One-tube multiplex PCR method for rapid identification of mycobacterium tuberculosis.

A rapid, inexpensive, simple, and accurate multiplex polymerase chain reaction (PCR) was developed in a single tube for identification of Mycobacterium tuberculosis. Assessment of sensitivity and specificity of simple PCR was performed with 116 strains of M. tuberculosis complex (MTC) and 144 strains of nontuberculous mycobacteria (NTM) compared with the biochemical method. Specific amplification of KS4, MTC-specific DNA fragment, was found in 98% (114/116) of MTC and not detected in 99% (143/144) of NTM. Amplification of the mtp40 gene revealed 95% sensitivity (100/105 strains of M. tuberculosis) and 77% specificity (not found in 119/155 mycobacterial strains). A multiplex PCR method based on the combination of KS4- and mtp40-derived primers was used for identification of M. tuberculosis. Crude DNA from slow growing mycobacteria with cream rough colonies that showed both 768-bp amplified product for KS4 and 396-bp for mtp40 was identified as M. tuberculosis whereas that from MTC gave only the 768-bp product.

Etiologic agents of cutaneous infectious granuloma: 2 year-experience from granuloma clinic, Siriraj Hospital.

Etiological diagnosis of cutaneous infectious granuloma is difficult to achieve and leads to difficult in patient management. We proposed to search for etiologic agents by special stain of histologic sections, cultural method and PCR technique for tuberculous and non-tuberculous mycobacteria. The skin biopsy specimens were obtained from dermatologic patients who attended Granuloma Clinic, Siriraj Hospital from January 1994 to December 1996. Sixty-nine cases of mixed cell granuloma were found during the 2-year-period. The causative agents had been found in 45 cases (65.22%). Mycobacterial infections were documented in 25 cases. Five cases of tuberculous infection were diagnosed by PCR method. Non-tuberculous mycobacterial infections were diagnosed by cultural method in 14 cases and the other 6 cases were diagnosed by acid-fast stain in histologic section alone. Actinomycotic mycetoma were diagnosed in 4cases. Fungal infections were documented in 16 cases, which caused by hyaline fungi 7 cases and dematiaceous fungi 9 cases. About one-third of all cases (24 cases) the etiologic agent could not be identified.