[EMA-pCR detection of enterohemorrhagic Eschrichia coli O157:H7].

OBJECTIVE: A rapid and effective method with ethidium monoazide bromide (EMA) in combination with PCR (EMA-PCR) was established to detect live Enterohemorrhagic Eschrichia Coli O157:H7. METHODS: The rfbE gene was used as the target gene for PCR detection of Eschrichia Coli O157:H7 by utilizing its pure isolates after the treatment of EMA as the template. The EMA concentration and reaction time was optimized. RESULTS: The use of 10 microg/mL or less EMA did not inhibit the PCR amplification of DNA derived from viable bacteria. The PCR amplification of DNA derived from 2 x 10(7) CFU/mL dead cells can be inhibited by 0.5 microg/mL EMA. The sensitivity of the method was 2 x 10(4) CFU/mL. The results demonstrated that it could detect 1% live bacteria from a mixed bacterial population. CONCLUSION: EMA-PCR can effectively detect live bacteria of O157:H7, it could be a potential rapid detection method applied in public health emergent events.

[Study on rapid detecting of live Pseudomonas aeruginosa by EMA-PCR].

OBJECTIVE: To establish a effective and rapid method by Ethidium Monoazide Bromide(EMA) in combination with PCR(EMA-PCR) for thedetection of live Pseudomrnonas aeruginosa. METHODS: The oprI gene was used as the target gene for PCR detection of Pseudomonas aeruginosa , and PCR amplification was carried out by utilizing its pure isolates as the template. Sensitivity, EMA concentration and exposure time were optimized. RESULTS: The sensitivity of PCR detection was 3 X 10(3) CFU/mIL, exposure time was 10 min. when the EMA concentration was not more than 5 µg/mLI, no obvious inhibition to the amplification of DNA derived from viable bacteria was observed. The PCR amplification of DNA derived from 3 X 10(6) CFU/mL dead cells could be inhibited effectively by EMA at the final concentration of 1 µg/mL. The results demonstrated the establised method could detect 1% live bacteria from a mixed bacterial population. CONCLUSION: EMA-PCR can detect live bacteria of Pseudomonas aeruginosa effectively and avoid false positive result of the PCR detection.

[Establishment and application of molecular beacon real-time PCR in the detection of Mycobacteriun tuberculosis].

OBJECTIVE: To establish a method for detection of M. tuberculosis by molecular beacon real-time PCR and evaluate its application in the clinical detection of M. tuberculosis. METHODS: The primers and molecular probe were designed to M. tuberculosis specific DNA sequence for Ag85B, and constitute a standards. 158 sputum specimens from patients with pulmonary tuberculosis were detected using molecular beacon real-time PCR, acid-fast stain and cultivation, and the detection rates of the three methods were compared. RESULTS: The lowest detection limit of the detected method is 2 genome copies per reaction, the linear standard curve was obtained between 2 x 10(2) and 2 x 10(8) DNA copies/mL. The method detected 5 strains of non-mycobacterium, 6 strains of non-tuberculosis mycobacterium and M. tuberculosis H37Rv, only M. tuberculosis H37Rv had 75 bp specific band. The positive rates of detection using molecular beacon real-time PCR, acid-fast stain and cultivation were 60.13%, 16.46%, 31.01% respectively, the positive rates of detection of real-time PCR was significantly higher than that of acid-fast stain and cultivation. CONCLUSION: Molecular beacon real-time PCR has high sensitivity and specificity in the detection of M. tuberculosis, which is clinical significance for the diagnosis of tuberculosis.