Indian Journal of Biotechnology & Biochemistry

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Evaluation of Different Methods of Mycobacterium Tuberculosis DNA Extraction for Getting better Yield from Clinical Samples


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1 Kilpest India Limited/ Dygnogene 7C Industrial Area, Govindpura, Bhopal, M.P, India
     

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In present study different clinical samples from suspected TB patients were collected from areas around Bhopal. For this five different methods were developed using different combination of Lysis Buffer (5.25 M GuSCN, Triton-X, GuSCN+ Triton-X, SDS/Lysozyme), Wash Buffers (20mM NaCl, 80% EtOH) Binding Buffer (GuHCl) and Elution Buffer for extraction of MTB-DNA by using silica column. Among all methods GuSCN and Triton-X when used together i.e. 5.25 M GuSCN+ Triton-X was found better as it gave comparatively higher DNA yield than other methods and it also showed maximum sensitivity in Nested-PCR targeting IS6110 plus the higher 260/280 ratio for the detection of MTB-DNA in clinical samples. Other methods like SDS/Lysozme lysis buffer used for extraction of DNA gave higher yield of MTB-DNA but the sensitivity in nested PCR was not that good. Incidence of TB was also studied among Extrapulmonary and Pulmonary TB cases, Pulmonary TB showed higher positivity rate (70.83%) as compare to Extrapulmonary.
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  • Evaluation of Different Methods of Mycobacterium Tuberculosis DNA Extraction for Getting better Yield from Clinical Samples

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Authors

Shaifali Gupta
Kilpest India Limited/ Dygnogene 7C Industrial Area, Govindpura, Bhopal, M.P, India
Deepika Choudhary
Kilpest India Limited/ Dygnogene 7C Industrial Area, Govindpura, Bhopal, M.P, India

Abstract


In present study different clinical samples from suspected TB patients were collected from areas around Bhopal. For this five different methods were developed using different combination of Lysis Buffer (5.25 M GuSCN, Triton-X, GuSCN+ Triton-X, SDS/Lysozyme), Wash Buffers (20mM NaCl, 80% EtOH) Binding Buffer (GuHCl) and Elution Buffer for extraction of MTB-DNA by using silica column. Among all methods GuSCN and Triton-X when used together i.e. 5.25 M GuSCN+ Triton-X was found better as it gave comparatively higher DNA yield than other methods and it also showed maximum sensitivity in Nested-PCR targeting IS6110 plus the higher 260/280 ratio for the detection of MTB-DNA in clinical samples. Other methods like SDS/Lysozme lysis buffer used for extraction of DNA gave higher yield of MTB-DNA but the sensitivity in nested PCR was not that good. Incidence of TB was also studied among Extrapulmonary and Pulmonary TB cases, Pulmonary TB showed higher positivity rate (70.83%) as compare to Extrapulmonary.

References