Evaluation of Nested Polymerase Chain Reaction targeting hsp65 of Mycobacterium tuberculosis for the Detection of Organism in the Sputum Samples
Yogaraje GC Varadaiah, Raghu K Chinnappa, Rakshitha M Nagaraj, Thejashwini NA, Robinson K Samuel, Devananda Devegowda
Citation Information :
Varadaiah YG, Chinnappa RK, Nagaraj RM, NA T, Samuel RK, Devegowda D. Evaluation of Nested Polymerase Chain Reaction targeting hsp65 of Mycobacterium tuberculosis for the Detection of Organism in the Sputum Samples. Indian J Med Biochem 2016; 20 (1):28-33.
The poor sensitivity of conventional smear microscopy and the delay in obtaining Mycobacterium culture results prevent the early diagnosis of Myobacterium tuberculosis (MTB). By using nucleic acid amplification techniques like polymerase chain reaction (PCR), one may be able to diagnose the disease on the day of arrival of specimen in the laboratory. The present study aimed to evaluate the applicability of the nested-PCR (nPCR) technique as a rapid and direct molecular method for the diagnosis of M. tuberculosis in sputum specimens of patients whose sputum smear was acid-fast bacilli (AFB) negative using heat shock protein (hsp65) as the gene target.
Materials and methods
Early morning sputum samples were collected in sterile containers respectively from about 40 suspected patients of pulmonary tuberculosis, attending the outpatient units of JSS Medical College and PKTB Hospital, Mysore and from 20 age and sex-matched healthy controls. Sputum samples were decontaminated by modified Petroff's method and DNA was isolated using QIAGEN DNA extraction kit. The nPCR was carried out for the detection of MTB using the target gene hsp65.
Nested-PCR showed specific amplification (165bp) of M. tuberculosis in 18 out of 20 sputum AFB positive samples and 9 out of 20 AFB negative samples. None of the healthy controls showed any amplification with nPCR. The nPCR when compared to that of Ziehl-Neelsen staining had a sensitivity of 90%, specificity of 77.5%, positive predictive value (PPV) of 66.6%, and negative predictive value (NPV) of 93.9%. The percentage of false positive was 33.3% and percentage of false negative was 6.1%.
The detection of M. tuberculosis with nPCR in smear negative patients provides the bacteriological data 4 to 8 weeks earlier. A molecular approach, based on the amplification of hsp65 gene by nPCR, showed that there is high probability of the disease being absent when the test is negative because of the high negative predictive value (NPV).
How to cite this article
Varadaiah YGC, Prashant A, Chinnappa RK, Nagaraj RM, Thejashwini, Samuel RK, Devegowda D, Vishwanath P. Evaluation of Nested Polymerase Chain Reaction targeting hsp65 of Mycobacterium tuberculosis for the Detection of Organism in the Sputum Samples. Indian J Med Biochem 2016;1(1):28-33.
Available from: http://www.who.int/tb/publications/global report/en/index.html.
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