TY - JOUR
T1 - Direct detection of Mycobacterium tuberculosis in clinical specimens using single-tube biotinylated nested polymerase chain reaction-enzyme linked immunoassay (PCR-ELISA)
AU - Yam, W. C.
AU - Cheng, V. C.C.
AU - Hui, W. T.
AU - Wang, L. N.
AU - Seto, W. H.
AU - Yuen, K. Y.
PY - 2004/4
Y1 - 2004/4
N2 - A biotinylated single-tube nested polymerase chain reaction (PCR) assay with microwell hybridization assay (bPCR-ELISA) was developed for detection of Mycobacterium tuberculosis in clinical specimens. A total of 659 specimens (601 respiratory specimens and 58 nonrespiratory specimens) were collected for evaluation using three DNA amplification techniques: newly designed bPCR-ELISA, in-house single-tube nested PCR for IS6110 gene sequence (nPCR), and commercial automated assays, the Cobas Amplicor System from Roche Diagnostic Systems (aPCR). Sixty-four (9.7%) specimens were culture-positive for M. tuberculosis. Eleven (1.7%) specimens culture-positive for nontuberculosis mycobacteria were negative by all three PCR assays. The resolved performance of bPCR-ELISA, nPCR, and aPCR was found at sensitivities of 97%, 94%, and 97%, respectively. All three PCR assays exhibited a 100% specificity. In evaluation of bPCR-ELISA, a clear distinction between PCR-positive and PCR-negative specimens when an OD405 value of 0.6 was chosen as cut-off. With serial dilutions of M. tuberculosis H37Rv DNA, the detection limit of bPCR-ELISA was found to be 0.75 cfu per reaction at OD405 value of 0.6. Our developed bPCR-ELISA provides a highly sensitive and low-costing molecular diagnosis suitable for developing countries with high prevalence of tuberculosis.
AB - A biotinylated single-tube nested polymerase chain reaction (PCR) assay with microwell hybridization assay (bPCR-ELISA) was developed for detection of Mycobacterium tuberculosis in clinical specimens. A total of 659 specimens (601 respiratory specimens and 58 nonrespiratory specimens) were collected for evaluation using three DNA amplification techniques: newly designed bPCR-ELISA, in-house single-tube nested PCR for IS6110 gene sequence (nPCR), and commercial automated assays, the Cobas Amplicor System from Roche Diagnostic Systems (aPCR). Sixty-four (9.7%) specimens were culture-positive for M. tuberculosis. Eleven (1.7%) specimens culture-positive for nontuberculosis mycobacteria were negative by all three PCR assays. The resolved performance of bPCR-ELISA, nPCR, and aPCR was found at sensitivities of 97%, 94%, and 97%, respectively. All three PCR assays exhibited a 100% specificity. In evaluation of bPCR-ELISA, a clear distinction between PCR-positive and PCR-negative specimens when an OD405 value of 0.6 was chosen as cut-off. With serial dilutions of M. tuberculosis H37Rv DNA, the detection limit of bPCR-ELISA was found to be 0.75 cfu per reaction at OD405 value of 0.6. Our developed bPCR-ELISA provides a highly sensitive and low-costing molecular diagnosis suitable for developing countries with high prevalence of tuberculosis.
KW - Enzyme-linked immunoassay
KW - Mycobacterium tuberculosis
KW - Polymerase chain reaction
UR - http://www.scopus.com/inward/record.url?scp=1942488391&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=1942488391&partnerID=8YFLogxK
U2 - 10.1016/j.diagmicrobio.2003.11.006
DO - 10.1016/j.diagmicrobio.2003.11.006
M3 - Article
C2 - 15062920
AN - SCOPUS:1942488391
SN - 0732-8893
VL - 48
SP - 271
EP - 275
JO - Diagnostic Microbiology and Infectious Disease
JF - Diagnostic Microbiology and Infectious Disease
IS - 4
ER -