A Study On Rapid Methods For Detection And Identification Of Mycobacteria From The Clinical Cutaneous Specimens And Their Applications

With the growing number of patients with AIDS and other immunosuppressive diseases, the incidence of mycobacterial infection has been increased. Mycobacteria can cause both systemic symptoms and cutaneous infection. Because of the nonspecific clinical and pathological presentation, the diagnosis of cutaneous mycobacterial infection mainly depends on detection of the organism. Direct microscopy on the lesional skin has lower sensitivity, while cultivation and biochemical tests of mycobacteria usually take a long time. So it is difficult for clinicians to acquire valid information immediately according to these conventional procedures. Rapid, sensitive and specific methods are essential for detection and identification of mycobacteria to the species level. Molecular techniques based on PCR make it possible for rapid and accurate diagnosis of mycobacterial infection. In this article, we developed PCR, multiplex PCR and PCR-RFLP to detect and differentiate the mycobacterial species that have been reported as pathogens of cutaneous infection. Three clinical isolates were identified from the results of morphological and biochemical characterization, PCR-RFLP and hsp65 gene sequencing. Chapter Ⅰ : Developing PCR for detection of five common mycobacterial species. First, serial diluted mycobacterial DNA of the five species were amplified respectively with their specific primers to testify the sensitivity of PCR for mycobacterial detection. Second, DNA of other 17 mycobacterial reference species were amplified individually with the above five pairs of primers to dectect the specificity of PCR. Third, detection of M. avium in imitated clinical cutaneous specimens was explored. The sensitivity of PCR for detection of M. avium, M. intracellulare, M. kansasii, M. tuberculosis was 1 × 10~2cells/mL, while for M. ulcerans, it was 1 × 10~0 cells/mL. The mycobacterial DNA could only be amplied with their own primers, and no products were found after other 17 species of mycobacterial DNA were amplified with any pair of these primers. The low limitation of PCR became 1 × 10~4 cells/mL when M. avium was mixed with homogenized skin specimen, which showed that there was intensive inhibiting factors for PCR. With the homogenized tissue diluted, the sensitivity became higher gradually. This result suggested that dilution and centrifugation with high speed to deposit the oranism in homogenized tissue might be helpful when PCR was used to detect mycobacteria in the infectious skin specimen directly.