Single-tube nested PCR in the diagnosis of tuberculosis

C M Chan, K Y Yuen, K S Chan, W C Yam, K H Yim, W F Ng, M H Ng
1996 Journal of Clinical Pathology  
Aims-To evaluate the usefulness of a single-tube nested polymerase chain reaction (PCR) assay in the diagnosis of tuberculosis in 1497 pulmonary and 536 extrapulmonary specimens. Methods-A single-tube nested PCR, utilising two sets of primers with different melting temperatures (88'C for external primers; 70°C for internal primers) to augment sensitivity and specificity without increasing the risk of amplicon contamination, was evaluated. Specimens were initially tested for the repetitive
more » ... sequences and if negative, retested for the universal 38 kilodalton sequence and for inhibitors. dUTP/Uracil-N-glycosylase and Instagene treatment were used to minimise contamination and the effect of inhibitors, respectively. Results-Using culture as the gold standard, the overall sensitivity of the assay was 89% for pulmonary and 42% for extrapulmonary specimens. Sensitivity varied greatly with respect to sample type (92% for follow up specimens from a chest hospital and 70% for non-follow up specimens from a general hospital). The smear positivity rates were 15% for extrapulmonary specimens, and 69% and 45%, respectively, SPECIMEN PROCESSING Pulmonary specimens All specimens were collected in gamma irradiated disposable plastic bottles. After a
doi:10.1136/jcp.49.4.290 pmid:8655703 pmcid:PMC500452 fatcat:njytqodkzreufdozpkz7kejmwu