嗜水气单胞菌TaqMan实时PCR检测方法的建立

Objective To develop a TaqMan real-time PCR for the detection of aeromonas hydrophila. Methods The conserved region of major adhesion gene of aeromonas hydrophila (aha) was used to design primers and TaqMan probe. A total of six concentration gradients for forward and reverse primers ranging from 200 -700 nmol/L were chosen, and four concentration gradients for probe ranging from I00 -400 nmol/L were chosen. And then the concentration of primers and probe were optimized by ANOVA of completely randomized design respectively. The specificity of the established method was evaluated by using bacteria as contrast, including 45 strains Vibrio cholerae,20 strains Vibrio parahemolyticus, 10 strains Vibrio fluvialis,4 strains Vibrio mimicus,5 strains Vibrio vulnificus, 1 strain Vibrio aiginoayticns, 1 strain Vibrio furnissii, 5 strains Salmonella, 10 strains Shigella and 2 strains Piesiomonas shigelloides. The sensitivity, bacterial sensitivity and DNA sensitivity included,were evaluated. The stool of healthy people was contaminated by aeromonas hydrephila artificially, and the ability of the established TaqMan real-time PCR system for detection of aeromonas hydrophila was also evaluated. Results The cycle threshold (Ct) value deserved from 6 groups of primers concentration gradient was (x±s) :20.69±0.33,20.72±0.21,20.81±0. 12,20.74±0.12,20.51±0. 16 and 20.69±0. 11, respectively, and the concentration of forward primer and reverse primer was determined to be 200 nmol/L (F=1.33, P=0. 28). The Ct value deserved from 4 groups of probe concentration gradient was (x±s) : 20.56±0. 08,20.82±0.05,20. 82±0. 11 and 20.93±0.09,respectively,and the concentration of probe was determined to be 100 nmol/L(F =5.26,P =O. 01 ). The bacterial sensitivity and DNA sensitivity were 80 CFU/ml and 100 fg/μl respectively,and the sensitivity to detect aeromonas hydrophila from stool was 8 × 103 CFU/ml, which might be 8 CFU/ml after 8 hours' enrichment. No amplification was observed in the templates of other bacterial. Conclusion The TaqMan real-time PCR method targeting the aha gene of aeromonas hydrophila had a high sensitivity and specificity and might be used to detect aeromonas hydrophila from pure bacterial and stool rapidly.

Development of TaqMan real-time PCR in detection of aeromonas hydrophila