A Study On Rapid And All-purpose Molecular Methods For Detection And Identification Of Mycobacteria From 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. We have primarily developed PCR, multiplex PCR and PCR-RFLP to detect and differentiate the several mycobacterial species that have been reported as pathogens of cutaneous infection. In this study, our mainly research works are (1) how to use PCR to detect mycobacteria in the infectious skin specimen directly with high sensitivity, (2) evaluating the PCR method to dectect and identify mycobacteria in the infectious skin specimen and comparing it with bacterial culture, (3) developing a PCR-microplate reverse hybridization method for the detection and identification of the several mycobacterial species that have been reported as pathogens of cutaneous infection, (4) the traditional and modern methods combines to determine and identify mycobacteria in the infectious skin specimen directly.Chapter Ⅰ : To develop PCR method for detection of mycobacteria in the infectious skin specimen directly, it is most important to improve its sensitivity and reduce the inhibiting factors for PCR. A series of simulant clinical specmen were formed by serial diluted M. smegmatis mixing with homogenized skin specimen. The sensitivity of PCR for serial diluted M. smegmatis with DDW is 1 × 10~2cells/mL. However, the sensitivity of PCR became 1 × 10~4cells/mL , when PCR method was used to detect simulant clinical specmen directly, which showed that there was intensive inhibiting factors for PCR. With the simulant clinical specmens diluted and centrifugation(14,000×g,15min) , the sensitivity became higher gradually and could reach 1×10~3 cells/mL; however, when the simulant clinical specmens were pretreated with SDS and then centrifugation, the sensitivity of PCR became 1 × 10~5 cells/mL. The sensitivity of PCR became 1 × 10~2 ~ 1×10~3 cells/mL , when proteinase K and centrifugation with high speed were used to pretreat the simulant clinical specmens. These results suggested that the preteatment for infectious skin specimen by dilution, centrifugation with high speed and proteinase K might be helpful, when PCR was used to detect mycobacteria in the infectious skin specimen directly. Chapter Ⅱ: To evaluating the PCR method to dectect and identify mycobacteria in the infectious...