WO2009087687A1

WO2009087687A1 - Nucleic acid based detection process and uses thereof

Info

Publication number: WO2009087687A1
Application number: PCT/IN2009/000015
Authority: WO
Inventors: Rajeev Soni; Nupur Mehrotra; Prabuddha Kumar Kundu; Kajal Arora
Original assignee: Premas Biotech Pvt.Ltd
Priority date: 2008-01-04
Filing date: 2009-01-05
Publication date: 2009-07-16

Abstract

The present invention provides a nucleic acid based detection process for detecting one or more than one region of a target nucleic acid and/or more than one target nucleic acid in a sample, wherein the target nucleic acid can be from a group of organisms comprising of bacteria, mammals, plants, viruses, fungi, protozoa and parasites. The process disclosed in the present invention detect the target nucleic acids inclusive of but not limited to microorganisms, pathogens, mutations, cancer and/or single nucleotide polymorphism (SNP) variations in a sample.

Description

NUCLEIC ACID BASED DETECTION PROCESS AND USES THEREOF TECHNICAL FIELD

The present invention relates to a process 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.

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 processs are becoming more popular due to their ease of performance, reproducibility, sensitivity and specificity of results obtained compared to traditional processs.

Various processs 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; PCR being the most commonly used technology currently.

Presently, the most practical and useful application of nucleic acid amplification tests (NAATs) is in detecting and identifying infectious agents, cancer where they supersede the routine growth-based culture and microscopy processs in terms of their ease of use and a low turn-around time. Apart from providing comparable and/or better confirmatory results in certain cases, the NAATs provide substantial time and cost saving over traditional culture processs 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.

Besides the advantages offered by NAATs as mentioned above, there is an increasing evidence in literature and from our own experience that the nucleic acid amplification may be inhibited in certain cases due to the quality of genomic DNA template (obtained by processs well known in the art) and various other reasons associated with the genomic DNA and/or reaction conditions, but not fully understood. A number of laboratories are reportedly engaged in research to eliminate this drawback and identify means to improve currently existing nucleic acid amplification technologies (S. Chakravorty, J. S. Tyagi FEMS Microbiology letters 205 (2001), 113-117).

SUMMARY OF THE INVENTION

The present invention relates to a nucleic acid based detection process for the diagnosis of disease and non disease related conditions inclusive of but not limited to microorganisms, pathogens, mutations, cancer and/or single nucleotide polymorphism (SNP) variations in a sample.

One aspect of the present invention relates to a process for detecting a target nucleic acid in a sample using multiplex dual isothermal nucleic acid amplification reaction, wherein the process comprises (a) providing a reaction mixture comprising a sample containing or suspected of containing one or more than one target nucleic acid; more than one set of oligonucleotides specific for one or more than one region of one or more than one target nucleic acids, wherein the set comprises at least four oligonucleotides, wherein a first oligonucleotide of the set is complementary to a third oligonucleotide and a second oligonucleotide is complementary to a fourth oligonucleotide; and thermostable ligase; (b) subjecting the reaction mixture under isothermal conditions to obtain a first resultant reaction mixture thereby amplifying the target nucleic acids, wherein the isothermal conditions comprises one cycle of denaturation at a temperature ranging from about 90⁰C to 99°C for 30 seconds to 10 minutes; and annealing and amplification at a temperature ranging from about 55⁰C to 74⁰C for 30 seconds to 3 minutes; and 8 to 25 cycles of denaturation at a temperature ranging from about 90⁰C to 99⁰C for 30 seconds to 3 minutes and annealing and amplification at a temperature ranging from about 55⁰C to 74⁰C for 30 seconds to 3 minutes; (c) subjecting a part or the whole of the first resultant reaction mixture to the following isothermal conditions using the set of oligonucleotides, and the thermostable ligase to obtain a second resultant reaction mixture, wherein the isothermal conditions comprise 8-32 cycles of denaturation at a temperature ranging from about 90⁰C to 99°C for 30 seconds to 3 minutes and annealing and amplification at a temperature ranging from about 55°C to 74⁰C for 30 seconds to 3 minutes; and (d) detecting the amplified nucleic acid.

Another aspect of the present invention relates to a process for detecting a target nucleic acid in a sample using multiplex dual isothermal nucleic acid amplification reactions, wherein the process comprises (a) providing a reaction mixture comprising a sample containing or suspected of containing one or more than one target nucleic acid; more than one set of oligonucleotides specific for one or more than one region of one or more than one target nucleic acids, wherein the set comprises at least four oligonucleotides, wherein a first oligonucleotide of the set is complementary to a third oligonucleotide and a second oligonucleotide is complementary to a fourth oligonucleotide; and thermostable ligase;

(b) subjecting the reaction mixture under isothermal conditions to obtain a first resultant reaction mixture, wherein the isothermal conditions are one cycle of denaturation at 95⁰C for 10 minutes; and annealing and amplification at 65⁰C for 3 minutes; and 14 cycles of denaturation at 95°C for 1 minute and annealing and amplification at 65⁰C for 1 minute;

(c) subjecting a part or whole of the first resultant reaction mixture to following isothermal conditions using the set of oligonucleotides, and the thermostable ligase to obtain a second resultant reaction mixture, wherein the isothermal conditions are 23 cycles at 95°C for 1 minute and 65°C for 1 minute; and (d) detecting the amplified nucleic acid.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWING

Figure 1 shows gel photograph for multiplex dual isothermal nucleic acid amplification reaction (M-DINAR) for detection of M. tuberculosis.

DESCRIPTION OF THE INVENTION

The present invention relates to a nucleic acid based detection process for the identification of disease and non disease related conditions inclusive of but not limited to microorganisms, pathogens, mutations, cancer and/or single nucleotide polymorphism (SNP) variations in a sample.

The nucleic acid based detection process for the diagnosis of infectious diseases, genetically transmitted diseases, cancer and/or SNP variations in samples is herein after also referred as Multiplex Dual Isothermal Nucleic Acid Amplification Reaction (M- DINAR) in which two similar but separate reactions with multiple oligonucleotide sets (each set containing at least 4 oligonucleotides), are performed wherein the product obtained from the first reaction is used as a template for the second reaction; product from the first reaction may or may not have undergone suitable processing. M-DINAR has an advantage over standard ligase chain reaction as the latter has been known to give non- reproducible results coupled with poor amplification signals. M-DINAR has an advantage over standard ligase chain reaction as the former helps in overcoming inhibition of amplification seen in certain samples by detecting different regions of the target nucleic acid in the same reaction mixture thereby enhancing the signal.

In another embodiment, M-DINAR finds application in the detection of multiple target nucleic acids in the same sample.

Another embodiment of the present invention provides process of detection of target nucleic acid, said process comprising providing sample for detection; providing oligonucleotide sequences specific for one or more than one region of a target nucleic acid and/or one or more than one target nucleic acid; performing the first isothermal nucleic acid amplification reaction under suitable conditions for appropriate number of cycles; performing the second isothermal nucleic acid reaction using the product of the first reaction as such or after suitable processing, and using the oligonucleotides under suitable conditions for appropriate number of cycles and detecting the presence of target nucleic acid. The reaction product may be analyzed using various modes, but not limited to one of the following namely 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, solid phase using radioactive detection.

The process described in the present invention for nucleic acid detection of one or more than one region of a target nucleic acid and/or one or more than one target nucleic acids in the sample (if present), leads to the enhancement of amplification signal by overcoming inhibition due to genomic DNA, other reaction constituents or reaction conditions. The process is also highly sensitive in eliminating the problem of non specific amplification that leads to false-positives. Another embodiment of the present invention provides nucleic acid based detection process i.e. Multiplex Dual Isothermal Nucleic Acid Amplification Reaction (M-DINAR) that is highly sensitive, specific, easy to use, capable of detecting very low titers, as exemplified by detection of upto 1.6 genomic equivalents of M. tuberculosis genomic DNA that corresponds to 8fg and significantly lower than attomole quantities of the nucleic acid present and yields consistent and reproducible results. The genomic DNA used can be extracted from the sample by using process well known in the art.

Yet another embodiment of the present invention provides M-DINAR for the detection target nucleic acid of disease or non-disease related conditions inclusive but not limiting to infectious diseases, genetically transmitted diseases, cancer and/or SNP variations in samples of human, veterinary and/or plant origin, wherein the sample may be selected from a group consisting of blood, sputum, tissue, saliva, cerebro-spinal fluid, pleural fluid, lymph, synovial fluid, semen and other body fluids, body secretions such as milk, urine and other body excretions, bronchoalveolar lavage and other washings from a / subject and plant extracts.

Yet another embodiment of the present invention provides a method for detection of a target nucleic acid of an organism, wherein the said organism is selected from a group consisting of bacteria, mammals, plants, viruses, fungi, parasites and protozoans.

Yet another embodiment of the present invention provides a method for detection of a target nucleic acid of bacteria, wherein said bacteria includes M. tuberculosis, M. bovis and M. avium.

Another embodiment of the present invention provides a method for detection of a target nucleic acid of bacteria, wherein said bacteria includes Mycobacterium species that include M. tuberculosis and/or M. bovis.

Another embodiment of the present invention provides a method for detection of a target nucleic acid of bacteria, wherein said bacteria is Mycobacterium tuberculosis.

Another embodiment of the present invention provides a method for detection of a target nucleic acid of bacteria, wherein said bacteria is Mycobacterium bovis.

Another embodiment of the present invention provides a method for detection of a target nucleic acid of bacteria, wherein said bacteria is Mycobacterium avium. Another embodiment of the present invention provides a method for detection of a target nucleic acid of bacteria, wherein said bacteria is M. avium subsp. paratuberculosis KlO.

Another embodiment of the present invention provides a method for detection of a target nucleic acid of bacteria, wherein said bacteria is M. avium 104.

Yet another embodiment of the present invention provides a method for detection of a target nucleic acid of viruses, wherein said virus includes Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus and/or Hepatitis E virus.

Yet another embodiment of the present invention provides a method for detection of a target nucleic acid of viruses, wherein said virus includes Hepatitis B and Hepatitis C virus.

Yet another embodiment of the present invention provides a method for detection of a target nucleic acid of viruses, wherein said virus is Hepatitis B virus.

Yet another embodiment of the present invention provides a method for detection of a target nucleic acid of viruses, wherein said virus is Hepatitis C virus.

Yet another embodiment of the present invention provides a method for detection of sequence specific regions that are implicated in susceptibility to prostate cancer.

Yet another embodiment of the present invention provides a method for detection of a target nucleic acid of mammals, wherein said mammals can be humans and/or animals.

Yet another embodiment of the present invention provides a method for detection of a target nucleic acid of plant species.

Another embodiment of the present invention provides a method of detection of nucleic acid in a sample, wherein the method is useful for detecting mutations in a sample, wherein the mutation may be mutation resulting in drug resistance.

In an embodiment, the present invention provides oligonucleotides useful for detection of nucleic acid, wherein the oligonucleotide set 1 as set forth in SEQ ID NO: 2-5 is 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: CAA CAT GTT TTC GAT GGG ACT GTA A SEQ ID NO: 2

IB: GCG GCA CAC ACA ATT TCG TCG CCA A SEQ ID NO: 3

1C: TTA CAG TCC CAT CGA AAA CAT GTT G SEQ ID NO: 4

ID: TTG GCG ACG AAA TTG TGT GTG CCG C SEQ ID NO: 5

In an embodiment, the present invention provides oligonucleotides useful for detection of nucleic acid, wherein the oligonucleotide set 2 as set forth in SEQ ID NO: 7-10 is 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. 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: AAA CCC AGA CAG ATT AGT GAA TGC G SEQ ID NO: 7

2B: TGG CTC GGC GTT GTA GGC GGT GGA A SEQ ID NO: 8

2C: CGC ATT CAC TAA TCT GTC TGG GTT T SEQ ID NO: 9

2D: TTC CAC CGC CTA CAA CGC CGA GCC A SEQ ID NO: 10

In an embodiment, the present invention provides oligonucleotides useful for detection of nucleic acid, wherein the oligonucleotide set 3 as set forth in SEQ ID NO: 12-15 is 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: CGT GCG CGT TTG GAG GTC CCT GAG C SEQ ID NO: 12

3B: GAT GGG CGA TCT GAG CAT TAG CCAG SEQ ID NO: 13

3C: GCT CAG GGA CCT CCA AAC GCG CAC G SEQ ID NO: 14

3D: CTG GCT AAT GCT CAG ATC GCC CAT C SEQ ID NO: 15 In an embodiment, the present invention provides oligonucleotides useful for detection ot nucleic acid, wherein the oligonucleotide set 4 as set forth in SEQ ID NO: 17-20 is designed from nucleotide position 2041-2088 (SEQ ID NO: 16) of the DNA sequence of Mycobacterium tuberculosis CDCl 551 (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 SEQIDNO: 16

4A: GAA AGG GCG CAA TGG ACG CGG CTA SEQ ID NO: 17

4B: CGA CAA GAG TTG GCC TCA CCG ACT SEQ ID NO: 18

4C: TAG CCG CGT CCA TTG CGC CCT TTC SEQ ID NO: 19

4D: AGT CGG TGA GGC CAA CTC TTG TCG SEQ ID NO: 20

In an embodiment, the present invention provides oligonucleotides useful for detection of nucleic acid, wherein the oligonucleotide set 5 as set forth in SEQ ID NO: 22-25 is 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: GAT CGC TGA GAT TCA CTT GTT CGG SEQ ID NO: 22

5B: CAC CCA GGA GTA TCG CTG GGT GCT SEQIDNO: 23

5C: CCG AAC AAG TGA ATC TCA GCG ATC SEQ ID NO: 24

5D: AGC ACC CAG CGA TAC TCC TGG GTG SEQ ID NO: 25

In an embodiment, the present invention provides oligonucleotides useful for detection of nucleic acid, wherein the oligonucleotide set 6 as set forth in SEQ ID NO: 27-30 is 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: TGG CGC CCA CGC TCT CGG TAA TGG SEQ ID NO: 27

6B: CCC AGG AAT CGT TGT CGG TCC GCG SEQ ID NO: 28

6C: CCA TTA CCG AGA GCG TGG GCG CCA SEQ ID NO: 29

6D: CGC GGA CCG ACA ACG ATT CCT GGG SEQ ID NO: 30

In an embodiment, the present invention provides oligonucleotides useful for detection of nucleic acid, wherein the oligonucleotide set 7 as set forth in SEQ ID NO: 32-35 is 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

AAACG SEQ ID NO: 31

7A: GCG ACG CAG TAA CGC CCG CAG CCG A SEQ ID NO: 32

7B: AGC ACG ACC TCC TCG ATG CTA AAC G SEQ ID NO: 33

7C: TCG GCT GCG GGC GTT ACT GCG TCG C SEQ ID NO: 34

7D: CGT TTA GCA TCG AGG AGG TCG TGC T SEQ ID NO: 35

In an embodiment, the present invention provides oligonucleotides useful for detection of nucleic acid, wherein the oligonucleotide set 8 as set forth in SEQ ID NO: 37-40 is designed from nucleotide position 3002631-3002680 (SEQ ID NO: 36) of the DNA sequence of Mycobacterium tuberculosis CDC1551 (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: GGC TGG GCA ACG TTA TCT CGG CTT C SEQ ID NO: 37

8B: CTC GGC CTG TTC CGC CAA CTC CCG G SEQ ID NO: 38

8C: GAA GCC GAG ATA ACG TTG CCC AGC C SEQ ID NO: 39

8D: CCG GGA GTT GGC GGA ACA GGC CGA G' SEQ ID NO: 40 In an embodiment, the present invention provides oligonucleotides useful for detection of nucleic acid, wherein the oligonucleotide set 9 as set forth in SEQ ID NO: 42-45 is designed from nucleotide position 50070 to 50119 (SEQ ID NO: 41) of the DNA sequence of Mycobacterium tuberculosis CDC1551 (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: GAT GCC GCG GTC CTT GGC GAT CTT G SEQ ID NO: 42 9B: GCC GCC CGC ACC GCG TCG ATG ATG A SEQ ID NO: 43 9C: CAA GAT CGC CAA GGA CCG CGG CAT C SEQ ID NO: 44 9D: TCA TCA TCG ACG CGG TGC GGG CGG C SEQ ID NO: 45

In an embodiment, the present invention provides oligonucleotides useful for detection of nucleic acid, wherein the oligonucleotide set 10 as set forth in SEQ ID NO: 47-50 is designed from nucleotide position 20679 to 20720 (SEQ ID NO: 46) of the DNA sequence of Mycobacterium aviumlO4 (Accession number CP000479). This region is specific to detect Mycobacterium avium 104 and Mycobacterium avium paratuberculosis KlO.

Region 10: SEQ ID NO: 46

CCGGTTGGTCATCGGGTTGTCGGGATCGTCGCGCAGCCGCTG 1 OA: CCG GTT GGT CAT CGG GTT GTC SEQ ID NO: 47

1 OB; GGG ATC GTC GCG CAG CCG CTG SEQ ID NO: 48

1 OC: GAC AAC CCG ATG ACC AAC CGG SEQ ID NO:49

1 OD: CAG CGG CTG CGC GAC GAT CCC SEQ ID NO: 50

In an embodiment, the present invention provides oligonucleotides useful for detection of nucleic acid, wherein the oligonucleotide set 1 1 as set forth in SEQ ID NO: 52-55 is designed from nucleotide position 2152482 to 2152432 nucleotide (SEQ ID NO: 51) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516). This region is specific to detect drug resistance against Isoniazid due to mutation in Kat G gene of Mycobacterium tuberculosis, Mycobacterium bovis. In case of absence of drug resistance the wild type KAT G gene has Serine (S) at position 315. Region HW:

AAC CGG TAA GGA CGC GAT CAC CAG CGG CAT CGA GGT CGT ATG GAC

GAAC SEQ ID NO: 51

HA 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

11D 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). Thus this mutation can be detected using the oligonucleotide set (SEQ ID NO: 57-60).

REGION 1 IM: 2152482 to 2152433 in CDC 1551 Accession no. AE000516

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 an embodiment, the present invention provides oligonucleotides useful for detection of nucleic acid, wherein the oligonucleotide set 12 as set forth in SEQ ID NO: 62-65 is designed from nucleotide position designed from 242 to 284 nucleotide (SEQ ID NO: 61) of the DNA sequence of hepatitis B virus (Accession number NC 003977). This region is specific to detect Hepatitis B Virus.

Region 12:

CAG AGT CTA GAC TCG TGG TGG ACT TCT CTC AAT TTT CTA GGG G

SEQ ID NO: 61

12A: CAG AGT CTA GAC TCG TGG TGG SEQ ID NO: 62

12B: ACT TCT CTC AAT TTT CTA GGG G SEQ ID NO: 63

12C: CCA CCA CGA GTC TAG ACT CTG SEQ ID NO: 64

12D: CCC CTA GAA AAT TGA GAG AAG T SEQ ID NO: 65

In an embodiment, the present invention provides oligonucleotides useful for detection of nucleic acid, wherein the oligonucleotide set 13 as set forth in SEQ ID NO: 67-70 is designed from nucleotide position from 672 to 717 nucleotide (SEQ ID NO: 66) of the DNA sequence of hepatitis B virus (Accession number NC_003977). This region is specific to detect Hepatitis B Virus

Region 13:

TCA GTT TAC TAG TGC CAT TTG TTC AGT GGT TCG TAG GGC TTT CCC

SEQ ID NO: 66

13A: TCA GTT TAC TAG TGC CAT TTG T SEQ ID NO: 67 13B: TC AGT GGT TCG TAG GGC TTT CCC SEQ ID NO: 68 13C: ACA AAT GGC ACT AGT AAA CTG A SEQ ID NO: 69 13D: GGG AAA GCC CTA CGA ACC ACT GA SEQ ID NO: 70

In an embodiment, the present invention provides oligonucleotides useful for detection of nucleic acid, wherein the oligonucleotide set 14 as set forth in SEQ ID NO: 72-75 is designed from nucleotide position 48-106 nucleotide (SEQ ID NO: 71) of the DNA sequence of hepatitis C virus (Accession number NC 004102). This region is specific to detect Hepatitis C Virus.

Region 14:

TGA GGA ACT ACT GTC TTC ACG CAG AAA GCG TCT AGC CAT GGC GTT

AGTATG AGT GTC GT SEQ ID NO: 71

14A: TGA GGA ACT ACT GTC TTC ACG CAG AAA GC SEQ ID NO: 72

14B: GTC TAG CCA TGG CGT TAG TAT GAG TGT CGT SEQ ID NO: 73

14C: GCT TTC TGC GTG AAG ACA GTA GTT CCT CA SEQ ID NO: 74

14D: ACG ACA CTCATA CTA ACG CCATGG CTAGAC SEQ ID NO: 75

In an embodiment, the present invention provides oligonucleotides useful for detection of nucleic acid, wherein the oligonucleotide set 15 as set forth in SEQ ID NO: 77-82 is designed from nucleotide position 127-176 nucleotide (SEQ ID NO: 76) of the DNA sequence of hepatitis C virus (Accession number NC 004102). This region is specific to detect Hepatitis C Virus.

Region 15:

TCC CGG GAG AGC CAT AGT GGT CTG CGG AAC CGG TGA GTA CAC CGG

AAT TG SEQ ID NO: 76

15A: TCC CGG GAG AGC CAT AGT GGT CTG C SEQ ID NO: 77 15B: GGA ACC GGT GAG TAC ACC GGA ATT G SEQ ID NO: 78

15C: GCA GAC CAC TAT GGC TCT CCC GGG A SEQ ID NO: 79

15D: CAA TTC CGG TGT ACT CAC CGG TTC C SEQ ID NO: 80

In an embodiment, the present invention provides oligonucleotides useful for detection of nucleic acid, wherein the oligonucleotide set 16 as set forth in SEQ ID NO: 82-85 is designed from nucleotide position 204-261 nucleotide (SEQ ID NO: 81) of the DNA sequence of hepatitis C virus (Accession number NC 004102). This region is specific to detect Hepatitis C Virus.

Region 16:

AAA CCC GCT CAA TGC CTG GAG ATT TGG GCG TGC CCC CGC AAG ACT

GCTAG SEQIDNO: 81

16A: AAA CCC GCT CAA TGC CTG GAG ATT T SEQ ID NO: 82

16B: GGG CGT GCC CCC GCA AGA CTG CTA G SEQ ID NO: 83

16C: AAA TCT CCA GGC ATT GAG CGG GTT T SEQ ID NO: 84

16D: CTA GCA GTC TTG CGG GGG CAC GCC C SEQ ID NO: 85

Another embodiment of the present invention provides a diagnostic kit for detecting presence of nucleic acid in samples associated with infectious diseases caused by microorganisms, genetically transmitted diseases, cancer and/or SNP variations.

In an embodiment, the present invention provides oligonucleotides useful for detection of nucleic acid, wherein the oligonucleotide set 17 as set forth in SEQ ID NO: 87-90 is designed from nucleotide position 484256 to 484207 (SEQ ID NO: 86) of the DNA sequence of Mycobacterium tuberculosis CDC 1551 (Accession number AE000516).

Region 17:

CCA TCG ACC TAC TAC GAC CAC ATC AAC CGG GAG CCC AGC CGC CGC

GAG CT SEQ ID NO:86

17A: CCA TCG ACC TAC TAC GAC CAC ATC A SEQ ID NO:87

17B: ACC GGG AGC CCA GCC GCC GCG AGC T SEQ ID NO:88

17C: TGA TGT GGT CGT AGT AGG TCG ATG G SEQ ID NO:89

17D: AGC TCG CGG CGG CTG GGC TCC CGG T SEQ ID NO:90

A phosphate group was attached to the 5' end of the oligonucleotides as set forth in SEQ ID NO: 88 and SEQ ID NO: 89. The oligonucleotide set as set forth in SEQ ID NO: 87-90 was tested with sputum, blood, ascitic fluid, bronchial washing samples, pleural fluid, urine, semen, tissue samples, milk, serum and pure culture of Mycobacterium species.

In an embodiment, the present invention provides oligonucleotides useful for detection of sequence specific regions that are implicated in susceptibility to prostate cancer (oligonucleotide set 18 and set 19) having nucleotide sequence as set forth in SEQ ID NO: 92-95 and SEQ ID NO: 97-100.

The oligonucleotide set 18 as set forth in SEQ ID NO: 92-95 is designed to include the INDELl region in the MSRl gene that corresponds to nucleotide position -14458bp (SEQ ID NO: 91) of the DNA sequence of Homo sapiens chromosome 8 (Accession number NT 030737). This region is specific to detect presence of marker INDELl which is an MSRl sequence variant and maybe implicated in association with prostate cancer risk.

Region 18:

AAA AAC CAA ACC AAA TTA TTG CTG ATA CAA TAA AGC ATT CAT CTC

ATC AT SEQ ID:91

INDEL1-18A: AAA AAC CAA ACC AAA TTA TTG CTG A SEQ ID:92

INDELlM-18B: TAC AAT AAA GCA TTC ATC TCA TCA T SEQ ID:93

INDEL 1-18C: TCA GCA ATA ATT TGG TTT GGT TTT T SEQ ID:94

INDELlM-18D: ATG ATG AGA TGA ATG CTT TAT TGT A SEQ ID:95

In this case, a 15 base pair sequence INDELl is present in the gene MSRl. The insertion of INDELl can be detected using the oligonucleotide set (SEQ ID NO: 92-95).

The oligonucleotide set 19 as set forth in SEQ ID NO: 97-100 is designed by deleting the INDELl region in the MSRl gene that corresponds to nucleotide position -14458bp (SEQ ID NO: 96) of the DNA sequence of Homo sapiens chromosome 8 (Accession number NT 030737). This region is specific to detect the absence of marker INDELl which is an MSRl sequence variant and maybe implicated in association with prostate cancer risk.

Region 19:

AAA AAC CAA ACC AAA TTA TTG CTG A ATC TCA TCA TAC ACA CAC AGA CAC A SEQ ID:96

INDEL1-19A: AAA AAC CAA ACC AAA TTA TTG CTG A SEQ ID:97

INDEL1N-19B: ATC TCA TCA TAC ACA CAC AGA CAC A SEQ ID:98 INDELl -19C: TCA GCA ATA ATT TGG TTT GGT TTT T SEQ ID:99 INDEL1N-19D: TGT GTC TGT GTG TGT ATG ATG AGA T SEQ ID:100

In this case, the 15 base pair sequence INDELl is deleted in the gene MSRl. This mutation by deletion can be detected using the oligonucleotide set (SEQ ID NO: 97-100).

A phosphate group was attached to the 5' end of the oligonucleotides as set forth in SEQ ID NO: 98 and SEQ ID NO: 99.

Another embodiment of the present invention provides a diagnostic kit comprising oligonucleotide sequences useful for detection of an organism, wherein the organism is selected from a group comprising of bacteria, mammals, plants, viruses, fungi, protozoa and parasites.

Another embodiment of the present invention provides a diagnostic kit comprising oligonucleotide set or primers used interchangeably herein, wherein the oligonucleotide set comprises at least four oligonucleotides, wherein the oligonucleotides having size ranging from 15-50 bp. The four oligonucleotides are designated as A, B, C, D, wherein first oligonucleotide (A) is complementary to third oligonucleotide (C) and second oligonucleotide (B) is complementary to fourth oligonucleotide (D). The second and third oligonucleotide (B and C) has a phosphate group attached to the 5' end. 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 modified by adding phosphate group at the 3' end. The modification may be carried out by adding phosphate, deoxy, alkyl or aryl group at the 3' end.

One embodiment relates to a probe that can be optionally attached to any of the four (A, B, C and D) oligonucleotides in each set wherein the probe is selected from a group consisting of radioactive, colorimetric, enzymatic, fluorescent, chemiluminiscent, and photo-active probe.

Another embodiment of the present invention provides a diagnostic kit comprising oligonucleotide sequences useful for nucleic acid detection of Mycobacterium species that include M. tuberculosis, M. avium and/or M. bovis.

Another embodiment of the present invention provides a diagnostic kit comprising oligonucleotide sequences useful for nucleic acid detection of Mycobacterium species that include M. tuberculosis and/or M. bovis. Another embodiment of the present invention provides a diagnostic kit comprising oligonucleotide sequences useful for nucleic acid detection of Mycobacterium tuberculosis.

Another embodiment of the present invention provides a diagnostic kit comprising oligonucleotide sequences useful for nucleic acid detection of Mycobacterium bovis.

Another embodiment of the present invention provides a diagnostic kit comprising oligonucleotide sequences useful for nucleic acid detection of Mycobacterium avium.

Another embodiment of the present invention provides a diagnostic kit comprising oligonucleotide sequences useful for nucleic acid detection of M. avium subsp. paratuberculosis KlO.

Another embodiment of the present invention provides a diagnostic kit comprising oligonucleotide sequences useful for nucleic acid detection of M. avium 104.

Another embodiment of the present invention provides a diagnostic kit comprising oligonucleotide sequences useful for nucleic acid detection of Hepatitis virus that include Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus and Hepatitis E virus.

Another embodiment of the present invention provides a diagnostic kit comprising oligonucleotide sequences useful for nucleic acid detection of Hepatitis B virus and/or Hepatitis C virus.

Another embodiment of the present invention provides a diagnostic kit comprising oligonucleotide sequences useful for nucleic acid detection of Hepatitis B virus.

Another embodiment of the present invention provides a diagnostic kit comprising oligonucleotide sequences useful for nucleic acid detection of Hepatitis C virus.

Another embodiment of the present invention relates to a kit for detection of sequence specific regions that are implicated in susceptibility to prostate cancer.

In accordance with the present invention, in one embodiment there is provided a process for detecting a target nucleic acid in a sample using multiplex dual isothermal nucleic acid amplification reaction, wherein the process comprises (a) providing a reaction mixture comprising a sample containing or suspected of containing one or more than one target nucleic acid; more than one set of oligonucleotides specific for one or more than one region of one or more than one target nucleic acids, wherein the set comprises at least four oligonucleotides, wherein a first oligonucleotide of the set is complementary to a third oligonucleotide and a second oligonucleotide is complementary to a fourth oligonucleotide; and thermostable ligase; (b) subjecting the reaction mixture under isothermal conditions to obtain a first resultant reaction mixture thereby amplifying the target nucleic acids, wherein the isothermal conditions comprises one cycle of denaturation at a temperature ranging from about 90⁰C to 99°C for 30 seconds to 10 minutes; and annealing and amplification at a temperature ranging from about 55⁰C to 74⁰C for 30 seconds to 3 minutes; and 8 to 25 cycles of denaturation at a temperature ranging from about 9O⁰C to 99°C for 30 seconds to 3 minutes and annealing and amplification at a temperature ranging from about 55⁰C to 74°C for 30 seconds to 3 minutes; (b) subjecting a part or the whole of the first resultant reaction mixture to the following isothermal conditions using the set of oligonucleotides, and the thermostable ligase to obtain a second resultant reaction mixture, wherein the isothermal conditions comprise 8-32 cycles of denaturation at a temperature ranging from about 90⁰C to 99°C for 30 seconds to 3 minutes and annealing and amplification at a temperature ranging from about 55°C to 74⁰C for 30 seconds to 3 minutes; and (d) detecting the amplified nucleic acid.

Another embodiment of the present invention provides a process for detecting a target nucleic acid in a sample using multiplex dual isothermal nucleic acid amplification reactions, wherein the process comprises (a) providing a reaction mixture comprising a sample containing or suspected of containing one or more than one target nucleic acid; more than one set of oligonucleotides specific for one or more than one region of one or more than one target nucleic acids, wherein the set comprises at least four oligonucleotides, wherein a first oligonucleotide of the set is complementary to a third oligonucleotide and a second oligonucleotide is complementary to a fourth oligonucleotide; and thermostable ligase; (b) subjecting the reaction mixture under isothermal conditions to obtain a first resultant reaction mixture, wherein the isothermal conditions are one cycle of denaturation at 95°C for 10 minutes; and annealing and amplification at 65⁰C for 3 minutes; and 15 cycles of denaturation at 95°C for 1 minute and annealing and amplification at 65⁰C for 1 minute; (c) subjecting a part or whole of the first resultant reaction mixture to following isothermal conditions using the set of oligonucleotides, and the thermostable ligase to obtain a second resultant reaction mixture, wherein the isothermal conditions are 23 cycles at 95⁰C for 1 minute and 65°C for 1 minute; and (d) detecting the amplified nucleic acid.

The process for detecting a target nucleic acid in a sample using multiplex dual isothermal nucleic acid amplification reaction as disclosed in the present invention, wherein step (c) optionally comprises passing the first resultant reaction mixture through a purification column to obtain flowthrough and subjecting a part or whole of the flowthrough under said isothermal conditions to obtain a second resultant reaction mixture.

The process for detecting a target nucleic acid in a sample using multiplex dual isothermal nucleic acid amplification reaction as disclosed in the present invention, wherein a phosphate group is attached to the 5' end of the second and third oligonucleotides.

The process for detecting a target nucleic acid in a sample using multiplex dual isothermal nucleic acid amplification reaction as disclosed in the present invention, wherein the 3' end of the second and third oligonucleotides are optionally modified.

The process for detecting a target nucleic acid in a sample using multiplex dual isothermal nucleic acid amplification reaction as disclosed in the present invention, wherein the modification is carried out by adding a phosphate, deoxy, alkyl or aryl group at the 3' end.

The process for detecting a target nucleic acid in a sample using multiplex dual isothermal nucleic acid amplification reaction as disclosed in the present invention, wherein the modification is carried out by adding a phosphate group at the 3' end.

The process for detecting a target nucleic acid in a sample using multiplex dual isothermal nucleic acid amplification reaction as disclosed in the present invention, wherein at least one oligonucleotide of the set of oligonucleotides is optionally attached to a probe selected from the group consisting of radioactive, colorimetric, enzymatic, fluorescent, chemiluminiscent, and photo-active probes.

The process for detecting a target nucleic acid in a sample using multiplex dual isothermal nucleic acid amplification reaction as disclosed in the present invention, wherein the process detects a target nucleic acid of disease or non-disease related conditions selected from a group consisting of infectious diseases, genetically transmitted diseases, cancer and/or SNP variations, mutations and drug resistance, and combinations thereof.

The process for detecting a target nucleic acid in a sample using multiplex dual isothermal nucleic acid amplification reaction as disclosed in the present invention, wherein the sample is of human, veterinary, plant or microorganism origin.

The process for detecting a target nucleic acid in a sample using multiplex dual isothermal nucleic acid amplification reaction as disclosed in the present invention, wherein the sample is selected from the group consisting of blood, sputum, tissue, saliva, cerebro-spinal fluid, pleural fluid, lymph, synovial fluid, semen, other body fluids, milk and other body secretions, urine, other body excretions, broncho-alveolar lavage and other washings from a subject.

The process for detecting a target nucleic acid in a sample using multiplex dual isothermal nucleic acid amplification reaction as disclosed in the present invention, wherein the sample is of plant origin.

The process for detecting a target nucleic acid in a sample using multiplex dual isothermal nucleic acid amplification reaction as disclosed in the present invention, wherein the cancer is prostate cancer.

The process for detecting a target nucleic acid in a sample using multiplex dual isothermal nucleic acid amplification reaction as disclosed in the present invention, wherein the micro-organism is selected from the group consisting of bacteria, viruses, fungi, archaea, protozoans, and combinations thereof.

The process for detecting a target nucleic acid in a sample using multiplex dual isothermal nucleic acid amplification reaction as disclosed in the present invention, wherein the micro-organism is selected from the group consisting of M. tuberculosis, M. bovis, M. avium, Hepatitis A virus, Hepatitis B^irus, Hepatitis C virus, Hepatitis D virus and Hepatitis E virus.

In one embodiment of the present invention there is provided a set of oligonucleotides for detection of Mycobacterium nucleic acid in a sample, wherein the nucleotide sequence of said set of oligonucleotides is selected from the group consisting of the nucleotide sequence as set forth in 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 and SEQ ID NO: 87-90, wherein Mycobacterium is selected from the group consisting of Mycobacterium tuberculosis, M. avium and M. bovis.

In another embodiment of the present invention there is provided a set of oligonucleotides for detection of nucleic acid of Mycobacterium in a sample, wherein said set of oligonucleotides is selected from the group consisting of nucleotide sequence as set forth in SEQ ID NO: 42-45 and SEQ ID NO: 47-50, wherein Mycobacterium is selected from the group consisting of Mycobacterium avium paratuberculosis KlO and Mycobacterium avium 104.

In another embodiment of the present invention there is provided a set of oligonucleotides for detection of nucleic acid of Mycobacterium tuberculosis nucleic acid in a sample, wherein said set of oligonucleotides is selected from the group consisting of nucleotide sequence as set forth in 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 and SEQ ID NO: 87-90.

In yet another embodiment of the present invention there is provided a set of oligonucleotides for detection of nucleic acid of Mycobacterium bovis in a sample, wherein said set of oligonucleotides is selected from the group consisting of the nucleotide sequence as set forth in 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 and SEQ ID NO: 87-90.

In yet another embodiment of the present invention there is provided a set of oligonucleotides for nucleic acid detection of Mycobacterium tuberculosis and Mycobacterium bovis in a sample, wherein the nucleotide sequence of said set of oligonucleotides is as set forth in 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 and SEQ ID NO: 87-90.

In yet another embodiment of the present invention there is provided a set of oligonucleotides for nucleic acid detection of Hepatitis virus in a sample, wherein the nucleotide sequence of said set of oligonucleotides is as set forth in SEQ ID NO: 62-65, SEQ ID NO: 67-70, SEQ ID NO: 72-75, SEQ ID NO: 77-80 or SEQ ID NO: 82-85, said Hepatitis virus being selected from the group consisting of Hepatitis B and C virus.

In yet another embodiment of the present invention there is provided a set of oligonucleotides for nucleic acid detection of Hepatitis B virus in a sample, wherein the nucleotide sequence of said set of oligonucleotides is as set forth in SEQ ID NO: 62-65 or SEQ ID NO: 67-70.

In yet another embodiment of the present invention there is provided a set of oligonucleotides for nucleic acid detection of Hepatitis C virus in a sample, wherein the nucleotide sequence of said set of oligonucleotides is as set forth in SEQ ID NO: 72-75, SEQ ID NO: 77-80 or SEQ ID NO: 82-85.

In still yet another embodiment of the present invention there is provided a set of oligonucleotides for detection of sequence specific regions implicated in the susceptibility to prostate cancer in a sample, wherein the nucleotide sequence of said set of oligonucleotides is selected from the group consisting of the nucleotide sequence as set forth in SEQ ID NO: 92-95 and SEQ ID NO: 97-100.

Yet another embodiment of the present invention relates to a kit for detection of a nucleic acid of a Mycobacterium species in a sample, wherein said kit comprises the set of oligonucleotides selected from the group consisting of the nucleotide sequence as set forth in 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 and SEQ ID NO: 87-90 wherein the Mycobacterium is selected from the group consisting of Mycobacterium tuberculosis, M. avium and M. bovis.

Yet another embodiment of the present invention relates to a kit for detection of a nucleic acid of a Mycobacterium species in a sample, wherein said kit comprises the set of oligonucleotides selected from the group consisting of nucleotide sequence as set forth in SEQ ID NO: 42-45 and SEQ ID NO: 47-50, wherein the Mycobacterium is selected from the group consisting of Mycobacterium avium par atuberculo sis KlO and Mycobacterium avium 104.

Yet another embodiment of the present invention relates to a kit for detection of a nucleic acid of Mycobacterium tuberculosis in a sample, wherein said kit comprises the set of oligonucleotides selected from the group consisting of nucleotide sequence as set forth in 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 and SEQ ID NO: 87-90.

Yet another embodiment of the present invention relates to a kit for detection of a nucleic acid of Mycobacterium bovis, in a sample, wherein the kit comprises the set of oligonucleotides selected from the group consisting of the nucleotide sequence as set forth in 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 and SEQ ID NO: 87-90.

Yet another embodiment of the present invention relates to a kit for detection of a nucleic acid of Mycobacterium tuberculosis and Mycobacterium bovis in a sample, wherein said kit comprises the set of oligonucleotides is as set forth in 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 and SEQ ID NO: 87-90.

Yet another embodiment of the present invention relates to a kit for detection of a nucleic acid of a Hepatitis virus, wherein said kit comprises the set of oligonucleotides is as set forth in SEQ ID NO: 62-65, SEQ ID NO: 67-70, SEQ ID NO: 72-75, SEQ ID NO: 77-80 or SEQ ID NO: 82-85, wherein the Hepatitis virus being selected from the group consisting of Hepatitis B and C virus.

Yet another embodiment of the present invention relates to a kit for detection of a nucleic acid of Hepatitis B virus, wherein said kit comprises the set of oligonucleotides is as set forth in SEQ ID NO: 62-65 or SEQ ID NO: 67-70.

Yet another embodiment of the present invention relates to a kit for detection of a nucleic acid of Hepatitis C virus, wherein said kit comprises the set of oligonucleotides is as set forth in SEQ ID NO: 72-75, SEQ ID NO: 77-80 or SEQ ID NO: 82-85.

Yet another embodiment of the present invention relates to a kit for detection of sequence specific regions that are implicated in susceptibility to prostate cancer, wherein said kit comprises the set of oligonucleotides selected from the group consisting of the nucleotide sequence as set forth in SEQ ID NO: 92-95 and SEQ ID NO: 97-100. 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 puφoses 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

Multiplex Dual isothermal nucleic acid amplification reaction (M-DINAR) for detecting Mycobacterium DNA using oligonucleotide sets 1(SEQ ID NO: 2-5) and set 2(SEQ ID NO: 7-10)

The M-DINAR for detecting Mycobacterium DNA was carried out in two steps as given below.

STEP l

M-DINAR is performed for detection of typically two or more different DNA sequences (these sequences can be from the same target template DNA or different target templates) wherein the template DNA for the amplification reaction and their corresponding primer sets are taken together in the same reaction mixture. The amplification reaction was performed using oligonucleotide having nucleotide sequences as set forth in SEQ ID NO: 2-5 and SEQ ID NO: 7-10 for detection of Mycobacterium tuberculosis in samples. The total volume of the reaction mixture was 20 μl. The final concentration of the components of the reaction mixture was 2 μl buffer (10X), 1 μl Thermostable ligase (5U/μl), 1 μl each of oligonucleotides IA, IB, 1C and ID (SEQ ID NO: 2-5) and 2A, 2B, 2C and 2D (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 cycling reaction employed is as follows: 1 cycle 95°C for 10 min and 60⁰C for 3 min followed by 14 cycles 95°C for 1 min and 60⁰C for 1 min. Various samples and controls were used for detection of Mycobaterium DNA. The description of the different reactions (reactions 1-8) 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 TB patient)

Reaction 3: PCl+PC2-Positive control- 3.2 Kb plasmid (pPBPCl) with 250 bp from the region of 62716-62765 of Mycobacterium genome having accession number AE000516 and 3.2 Kb plasmid (pPBPC2) with 250 bp from the region of 85252-85301 of Mycobacterium genome having accession number AE000516

Reaction 4: S12-template DNA from sputum sample of confirmed Mycobacterium infected patient

Reaction 5: S 13- template DNA from sputum sample of confirmed Mycobacterium infected patient

Reaction 6: S 14- template DNA from sputum sample of suspected Mycobacterium infected patient

Reaction 7: S 16- template DNA from sputum sample of suspected Mycobacterium infected patient

Reaction 8: Sl 7- template DNA from bronchial washing sample of suspected Mycobacterium infected patient.

After completion of 15 cycles of reaction in the thermocycler, the resultant reaction mixture was processed through a purification column and the flow through obtained was used as a template for the second isothermal nucleic acid amplification reaction.

STEP 2

In the second step, isothermal nucleic acid amplification reaction was performed using the same primer sets as used in the first step (SEQ ID NO: 2-5 and SEQ ID NO: 7-10). The reaction mixture used for performing the second isothermal nucleic acid amplification reaction was 8 μl of the flow through obtained by processing the final reaction mixture of reactions 1-7 of step 1, 2 μl buffer (10X), 1 μl Thermostable ligase (5U/ μl), lμl each of oligonucleotides IA, IB, 1C and ID (SEQ ID NO: 2-5) and 2A, 2B, 2C and 2D (SEQ ID NO: 7-10) at concentration 2.5ng each and ultrapure water to bring the reaction volume to 20 μl. The cycling reaction was performed as follows: 23 cycles 95°C for 1 min and 60⁰C for 1 min.

After completion of the 23 cycles of reaction in the thermocycler, the entire reaction mixture of each reaction 1-8 was electrophoresed on a 2.5% agarose gel at 100V for 45 min and the DNA products were identified using process known in the art. The gel picture was captured on a gel documentation system as shown in Figure 1.

The amplified DNA product obtained can also be detected by various other process known in the art such as 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 process.

The results (Figure 1) indicate that there was no non-specific amplification of 50 bp fragment observed in the reaction 1 marked as lane NC (negative control sample) and reaction 2 marked as lane HGD (template DNA from healthy subject). A fragment of 25bp was obtained in reaction 1 and 2 and these correspond to primer annealing as expected. There was amplification of 50 bp fragment observed in reaction 3 marked as lane PC1+PC2 (positive control) and the reaction 4 marked as lane S12 (sample containing Mycobacterium DNA). Amplification of 50 bp fragment was observed in reactions 5-8 marked as lane S 13, S 14, S16 and S17 confirming presence of Mycobacterium DNA in the suspected patients. This clearly indicates that the primer sets having nucleotide sequence as set forth in SEQ ID NO: 2-5 and SEQ ID NO: 7-10 are useful in the detection of Mycobacterium infection. M-DINAR assay employing primer sets having nucleotide sequence as set forth in SEQ ID NO: 2-5 and SEQ ID NO: 7-10 is sensitive and easy to use for the detection of Mycobacterium infection in patients.

Further experiments were conducted and it was observed that the primer sets SEQ ID NO: 2-5 and SEQ ID NO: 7-10 amplify DNA Mycobacterium tuberculosis and Mycobacterium bovis but not Mycobacterium avium strain. Other reaction conditions employed for the above described process employed annealing and ligation temperatures varying from 55°C to 74⁰C for duration of 30seconds-3minutes and using different primer sets. The results obtained were similar as for the above described reaction set up. Other primer sets were tested with the same set of samples with similar results. The enzyme concentration for thermostable ligase was varied from 1-10 units with consistent and reproducible results.

The results obtained above have also been corroborated by MTb culture on LJ media and PCR using IS6110 region.

A) Culture process: The decontaminated samples were inoculated on Lowenstein-Jensen slants and 7H9 Middlebrook's liquid media for growth and incubated at 37°C for 3-4 weeks. Growth of MTB colonies was evident in all cases.

B) PCR process: Reference (Ogusku, Mauricio Analise de diferent Meso prriismhie, rest 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 IS6110 gene were used. The samples were found positive basis PCR amplification results analysis by 1.2% agarose gel electrophoresis.

Example 2

Multiplex Dual isothermal nucleic acid amplification reaction (M-DINAR) for detecting Mycobacterium DNA using oligonucleotide set 3 (SEQ ID NO: 12-15) and set 4 (SEQ ID NO: 17-20)

STEP 1

M-DINAR is performed for detection of typically two or more, different DNA sequences (these sequences can be from the same target template DNA or different target templates) wherein the template DNA for the amplification reaction and their corresponding primer sets are taken together in the same reaction mixture. The amplification reaction was performed using oligonucleotide having nucleotide sequences as set forth in SEQ ID NO: 12-15 and SEQ ID NO: 17-20 for detection of Mycobacterium tuberculosis in samples. The total volume of the reaction mixture was 20 μl. The final concentration of the components of the reaction mixture was 2 μl buffer (10X), 1 μl Thermostable ligase (5U/μl), 1 μl each of oligonucleotides 3A, 3B, 3C and 3D (SEQ ID NO: 12-15) and 4A, 4B, 4C and 4D (SEQ ID NO: 17-20) at concentration 2.5ng each, lOOng of template DNA and ultrapure water to bring the reaction volume to 20 μl. The cycling reaction employed is as follows: 1 cycle 95⁰C for 10 min and 6O⁰C for 3 min followed by 14 cycles 95°C for 1 min and 6O⁰C for 1 min. Various samples and controls were used for detection of Mycobacterium DNA. The description of the different reactions (reactions 1- 8) used as template DNA is given below.

Reaction 1 : NC-Negative control- no template DNA in the reaction mixture

Reaction 3: PC3+PC4-Positive control- 3.2 Kb plasmid (pPBPC3) with 250 bp from the region of 82712-82761 of Mycobacterium genome having accession number AE000516 and 3.2 Kb plasmid (pPBPC4) with 250 bp from the region of 2041-2088 of Mycobacterium genome having accession number AE000516

Reaction 8: S 17- template DNA from bronchial washing sample of suspected Mycobacterium infected patient

After completion of 23 cycles of reaction in the thermocycler, the resultant reaction mixture was used as such without any processing as a template for the second isothermal nucleic acid amplification reaction.

STEP 2

In the second step, isothermal nucleic acid amplification reaction was performed using the same primer sets as used in the first step (SEQ ID NO: 12-15 and SEQ ID NO: 17-20). The reaction mixture used for carrying out the second isothermal nucleic acid amplification reaction was 8 μl of the final reaction mixture of reactions 1-8 taken as such obtained from resulting reaction mixture of step 1, 2 μl buffer (10X), 1 μl Thermostable ligase (5 U/ μl), lμl each of oligonucleotides 3 A, 3B, 3C and 3D (SEQ ID NO: 12-15) and 4A, 4B, 4C and 4D (SEQ ID NO: 17-20) at concentration 2.5ng each and ultrapure water to bring the reaction volume to 20 μl. The cycling reaction was performed as follows: 23 cycles 95°C for 1 min and 60⁰C for 1 min.

After completion of the 23 cycles of reaction in the thermocycler, the entire reaction mixture of each reaction 1-8 was electrophoresed on a 2.5% agarose gel at 100V for 45 min and the DNA products were identified using processs known in the art. The gel picture was captured on a gel documentation system (data not shown).

The amplified DNA product obtained can also be detected by various other processs known in the art such as 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 process.

The results (data not shown) indicate that there was no non-specific amplification of 50 bp fragment observed in the reaction 1 marked as lane NC (negative control sample) and reaction 2 marked as lane HGD (template DNA from healthy subject). A fragment of 25bp was obtained in reaction 1 and 2 and these correspond to primer annealing as expected. There was amplification of 50 bp fragment observed in reaction 3 marked as lane PC3+PC4 (positive control) and the reaction 4 marked as lane Sl 2 (sample containing Mycobacterium DNA). Amplification of 50 bp fragment was observed in reactions 5-8 marked as lane Sl 3, Sl 4, S16 and S17 confirming presence of Mycobacterium DNA in the suspected patients. This clearly indicates that the primer sets having nucleotide sequence as set forth in SEQ ID NO: 12-15 and SEQ ID NO: 17-20 are useful in the detection of Mycobacterium infection. M-DINAR assay employing primer sets having nucleotide sequence as set forth in SEQ ID NO: 12-15 and SEQ ID NO: 17- 20 is sensitive and easy to use for the detection of Mycobacterium infection in patients. Further experiments were conducted and it was observed that the primer sets SEQ ID NO: 12-15 and SEQ ID NO: 17-20 amplify DNA Mycobacterium tuberculosis and Mycobacterium bovis but not Mycobacterium avium strain.

Other reaction conditions employed for the above described process employed annealing and ligation temperatures varying from 55°C to 74° C for duration of 30 seconds to 3 min and using different primer sets. The results obtained were similar as for the above described reaction set up. Other primer sets were tested with the same set of samples with similar results. Example 3

Multiplex Dual isothermal nucleic acid amplification reaction (M-DINAR) for detecting Hepatitis B virus DNA using oligonucleotide set 12 (SEQ ID NO: 62-65) and set 13 (SEQ ID NO: 67-70)

The M-DINAR for detecting Hepatitis B virus DNA was carried out in two steps as given below. STEP l

The isothermal nucleic acid amplification reaction was performed using oligonucleotide having nucleotide sequences as set forth in SEQ ID NO: 2-5 and SEQ ID NO: 7-10 for detection of Hepatitis B virus DNA in samples. The total volume of the reaction mixture was 25 μl. The final concentration of the components of the reaction mixture was 2.5 μl buffer (10X), 1 μl Thermostable ligase (5U/μl), 1 μl each of oligonucleotide IA, IB, 1C and ID (SEQ ID NO: 2-5) and 2A, 2B, 2C, 2D (SEQ ID NO: 7-10) at concentration 2.5ng each, lOOng of template DNA and ultrapure water to bring the reaction volume to 25 μl. The cycling reaction employed is as follows: first cycle at 95°C for 10 min and 65°C for 3 min followed by 14 cycles at 95°C for 1 min and 65°C for 1 min. Various samples and controls were used for detection of Hepatitis B viral DNA. The description of the different reactions (reactions 1-9) used as template DNA is given below: Sample 1 : Experimental Human Sample Bl (unknown blood Hepatitis sample) Sample 2: Experimental Human Sample B2 (unknown blood Hepatitis sample) Sample 3: Experimental Human Sample B3 (unknown blood Hepatitis sample) Sample 4: Experimental Human Sample B4 (unknown blood Hepatitis sample) Sample 5: Experimental Human Sample B5 (unknown serum Hepatitis sample) Sample 6: Experimental Human sample B6 (unknown serum Hepatitis sample) Sample 7: Experimental Human sample B7 (unknown serum Hepatitis sample) Sample 8: Negative control Human DNA (known Hepatitis negative sample)

Sample 9: Synthetic Positive control (plasmid containing 513bp fragment from Hepatitis B virus; Accession number NC 003977)

After completion of 15 cycles of reaction in the thermocycler, the resultant reaction mixture so obtained was subjected to the second step.

STEP 2

In the second step, isothermal nucleic acid amplification reaction was performed using the same primer set as used in the first step (SEQ ID NO: 2-5 and SEQ ID NO: 7-10). The reaction mixture used for performing the second isothermal nucleic acid amplification reaction was 10 μl of the final reaction mixture of reactions 1-9 of step 1, 2.5 μl buffer (10X), 1 μl Thermostable ligase (5U/ μl), lμl each of oligonucleotides IA, IB, 1C and ID (SEQ ID NO: 2-5) and 2A, 2B, 2C, 2D (SEQ ID NO: 7-10) at concentration 2.5ng each and ultrapure water to bring the reaction volume to 25 μl. The cycling reaction was carried out as follows: 23 cycles 95°C for 1 min and 65°C for 1 min.

After completion of the 23 cycles of reaction in the thermocycler, the entire reaction mixture of reactions 1-9 was electrophoresed on a 2.5% agarose gel at 100V for 45 min and the DNA products were identified using methods known in the art. The gel picture was captured on a gel documentation system.

The amplified DNA products obtained can also be detected by various other methods known in the art such as 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.

Amplification of expected fragment of 43bp for region 1 as set forth in SEQ ID NO: 1 and 45 bp for region 2 as set forth in SEQ ID NO: 6 was observed in positive control sample i.e. plasmid containing the 513 bp of hepatitis B nucleotide sequence, in Bl, B2, B4, B5 and B7. Fragment of 23 bp due to primer annealing only and no ligation in absence of target template was obtained in negative control (NC) sample. Fragment of 23 bp due to primer annealing and no fragment of 43 bp was obtained in B3 and B6 samples. This observation suggests that samples Bl, B2, B4, B5, and B7 are positive for the presence of hepatitis B virus in the isolated sample whereas samples B3 and B6 are not infected by hepatitis B virus.

Other reaction conditions employed for the above described process employed annealing and ligation temperatures varying from 55°C to 74° C for duration of 30 seconds to 3 min and using different primer sets. The results obtained were similar as for the above described reaction set up. Other primer sets were tested with the same set of samples with similar results. The enzyme concentration for thermostable ligase was varied from 1-10 units with consistent and reproducible results. These results have been confirmed by the ELISA and PCR tests.

Claims

IAVe Claim:

1. A process for detecting a target nucleic acid in a sample using multiplex dual isothermal nucleic acid amplification reactions, said process comprising: a. providing a reaction mixture comprising a sample containing or suspected of containing one or more than one target nucleic acid; more than one set of oligonucleotides specific for one or more than one region of one or more than one target nucleic acids, wherein said set comprises of at least four oligonucleotides, wherein a first oligonucleotide of said set is complementary to a third oligonucleotide and a second oligonucleotide is complementary to a fourth oligonucleotide; and thermostable ligase; b. subjecting said reaction mixture under isothermal conditions to obtain a first resultant reaction mixture thereby amplifying said target nucleic acids, wherein said isothermal conditions comprises one cycle of denaturation at a temperature ranging from about 90⁰C to 99°C for 30 seconds to 10 minutes; and annealing and amplification at a temperature ranging from about 55°C to 74°C for 30 seconds to 3 minutes; and 8 to 25 cycles of denaturation at a temperature ranging from about 9O⁰C to 99°C for 30 seconds to 3 minutes and annealing and amplification at a temperature ranging from about 55°C to 74°C for 30 seconds to 3 minutes; and c. subjecting a part or the whole of the said first resultant reaction mixture to the following isothermal conditions using said set of oligonucleotides, and said thermostable ligase to obtain a second resultant reaction mixture, wherein said isothermal conditions comprise 8-32 cycles of denaturation at a temperature ranging from about 90⁰C to 99°C for 30 seconds to 3 minutes and annealing and amplification at a temperature ranging from about 55°C to 74°C for 30 seconds to 3 minutes; and d. detecting the amplified nucleic acid.

2. A process for detecting a target nucleic acid in a sample using multiplex dual isothermal nucleic acid amplification reactions, said process comprising: a. providing a reaction mixture comprising a sample containing or suspected of containing one or more than one target nucleic acid; more than one set of oligonucleotides specific for one or more than one region of one or more than one target nucleic acids, wherein said set comprises at least four oligonucleotides, wherein a first oligonucleotide of said set is complementary to a third oligonucleotide and a second oligonucleotide is complementary to a fourth oligonucleotide; and thermostable ligase; b. subjecting said reaction mixture under isothermal conditions to obtain a first resultant reaction mixture, wherein said isothermal conditions are one cycle of denaturation at 95°C for 10 minutes; and annealing and amplification at 65⁰C for 3 minutes; and 14 cycles of denaturation at 95°C for 1 minute and annealing and amplification at 65°C for 1 minute; c. subjecting a part or whole of the said first resultant reaction mixture to following isothermal conditions using said set of oligonucleotides, and said thermostable ligase to obtain a second resultant reaction mixture, wherein said isothermal conditions are 23 cycles at 95°C for 1 minute and 65°C for 1 minute; and d. detecting the amplified nucleic acid.

3. The process as claimed in claim 1 or 2, wherein step (c) optionally comprises passing said first resultant reaction mixture through a purification column to obtain flowthrough and subjecting said flowthrough under said isothermal conditions to obtain a second resultant reaction mixture.

4. The process as claimed in claim 1 or 2, wherein a phosphate group is attached to the 5' end of said second and third oligonucleotides.

5. The process as claimed in claim 1 or 2, wherein the 3' end of said second and third oligonucleotides are optionally modified.

6. The process as claimed in claim 5, wherein said modification is carried out by adding a phosphate, deoxy, alky! or aryl group at the 3' end.

7. The process as claimed in claim 5, wherein said modification is carried out by adding a phosphate group at the 3' end.

8. The process as claimed in claim 1 or 2, wherein at least one oligonucleotide of said set of oligonucleotides is optionally attached to a probe selected from the group consisting of radioactive, colorimetric, enzymatic, fluorescence, chemiluminiscence, and photo-active probes.

9. The process as claimed in claim 1 or 2, wherein said process detects a target nucleic acid of disease or non-disease related conditions selected from a group consisting of infectious diseases, genetically transmitted diseases, cancer and/or SNP variations, mutations and drug resistance, and combinations thereof.

10. The process as claimed in claim 1 or 2, wherein said sample is of human, veterinary, plant or microorganism origin.

11. The process as claimed in claim 1 or 2, wherein said sample is selected from the group consisting of blood, sputum, tissue, saliva, cerebro-spinal fluid, pleural fluid, lymph, Synovial fluid, semen, other body fluids, milk and other body secretions, urine, other body excretions, broncho-alveolar lavage and other washings from a subject.

12. The process as claimed in claim 1 or 2, wherein said sample is of plant origin.

13. The process as claimed in claim 9, wherein said cancer is prostate cancer.

14. The process as claimed in claim 10, wherein said micro-organism is selected from the group consisting of bacteria, viruses, fungi, archaea, protozoans, and combinations thereof.

15. The process as claimed in claim 10, wherein said micro-organism is selected from the group consisting of M. tuberculosis, M. bovis, M. avium, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus and Hepatitis E virus.