Compositions and methods for identifying drug resistant tuberculosis
First Claim
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1. A method of detecting the presence and antibiotic resistance of Mycobacterium complex (MTB) in a subject, comprising:
- a) contacting a sample from said subject with first reagents for detecting the presence of MTB wherein said first reagents comprise primer SEQ ID NOs;
22-25, probe SEQ ID NOs;
26 and 27, and a first activation reagent comprising MgCl2, Tris, KCl, Proclin 950, sodium azide and ultrapure water;
b) performing an MTB detection assay with said first reagents, comprising;
i) amplifying said sample from said subject with SEQ ID NO;
22 and SEQ ID NO;
23 to generate an IS6110 amplicon using PCR cycling conditions;
ii) detecting said IS6110 amplicon with SEQ ID NO;
26;
iii) amplifying said sample from said subject with SEQ ID NO;
24 and 25 to generate a PAB amplicon using PCR cycling conditions;
iv) detecting said PAB amplicon with SEQ ID NO;
27; and
c) contacting a sample from said subject with second reagents for detecting the presence of MTB rifampicin and isoniazid resistance genes wherein said second reagents comprise primer SEQ ID NOs;
1-6, probe SEQ ID NOs;
7-18, and a second activation reagent comprising MgCl2, Tris, KCl, Proclin 950, sodium azide and ultrapure water; and
d) performing a rifampicin and isoniazid resistance assay with said second reagents, comprising;
i) amplifying said sample from said subject with SEQ ID NO;
1 and SEQ ID NO;
2 to generate a rpoB RRDR amplicon using PCR cycling conditions;
ii) detecting said rpoB RRDR amplicon with SEQ ID NOs;
7-14;
iii) amplifying said sample from said subject with SEQ ID NO;
3 and SEQ ID NO;
4 to generate a katG amplicon using PCR cycling conditions;
iv) detecting said katG amplicon with SEQ ID NOs;
15 and 16;
v) amplifying said sample from said subject with SEQ ID NO;
5 and SEQ ID NO;
6 to generate an inhA upper stream promoter amplicon using PCR cycling conditions; and
vi) detecting said inhA upper stream promoter amplicon using SEQ ID NOs;
17 and 18; and
wherein said first activation reagent and said second activation reagent are the same activation reagents, and wherein said PCR cycling conditions are the same PCR cycling conditions.
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Abstract
Provided herein are compositions and methods for diagnosing and characterizing tuberculosis infection. In particular, provided herein are compositions and methods for identifying drug resistant tuberculosis.
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Citations
10 Claims
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1. A method of detecting the presence and antibiotic resistance of Mycobacterium complex (MTB) in a subject, comprising:
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a) contacting a sample from said subject with first reagents for detecting the presence of MTB wherein said first reagents comprise primer SEQ ID NOs;
22-25, probe SEQ ID NOs;
26 and 27, and a first activation reagent comprising MgCl2, Tris, KCl, Proclin 950, sodium azide and ultrapure water;b) performing an MTB detection assay with said first reagents, comprising; i) amplifying said sample from said subject with SEQ ID NO;
22 and SEQ ID NO;
23 to generate an IS6110 amplicon using PCR cycling conditions;ii) detecting said IS6110 amplicon with SEQ ID NO;
26;iii) amplifying said sample from said subject with SEQ ID NO;
24 and 25 to generate a PAB amplicon using PCR cycling conditions;iv) detecting said PAB amplicon with SEQ ID NO;
27; andc) contacting a sample from said subject with second reagents for detecting the presence of MTB rifampicin and isoniazid resistance genes wherein said second reagents comprise primer SEQ ID NOs;
1-6, probe SEQ ID NOs;
7-18, and a second activation reagent comprising MgCl2, Tris, KCl, Proclin 950, sodium azide and ultrapure water; andd) performing a rifampicin and isoniazid resistance assay with said second reagents, comprising; i) amplifying said sample from said subject with SEQ ID NO;
1 and SEQ ID NO;
2 to generate a rpoB RRDR amplicon using PCR cycling conditions;ii) detecting said rpoB RRDR amplicon with SEQ ID NOs;
7-14;iii) amplifying said sample from said subject with SEQ ID NO;
3 and SEQ ID NO;
4 to generate a katG amplicon using PCR cycling conditions;iv) detecting said katG amplicon with SEQ ID NOs;
15 and 16;v) amplifying said sample from said subject with SEQ ID NO;
5 and SEQ ID NO;
6 to generate an inhA upper stream promoter amplicon using PCR cycling conditions; andvi) detecting said inhA upper stream promoter amplicon using SEQ ID NOs;
17 and 18; andwherein said first activation reagent and said second activation reagent are the same activation reagents, and wherein said PCR cycling conditions are the same PCR cycling conditions. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A method of detecting the antibiotic resistance of Mycobacterium complex (MTB) in a subject, comprising:
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a) contacting a sample from said subject with reagents for detecting the presence of MTB rifampicin and isoniazid resistance genes wherein said reagents comprise primer SEQ ID NOs;
1-6, probe SEQ ID NOs;
7-18, and an activation reagent comprising MgCl2, Tris, KCl, Proclin 950, sodium azide and ultrapure water; andb) performing a rifampicin and isoniazid resistance assay with said reagents, comprising; i) amplifying said sample from said subject with SEQ ID NO;
1 and SEQ ID NO;
2 to generate a rpoB RRDR amplicon using PCR cycling conditions;ii) detecting said rpoB RRDR amplicon with SEQ ID NOs;
7-14;iii) amplifying said sample from said subject with SEQ ID NO;
3 and SEQ ID NO;
4 to generate a katG amplicon using PCR cycling conditions;iv) detecting said katG amplicon with SEQ ID NOs;
15 and 16;v) amplifying said sample from said subject with SEQ ID NO;
5 and SEQ ID NO;
6 to generate an inhA upper stream promoter amplicon using PCR cycling conditions; andvi) detecting said inhA upper stream promoter amplicon using SEQ ID NOs;
17 and 18; andwherein said PCR cycling conditions are the same PCR cycling conditions.
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Specification