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Gupta, Shaifali
- 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, IN
1 Kilpest India Limited/ Dygnogene 7C Industrial Area, Govindpura, Bhopal, M.P, IN
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Indian Journal of Biotechnology & Biochemistry, Vol 1, No 1 (2013), Pagination: 57-62Abstract
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
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- Comparative Study of ZN Staining, Culture and Nested PCR in Detection of Mycobacterium Tuberculosis from Patients Suspected to Genitourinary Tuberculosis
Abstract Views :597 |
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Authors
Affiliations
1 Kilpest India Limited/Dygnogene 7C Industrial Area, Govindpura, Bhopal, M.P, IN
1 Kilpest India Limited/Dygnogene 7C Industrial Area, Govindpura, Bhopal, M.P, IN
Source
Indian Journal of Biotechnology & Biochemistry, Vol 1, No 1 (2013), Pagination: 81-86Abstract
The aim of the present study was to evaluate the diagnostic value of nested PCR in genitourinary tuberculosis (GUTB) compared with acid fast staining and culture method. In total 200 different types of clinical samples from suspected cases of GUTB were collected during the period of study. After NALC Treatment pellets were used for smear preparation, culture and DNA extraction was done by using Nucleo spin kit. Nested PCR was performed according to standard protocol using primers based on IS6110 gene fragment. The results obtained by PCR were compared with those obtained by standard acid-fast bacilli stain and culture method. Based on obtained results, the positivity rate of samples in this study was 17.5% and 22.5% by using culture and PCR methods respectively and 7.5% for acid fast staining. Fifteen out of total samples showed positive results in all three methods (7.5%). The sensitivity of PCR in this study was estimated as 90% which was higher than that of culture 70%, while the sensitivity for direct smear staining was 30%. In conclusion, the obtained rate of GUTB in our study was 25%. Since the detection rate of culture was much lower than nested PCR, we could suggest PCR as a rapid and sensitive test for early diagnosis of genitourinary tuberculosis and has great utility in providing diagnosis in clinically relevant time.References
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