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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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References

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