ ID NO: 47-50.
Another embodiment of the present invention provides oligonucleotide set for detection of Mycobacterium avium subsp. paratuberculosis KlO avium in a sample, wherein the oligonucleotide set is having nucleotide sequence as set forth in SEQ ID NO: 47-50.
The present invention further provides oligonucleotide set having nucleotide sequence as set forth in SEQ ID NO: 42-45, wherein the oligonucleotide set detects the target nucleic acid of Mycobacterium tuberculosis, Mycobacterium bovis and M. avium in a sample. The present invention provides oligonucleotide set having nucleotide sequence as set forth in SEQ ID NO: 57-60, wherein said oligonucleotide set detects mutation at position 315 of KAT G gene of Mycobacterium.
In an embodiment, the present invention provides a kit for detection of a target nucleic acid of Mycobacterium in a sample, said kit comprising the oligonucleotide set selected from a group consisting of set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 17-20), set 5 (SEQ ID NO: 22-25), set 6 ( SEQ ID NO: 27-30), set 7 ( SEQ ID NO: 32-35), set 8 (SEQ ID NO: 37-40), set 9 (SEQ ID NO: 42-45), set 10 (SEQ ID NO: 47-50), and set 13 (SEQ ID NO: 62-65). In an embodiment, the present invention provides a kit for detection of a target nucleic acid in a sample, said kit comprising the oligonucleotide set selected from a group consisting of set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 17-20), set 5 (SEQ ID NO: 22-25), set 6 ( SEQ ID NO: 27-30), set 7 ( SEQ ID NO: 32-35), set 8 (SEQ ID NO: 37-40), set 9 (SEQ ID NO: 42-45) and set 13 (SEQ ID NO: 62- 65) wherein the said kit is useful for detection of the target nucleic acid of Mycobacterium tuberculosis and/or Mycobacterium bovis in a sample.
In an embodiment, the present invention provides a kit for detection of a target nucleic acid in a sample, said kit comprising the oligonucleotide set selected from a group consisting of set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 17-20), set 5 (SEQ ID NO: 22-25), set 6 ( SEQ ID NO: 27-30), set 7 ( SEQ ID NO: 32-35), set 8 (SEQ ID NO: 37-40), set 9 (SEQ ID NO: 42-45) and set 13 (SEQ ID NO: 62- 65) wherein the said kit is useful for detection of the target nucleic acid of Mycobacterium tuberculosis in a sample.
In an embodiment, the present invention provides a kit for detection of a target nucleic acid in a sample, said kit comprising the oligonucleotide set selected from a group consisting of set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ
ID NO: 17-20), set 5 (SEQ ID NO: 22-25), set 6 ( SEQ ID NO: 27-30), set 7 ( SEQ ID NO:
32-35), set 8 (SEQ ID NO: 37-40), set 9 (SEQ ID NO: 42-45) and set 13 (SEQ ID NO: 62-
65) wherein the said kit is useful for detection of the target nucleic acid of Mycobacterium bovis in a sample.
Another embodiment of the present invention relates to a kit for detection of a target nucleic acid of Mycobacterium tuberculosis, Mycobacterium bovis and M. avium in a sample, the kit comprises the oligonucleotide set having nucleotide sequence as set forth in SEQ ID NO: 42-45. Yet another embodiment of the present invention relates to a kit for detection of a target nucleic acid of Mycobacterium avium in a sample, the kit comprises the set having nucleotide sequence as set forth in SEQ ID NO: 47-50.
Another embodiment of the present invention relates to a kit for detection of Mycobacterium avium subsp. avium 104 in a sample, the kit comprises the set having nucleotide sequence as set forth in SEQ ID NO: 47-50.
Another embodiment of the present invention relates to a kit for detection of Mycobacterium avium subsp. paratuberculosis KlO avium in a sample, the kit comprises the set having nucleotide sequence as set forth in SEQ ID NO: 47-50. In another embodiment, the present invention provides a diagnostic kit for detection of mutation resulting in drug resistance, said kit comprising oligonucleotide sequences having nucleotide sequence as set forth in SEQ ID NO: 52-55 and SEQ ID NO: 57-60.
Each oligonucleotide set provided in the present invention consists of at least 4 oligonucleotides (A, B, C and D) of the size ranging from 15-50 bases, wherein first oligonucleotide (A) is complementary to third oligonucleotide (C) and second oligonucleotide (B) is complementary to fourth oligonucleotide (D). A phosphate group is attached to the 5' end of second and third oligonucleotide (B and C). The 3' end of the second and third oligonucleotide (B and C) is optionally modified. The second and third oligonucleotide (B and C) of each oligonucleotide set disclosed in the present invention are optionally modified by adding phosphate group at the 3' end. The modification can be carried out by adding phosphate, deoxy, alkyl or aryl group at the 3' end.
One embodiment relates to labeling agent or a probe that can be optionally attached to 5' end and/or 3' end of any of the four (A, B, C and D) oligonucleotides in each set wherein the labeling agent or a probe is radioactive, enzymatic, colorimetric, fluorescent, chemiluminiscent and/or photo-active.
The amplified product obtained using the method disclosed in the present invention can be detected by various methods known in the art such as agarose gel electrophoresis, solution phase colorimetric detection using enzymes and photoactivity assays, solid phase colorimetric detection using enzymes and photoactivity assays, solution phase fluorimetric detection, solid phase fluorimetric detection, solution phase chemiluminiscent detection, solid phase chemiluminiscent detection, solution phase using nanoparticles, solid phase using nanoparticles, solution phase using radioactive detection and solid phase using radioactive detection method. The kit disclosed in the present invention is based on INAR for the detection of a target nucleic acid of an infectious agent in a sample in one assay wherein the infectious agent may be present individually or in combination with each other.
The method of detection disclosed in the present invention is also useful for detecting cancer, mutations, single nucleotide polymorphism (SNP) and mutations resulting in drug resistance.
An embodiment of the present invention is to provide a kit containing all the necessary reagents to perform the methods of detection disclosed herein. The kit may contain specific oligonucleotide sequence sets optionally attached to a label, a suitable buffer and a thermostable ligase. The kit may further contain a set of printed instructions indicating that the kit is useful for detection of the specific disease and/or non disease related conditions as disclosed in the present invention.
EXAMPLES
It should be understood that the following examples described herein are for illustrative purposes only and that various modifications or changes in light will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. Example 1
Isothermal Nucleic acid Amplification Reaction (INAR) using oligonucleotide set as set forth in SEQ ID NO: 2-5
The oligonucleotide set (Set 1) having nucleotide sequence as set forth in SEQ ID NO: 2-5 were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 62716-62765 (SEQ ID NO: 1) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis. Region 1 : CAA CAT GTT TTC GAT GGG ACT GTA AGC GGC ACA CAC AAT TTC GTC GCC AA SEQ ID NO: 1
IA: 5' CAA CAT GTT TTC GAT GGG ACT GTA A 3' SEQ ID NO: 2
IB 5' GCG GCA CAC ACA ATT TCG TCG CCA A 3' SEQ ID NO: 3 1C 5' TTA CAG TCC CAT CGA AAA CAT GTT G S' SEQ ID NO: 4
ID 5' TTG GCG ACG AAA TTG TGT GTG CCG C S' SEQ ID NO: 5
A phosphate group was attached to the 5' end of the oligonucleotides as set forth in SEQ ID NO: 3 and SEQ ID NO: 4. The oligonucleotides as set forth in SEQ ID NO: 3 and SEQ ID NO: 4 were modified at 3' end by adding phosphate group. The oligonucleotide set (Set 1) as set forth in SEQ ID NO: 2-5 was tested with blood, sputum, lung tissue, cotyledons, ascitic fluid, pleural fluid, semen, urine, stool sample, milk, bronchoalveolar lavage, FNAC samples and pure culture of Mycobacterium species. The amplification reaction was carried out in a total volume of 20 μl containing 2 μl buffer (10X), 1 μl Thermostable ligase (5U/μl), lμl of each oligonucleotide (SEQ ID NO: 2-5) at concentration 2.5ng each, lOOng of template DNA and ultrapure water to bring the reaction volume to 20 μl.
The reaction conditions employed was as follows: first cycle- 95°C for 10 min and 650C for 2 min followed by 31 cycles - 95°C for 1 min and 65°C for 2 min. Various samples and controls were used for detection of Mycobacterium DNA. The description of the different reactions (reactions 1-7) used as template DNA is given below.
Reaction 1 : NC-Negative control- no template DNA in the reaction mixture
Reaction 2: HGD-Negative control- template DNA from healthy subject (not infected with
TB)
Reaction 3: Positive control- 3.2 Kb plasmid (pPBPCl) with 250 bp from the region of 62716-62765 of Mycobacterium genome having accession number AE000516.
Reaction 4: Sl -template DNA from sputum sample of known Mycobacterium tuberculosis positive patient
Reaction 5: S2-template DNA from sputum sample of known Mycobacterium tuberculosis infected patient Reaction 6: S3- template DNA from sputum sample of suspected Mycobacterium tuberculosis infected patient Reaction 7: S4- template DNA from sputum sample of suspected Mycobacterium tuberculosis infected patient
The reactions were electrophoresed on a 2.5% agarose gel at 100V for 45 min and captured on a gel documentation system. Amplification of expected fragment of 50 bp was observed in positive control i.e. plasmid containing 250 bp of Mycobacterium nucleotide sequence, in samples Sl and S2 known to be positive for tuberculosis and in unknown samples S3 and S4. Fragment of 25 bp due to primer annealing was obtained in negative control in the absence of the template nucleic acid. The oligonucleotide set 1 (SEQ ID NO: 2-5) detects Mycobacterium tuberculosis and M bovis in a sample and does not detect Mycobacterium avium.
The results obtained above have also been corroborated by MTb culture on LJ media and PCR using IS61 10 region as described before in detailed description of the invention. The annealing and ligation temperatures were varied from 55° to 74° C for 30seconds-5 min. with similar results. The enzyme concentration for thermostable ligase was varied from 1 - 10 units with consistent and reproducible results. Example 2
Isothermal Nucleic acid Amplification Reaction (INAR) using oligonucleotide set as set forth in SEQ ID NO: 7- 10 The oligonucleotide set (Set 2) having nucleotide sequence as set forth in SEQ ID NO: 7- 10 were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 85252-85301 (SEQ ID NO: 6) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis. Region: 2 AAA CCC AGA CAG ATT AGT GAA TGC GTG GCT CGG CGT TGT AGG CGG TGG AA SEQ ID NO: 6
2A: 5' AAA CCC AGA CAG ATT AGT GAA TGC G 3' SEQ ID NO: 7 2B: 5' TGG CTC GGC GTT GTA GGC GGT GGA A S' SEQ ID NO: 8
2C: 5' CGC ATT CAC TAA TCT GTC TGG GTT T 3' SEQ ID NO: 9
2D: 5' TTC CAC CGC CTA CAA CGC CGA GCC A S' SEQ ID NOMO A phosphate group was attached to the 5' end of the oligonucleotides as set forth in SEQ ID
NO: 8 and SEQ ID NO: 9. The oligonucleotides as set forth in SEQ ID NO: 8 and SEQ ID
NO: 9 were modified at 3' end by adding phosphate group.
The amplification reaction was carried out in a total volume of 20 μl containing 2 μl buffer (10X), 1 μl Thermostable ligase (5U/μl), 1 μl of each oligonucleotide (SEQ ID NO: 7-10) at concentration 2.5ng each, lOOng of template DNA and ultrapure water to bring the reaction volume to 20 μl.
The reaction conditions employed was as follows: first cycle -950C for 10 min and 68°C for
2 min followed by 31 cycles - 950C for 1 min and 68°C for 2 min. Various samples and controls were used for detection of Mycobacterial DNA. The description of the different reactions (reactions 1-7) used as template DNA is given below.
Reaction 1 : NC-Negative control- no template DNA in the reaction mixture
Reaction 2: HGD-Negative control- template DNA from healthy subject (not infected with
TB) Reaction 3: Positive control- 3.2 Kb plasmid (pPBPC2) with 250 bp from the region of
85252-85301 of Mycobacterium genome having accession number AE000516
Reaction 4: Sl -template DNA from sputum sample of known Mycobacterium tuberculosis positive patient
Reaction 5: S2-template DNA from sputum sample of known Mycobacterium tuberculosis infected patient
Reaction 6: S3- template DNA from sputum sample of suspected Mycobacterium tuberculosis infected patient
Reaction 7: S4- template DNA from sputum sample of suspected Mycobacterium tuberculosis infected patient The reactions were electrophoresed on a 2.5% agarose gel at 100V for 45 min and captured on a gel documentation system. Amplification of expected fragment of 50 bp was observed in positive control i.e. plasmid containing 250 bp of Mycobacterium nucleotide sequence, in samples Sl and S2 known to be positive for tuberculosis and in unknown samples S3 and S4. Fragment of 25 bp due to primer annealing was obtained in negative control in the absence of the template nucleic acid (Figure 1). The oligonucleotide set 2 (SEQ ID NO: 7-10) detects Mycobacterium tuberculosis and M. bovis in a sample and do not detect Mycobacterium avium.
Other samples such as blood, sputum, lung tissue, cotyledons, ascitic fluid, pleural fluid, semen, urine, stool sample, milk, bronchoalveolar lavage, FNAC samples and pure culture of Mycobacterium species have been tested with oligonucleotide set (Set 2) as set forth in SEQ ID NO: 7-10 under similar reaction conditions and obtained similar results. The results obtained above have also been corroborated by MTb culture on LJ media and PCR using IS6110 region as described before in detailed description of the invention. The annealing and ligation temperatures were varied from 50° to 74° C for 30seconds to 5 min. with similar results. The enzyme concentration for thermostable ligase was varied from 1-10 units with consistent and reproducible results. Example 3
Isothermal Nucleic acid Amplification Reaction (INAR) using oligonucleotide set as set forth in SEQ ID NO: 12-15 The oligonucleotide set (Set 3) having nucleotide sequence as set forth in SEQ ID NO: 12- 15 were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 82712-82761 (SEQ ID NO: 11) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis. Region: 3 CGT GCG CGT TTG GAG GTC CCT GAG CGA TGG GCG ATC TGA GCA TTA GCC AG SEQ ID NO : 11
3A 5' CGT GCG CGT TTG GAG GTC CCT GAG C 3' SEQ ID NO: 12
3B 5' GAT GGG CGA TCT GAG CAT TAG CCA G 3' SEQ ID NO: 13
3C 5' GCT CAG GGA CCT CCA AAC GCG CAC G 3' SEQ ID NO: 14 3D 5' CTG GCT AAT GCT CAG ATC GCC CAT C 3' SEQ ID NO: 15
A phosphate group was attached to the 5' end of the oligonucleotides as set forth in SEQ ID NO: 13 and SEQ ID NO: 14. The oligonucleotides as set forth in SEQ ID NO: 13 and SEQ ID NO: 14 were modified at 3' end by adding phosphate group. The oligonucleotide set as set forth in SEQ ID NO: 12-15 was tested with blood, sputum, lung tissue, cotyledons, ascitic fluid, pleural fluid, semen, urine, stool sample, milk, bronchoalveolar lavage, FNAC samples and pure culture of Mycobacterium species. The reaction used for detection of the target nucleic acid was performed under similar conditions as described in Example 1. Similar results as described in the Example 1 were obtained in said samples with the oligonucleotide set as set forth in SEQ ID NO: 12-15. The oligonucleotide set 3 (SEQ ID NO: 12-15) detects Mycobacterium tuberculosis and M. bovis in a sample and does not detect Mycobacterium avium.
The annealing and ligation temperatures were varied from 50° to 74° C for 30seconds to 5 min. with similar results. The enzyme concentration for thermostable ligase was varied from 1-10 units with consistent and reproducible results. Example 4 Isothermal Nucleic acid Amplification Reaction (INAR) using oligonucleotide set as set forth in SEQ ID NO: 17-20
The oligonucleotide set (Set 4) having nucleotide sequence as set forth in SEQ ID NO: 17- 20 were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 2041-2088 (SEQ ID NO: 16) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This r ( egion is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis.
Region 4: GAA AGG GCG CAA TGG ACG CGG CTA CGA CAA GAG TTG GCC TCA
CCG ACT SEQ ID NO: 16
4A 5' GAA AGG GCG CAA TGG ACG CGG CTA 3' SEQ ID NO: 17 4B 5' CGA CAA GAG TTG GCC TCA CCG ACT 3' SEQ ID NO: 18
4C 5' TAG CCG CGT CCA TTG CGC CCT TTC 3' SEQ ID NO: 19
4D 51 AGT CGG TGA GGC CAA CTC TTG TCG 3' SEQ ID NO: 20
A phosphate group was attached to the 5' end of the oligonucleotides as set forth in SEQ ID NO: 18 and SEQ ID NO: 19. The oligonucleotides as set forth in SEQ ID NO: 18 and SEQ ID NO: 19 were modified at 3' end by adding phosphate group.
The oligonucleotide set as set forth in SEQ ID NO: 17-20 was tested with blood, sputum, lung tissue, cotyledons, ascitic fluid, pleural fluid, semen, urine, stool sample, milk, bronchoalveolar lavage, FNAC samples and pure culture of Mycobacterium species. The reaction used for detection of the target nucleic acid was performed under similar conditions as described in Example 1. Similar results as described in the Example 1 were obtained in said samples with the oligonucleotide set as set forth in SEQ ID NO: 17-20. The oligonucleotide set 4 (SEQ ID NO: 17-20) detects Mycobacterium tuberculosis and M. bovis in a sample and does not detect Mycobacterium avium.
The annealing and ligation temperatures were varied from 50° to 74° C for 30seconds to 5 min. with similar results. The enzyme concentration for thermostable ligase was varied from 1-10 units with consistent and reproducible results. Example 5
Isothermal Nucleic acid Amplification Reaction (INAR) using oligonucleotide set as set forth in SEQ ID NO: 22-25 The oligonucleotide set (Set 5) having nucleotide sequence as set forth in SEQ ID NO: 22- 25 were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 79850 to 79897 (SEQ ID NO: 21) of the DNA sequence of Mycobacterium tuberculosis CDC1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis. Region 5: GAT CGC TGA GAT TCA CTT GTT CGG CAC CCA GGA GTA TCG CTG GGT GCT SEQ ID NO : 21
5A 5' GAT CGC TGA GAT TCA CTT GTT CGG 3' SEQ ID NO: 22
5B 5' CAC CCA GGA GTA TCG CTG GGT GCT 3' SEQ ID NO: 23
5C 5' CCG AAC AAG TGA ATC TCA GCG ATC 3' SEQ ID NO: 24
5D 5' AGC ACC CAG CGA TAC TCC TGG GTG 31 SEQ ID NO: 25 A phosphate group was attached to the 5' end of the oligonucleotides as set forth- in SEQ ID NO: 23 and SEQ ID NO: 24. The oligonucleotides as set forth in SEQ ID NO: 23 and SEQ ID NO: 24 were modified at 3' end by adding phosphate group.
The oligonucleotide set as set forth in SEQ ID NO: 22-25 was tested with blood, sputum, lung tissue, cotyledons, ascitic fluid, pleural fluid, semen, urine, stool sample, milk, bronchoalveolar lavage, FNAC samples and pure culture of Mycobacterium species. The reaction used for detection of the target nucleic acid was performed under similar conditions as described in Example 1. Similar results as described in the Example 1 were obtained in said samples with the oligonucleotide set as set forth in SEQ ID NO: 22-25. The oligonucleotide set 5 (SEQ ID NO: 22-25) detects Mycobacterium tuberculosis and M bovis in a sample and does not detect Mycobacterium avium. The annealing and ligation temperatures were varied from 50° to 74° C for 30seconds to 5 min. with similar results. The enzyme concentration for thermostable ligase was varied from 1-10 units with consistent and reproducible results. Example 6 Isothermal Nucleic acid Amplification Reaction (INAR) using oligonucleotide set as set forth in SEQ ID NO: 27-30
The oligonucleotide set (Set 6) having nucleotide sequence as set forth in SEQ ID NO: 27- 30 were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 4206202-4206249 (SEQ ID NO: 26) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis.
Region 6: TGG CGC CCA CGC TCT CGG TAA TGG CCC AGG AAT CGT TGT CGG TCC GCG SEQ ID NO: 26 6A 5' TGG CGC CCA CGC TCT CGG TAA TGG 3' SEQ ID NO: 27 6B 5' CCC AGG AAT CGT TGT CGG TCC GCG 3' SEQ ID NO: 28 6C 51 CCA TTA CCG AGA GCG TGG GCG CCA S' SEQ ID NO: 29 6D 5' CGC GGA CCG ACA ACG ATT CCT GGG 3' SEQ ID NO: 30 A phosphate group was attached to the 5' end of the oligonucleotides as set forth in SEQ ID NO: 28 and SEQ ID NO: 29. The oligonucleotides as set forth in SEQ ID NO: 28 and SEQ ID NO: 29 were modified at 3' end by adding phosphate group.
The oligonucleotide set as set forth in SEQ ID NO: 27-30 was tested with blood, sputum, lung tissue, cotyledons, ascitic fluid, pleural fluid, semen, urine, stool sample, milk, bronchoalveolar lavage, FNAC samples and pure culture of Mycobacterium species. The reaction used for detection of the target nucleic acid was performed under similar conditions as described in Example 1. Similar results as described in the Example 1 were obtained in said samples with the oligonucleotide set as set forth in SEQ ID NO: 27-30. The oligonucleotide set 6 (SEQ ID NO: 27-30) detects Mycobacterium tuberculosis and M bovis in a sample and does not detect Mycobacterium avium. The annealing and ligation temperatures were varied from 50° to 74° C for 30seconds to 5 min. with similar results. The enzyme concentration for thermostable ligase was varied from 1-10 units with consistent and reproducible results. Example 7 Isothermal Nucleic acid Amplification Reaction (INAR) using oligonucleotide set as set forth in SEQ ID NO: 32-35
The oligonucleotide set (Set 7) having nucleotide sequence as set forth in SEQ ID NO: 32- 35 were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 4203720-4203769 (SEQ ID NO: 31) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis.
Region 7: GCG ACG CAG TAA CGC CCG CAG CCG AAG CAC GAC CTC CTC GAT GCT AAA CG SEQ ID NO : 31 7A 51 GCG ACG CAG TAA CGC CCG CAG CCG A S' SEQ ID NO: 32
7B 5' AGC ACG ACC TCC TCG ATG CTA AAC G 3' SEQ ID NO: 33
7C 5' TCG GCT GCG GGC GTT ACT GCG TCG C 3' SEQ ID NO: 34
7D 5' CGT TTA GCA TCG AGG AGG TCG TGC T 3' SEQ ID NO: 35
A phosphate group was attached to the 5' end of the oligonucleotides as set forth in SEQ ID NO: 33 and SEQ ID NO: 34. The oligonucleotides as set forth in SEQ ID NO: 33 and SEQ ID NO: 34 were modified at 3' end by adding phosphate group.
The oligonucleotide set as set forth in SEQ ID NO: 32-35 was tested with blood, sputum, lung tissue, cotyledons, ascitic fluid, pleural fluid, semen, urine, stool sample, milk, bronchoalveolar lavage, FNAC samples and pure culture of Mycobacterium species. The reaction used for detection of the target nucleic acid was performed under similar conditions as described in Example 1. Similar results as described in the Example 1 were obtained in said samples with the oligonucleotide set as set forth in SEQ ID NO: 32-35. The oligonucleotide set 7 (SEQ ID NO: 32-35) detects Mycobacterium tuberculosis and M. bovis in a sample and does not detect Mycobacterium avium. The annealing and ligation temperatures were varied from 50° to 74° C for 30seconds to 5 min. with similar results. The enzyme concentration for thermostable ligase was varied from 1-10 units with consistent and reproducible results. Example 8 Isothermal Nucleic acid Amplification Reaction (INAR) using oligonucleotide set as set forth in SEQ ID NO: 37-40
The oligonucleotide set (Set 8) having nucleotide sequence as set forth in SEQ ID NO: 37- 40 were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 3002631-3002680 (SEQ ID NO: 36) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis and Mycobacterium bovis. Region 8: GGC TGG GCA ACG TTA TCT CGG CTT C C TCG GCC TGT TCC GCC AAC TCC CGG SEQ ID NO: 36
8A 5'GGC TGG GCA ACG TTA TCT CGG CTT C 3' SEQ ID NO: 37 8B 5' CTC GGC CTG TTC CGC CAA CTC CCG G 3' SEQ ID NO: 38
8C 5' GAA GCC GAG ATA ACG TTG CCC AGC C 3' SEQ ID NO: 39
8D 5'CCG GGA GTT GGC GGA ACA GGC CGA G 3' SEQ ID NO: 40
A phosphate group was attached to the 5' end of the oligonucleotides as set forth in SEQ ID NO: 38 and SEQ ID NO: 39. The oligonucleotides as set forth in SEQ ID NO: 38 and SEQ ID NO: 39 were modified at 3' end by adding phosphate group.
The oligonucleotide set as set forth in SEQ ID NO: 37-40 was tested with blood, sputum, lung tissue, cotyledons, ascitic fluid, pleural fluid, semen, urine, stool sample, milk, bronchoalveolar lavage, FNAC samples and pure culture of Mycobacterium species. The reaction used for detection of the target nucleic acid was performed under similar conditions as described in Example 1. Similar results as described in the Example 1 were obtained in said samples with the oligonucleotide set as set forth in SEQ ID NO: 37-40. The oligonucleotide set 8 (SEQ ID NO: 37-40) detects Mycobacterium tuberculosis and M bovis in a sample and does not detect Mycobacterium avium. The annealing and ligation temperatures were varied from 50° to 74° C for 30seconds to 5 min. with similar results. The enzyme concentration for thermostable ligase was varied from 1-10 units with consistent and reproducible results. Example 9
Isothermal Nucleic acid Amplification Reaction (INAR) using oligonucleotide set as set forth in SEQ ID NO: 42-45
The oligonucleotide set (Set 9) having nucleotide sequence as set forth in SEQ ID NO: 42- 45 were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 50070 to 50119 (SEQ ID NO: 41) of the DNA sequence of Mycobacterium tuberculosis CDCl 551 (Accession number AE000516). This region is specific to detect Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium avium paratuberculosis KlO and Mycobacterium avium 104. Region: 9
GATGCCGCGGTCCTTGGCGATCTTGGCCGCCCGCACCGCGTCGATGATGA
SEQ ID NO: 41
9A 5'GAT GCC GCG GTC CTT GGC GAT CTT G S' SEQ ID NO: 42
9B 5' GCC GCC CGC ACC GCG TCG ATG ATG A 3' SEQ ID NO: 43 9C 5' CAA GAT CGC CAA GGA CCG CGG CAT C S' SEQ ID NO: 44
9D 5' TCA TCA TCG ACG CGG TGC GGG CGG C 3' SEQ ID NO: 45
A phosphate group was attached to the 5' end of the oligonucleotides as set forth in SEQ ID NO: 43 and SEQ ID NO: 44. The oligonucleotides as set forth in SEQ ID NO: 43 and SEQ ID NO: 44 were modified at 3' end by adding phosphate group. The oligonucleotide set as set forth in SEQ ID NO: 42-45 was tested with blood, sputum, lung tissue, cotyledons, ascitic fluid, pleural fluid, semen, urine, stool sample, milk, bronchoalveolar lavage, FNAC samples and pure culture of Mycobacterium species. The reaction used for detection of the target nucleic acid was performed under similar conditions as described in Example 1. Similar results as described in the Example 1 were obtained in said samples with the oligonucleotide set as set forth in SEQ ID NO: 42-45
The oligonucleotide set 9 (SEQ ID NO: 42-45) detects Mycobacterium tuberculosis M. bovis and/or Mycobacterium avium in a sample.
The annealing and ligation temperatures were varied from 50° to 74° C for 30seconds to 5 min. with similar results. The enzyme concentration for thermostable ligase was varied from 1-10 units with consistent and reproducible results. Example 10 Isothermal Nucleic acid Amplification Reaction (INAR) using oligonucleotide set as set forth in SEQ ID NO: 47-50
The oligonucleotide set (Set 10) having nucleotide sequence as set forth in SEQ ID NO: 47- 50 were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 20679 to 20720 (SEQ ID NO: 46) of the DNA sequence of Mycobacterium aviumlQA (Accession number CP000479). This region is specific Io detect Mycobacterium avium 104 and Mycobacterium avium paratuberculosis KlO. Region 10: CCG GTT GGT CAT CGG GTT GTC GGG ATC GTC GCG CAG CCG CTG
SEQ ID NO: 46 1 OA 5' CCG GTT GGT CAT CGG GTT GTC 3' SEQ ID NO: 47
1OB 5' GGG ATC GTC GCG CAG CCG CTG 3' SEQ ID NO: 48
1OC 5' GAC AAC CCG ATG ACC AAC CGG 3 ' SEQ ID NO : 49
1OD 5' CAG CGG CTG CGC GAC GAT CCC S' SEQ ID NO: 50
A phosphate group was attached to the 5' end of the oligonucleotides as set forth in SEQ ID NO: 48 and SEQ ID NO: 49. The oligonucleotides as set forth in SEQ ID NO: 48 and SEQ ID NO: 49 were modified at 3' end by adding phosphate group.
The oligonucleotide set as set forth in SEQ ID NO: 47-50 was tested with blood, sputum, lung tissue, cotyledons, ascitic fluid, pleural fluid, semen, urine, stool sample, milk, bronchoalveolar lavage, FNAC samples and pure culture of Mycobacterium species. The reaction used for detection of the target nucleic acid was performed under similar conditions as described in Example 1. Similar results as described in the Example 1 were obtained in said samples with the oligonucleotide set as set forth in SEQ ID NO: 47-50. The oligonucleotide set 10 (SEQ ID NO: 47-50) detects Mycobacterium avium in a sample and does not detect Mycobacterium tuberculosis and M. bovis. The annealing and ligation temperatures were varied from 50° to 74° C for 30seconds to 5 min. with similar results. The enzyme concentration for thermostable ligase was varied from 1-10 units with consistent and reproducible results. Example 11 Isothermal nucleic acid amplification reaction (INAR) for detecting Mycobacterium DNA using oligonucleotide set (Set 13) The oligonucleotide set (Set 13) having nucleotide sequence as set forth in SEQ ID NO: 62- 65 were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 484256 to 484207 (SEQ ID NO: 61) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). Region 13:
CCA TCG ACC TAC TAC GAC CAC ATC AAC CGG GAG CCC AGC CGC CGC GAG
CT SEQ ID NO:61
13A 5' CCA TCG ACC TAC TAC GAC CAC ATC A 3' SEQ ID NO:62
13B 5' ACC GGG AGC CCA GCC GCC GCG AGC T 3' SEQ ID NO:63 13C 5' TGA TGT GGT CGT AGT AGG TCG ATG G 3' SEQ ID NO:64
13D 5' AGC TCG CGG CGG CTG GGC TCC CGG T 3' SEQ ID NO:65
A phosphate group was attached to the 5' end of the oligonucleotides as set forth in SEQ ID NO: 63 and SEQ ID NO: 64. The oligonucleotide set as set forth in SEQ ID NO: 62-65 was tested with sputum, blood, ascitic fluid, bronchial washing samples, pleural fluid, urine, semen, tissue.
The reaction used for detection of the target nucleic acid was performed under similar conditions as described in Example 1. Similar results as described in the Example 1 were obtained in said samples with the oligonucleotide set as set forth in SEQ ID NO: 62-65 , The oligonucleotide set 13 (SEQ ID NO: 62-65) detects Mycobacterium tuberculosis and M. bovis in a sample and does not detect Mycobacterium avium.
The annealing and ligation temperatures were varied from 50° to 74° C for 30seconds to 5 min. with similar results. The enzyme concentration for thermostable ligase was varied from 1-10 units with consistent and reproducible results.

Claims

I/We Claim:
1. A process for detecting a target nucleic acid of Mycobacterium in a sample, said process comprising: a. providing a sample; b. providing a set of 4 oligonucleotides, wherein said set of oligonucleotides is selected from a group consisting of set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 17-20), set 5 (SEQ ID NO: 22-25), set 6 ( SEQ ID NO: 27-30), set 7 ( SEQ ID NO: 32-35), set 8 (SEQ ID NO: 37-40), set 9 (SEQ ID NO: 42-45), set 10 (SEQ ID NO: 47-50), and set 13 (SEQ ID NO: 62-65); c. providing thermostable ligase; d. mixing the above to obtain a reaction mixture; e. subjecting said reaction mixture to series of cycles comprising denaturation, annealing and ligation; and f. detecting the presence of said amplified nucleic acid of Mycobacterium.
2. The process as claimed in claim 1, wherein Mycobacterium is selected from a group consisting of Mycobacterium tuberculosis, M. bovis and M. avium.
3. The process as claimed in claim 1, wherein at least one oligonucleotide of said set of oligonucleotides is optionally attached to a label.
4. The process as claimed in claim 3, wherein said label is selected from a group consisting of chromophores, fluorescent moieties, enzymes, antigens, and chemiluminescent moieties.
5. The process as claimed in claim 1, wherein at least two oligonucleotides of said set of oligonucleotides are modified at 5' end with phosphate group.
6. The process as claimed in claim 1, wherein at least two oligonucleotides of said set of oligonucleotides are modified at 3' end with phosphate, deoxy, alkyl or aryl group.
7. The process as claimed in claim 1 , wherein at least two oligonucleotides of said set of oligonucleotides are modified at 3' end with phosphate group.
8. The process as claimed in claim 1, said amplification mixture was subjected to 18-40 cycles; preferably 32 cycles.
9. The process as claimed in claim 1, wherein annealing and ligation is carried out at a temperature ranging from about 55°C-74°C for 30 seconds-5 minutes preferably at 680C for 2 minutes.
10. Oligonucleotide set selected from a group consisting of set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 17-20), set 5 (SEQ
ID NO: 22-25), set 6 ( SEQ ID NO: 27-30), set 7 ( SEQ ID NO: 32-35), set 8 (SEQ ID NO: 37-40), set 9 (SEQ ID NO: 42-45), set 10 (SEQ ID NO: 47-50), and set 13 (SEQ ID NO: 62-65); wherein said oligonucleotide set is useful for detection of the target nucleic acid of Mycobacterium tuberculosis and/or Mycobacterium bovis in a sample.
1 1. Oligonucleotide set having nucleotide sequence as set forth in SEQ ID NO: 42-45, wherein said oligonucleotide set detects the target nucleic acid of Mycobacterium tuberculosis, Mycobacterium bovis and M. avium in a sample.
12. Oligonucleotide set having nucleotide sequence as set forth in SEQ ID NO: 47-50, wherein said oligonucleotide set detects the target nucleic acid of M. avium in a sample.
13. A kit for detection of a target nucleic acid of Mycobacterium in a sample, said kit comprising the oligonucleotide set selected from a group consisting of set 1 (SEQ ID NO: 2-5), set 2 (SEQ ID NO: 7-10), set 3 ( SEQ ID NO: 12-15), set 4 (SEQ ID NO: 17- 20), set 5 (SEQ ID NO: 22-25), set 6 ( SEQ ID NO: 27-30), set 7 ( SEQ ID NO: 32-35), set 8 (SEQ ID NO: 37-40), set 9 (SEQ ID NO: 42-45), set 10 (SEQ ID NO: 47-50) and set 13 (SEQ ID NO: 62-65).
14. A kit for detection of a target nucleic acid of Mycobacterium tuberculosis, Mycobacterium bovis and M. avium in a sample, said kit comprising the oligonucleotide set having nucleotide sequence as set forth in SEQ ID NO: 42-45.
15. A kit for detection of a target nucleic acid of Mycobacterium avium in a sample, said kit comprising the set having nucleotide sequence as set forth in SEQ ID NO: 47-50.
PCT/IN2009/000016 2008-01-04 2009-01-05 Method for detection of nucleic acid of mycobacterium and uses thereof WO2009087688A2 (en)

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