Journal
DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE
Volume 48, Issue 4, Pages 271-275Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.diagmicrobio.2003.11.006
Keywords
Mycobacterium tuberculosis; polymerase chain reaction; enzyme-linked immunoassay
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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 Lit 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. (C) 2004 Elsevier Inc. All rights reserved.
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