Wang Hai-Bo, Wang Duo-Chun, Bi Zhen-Qiang, Kan Biao
Department of Epidemiology and Health Statistics, School of Public Health, Shandong University, Jinan, China.
Zhonghua Yu Fang Yi Xue Za Zhi. 2009 Jul;43(7):611-4.
To develop a TaqMan real-time PCR for the detection of Aeromonas hydrophila.
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 100 - 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 parahaemolyticus, 10 strains Vibrio fluvialis, 4 strains Vibrio mimicus, 5 strains Vibrio vulnificus, 1 strain Vibrio alginolyticus, 1 strain Vibrio furnissii, 5 strains Salmonella, 10 strains Shigella and 2 strains Plesiomonas shigelloides. The sensitivity, bacterial sensitivity and DNA sensitivity included,were evaluated. The stool of healthy people was contaminated by Aeromonas hydrophila artificially, and the ability of the established TaqMan real-time PCR system for detection of Aeromonas hydrophila was also evaluated.
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 = 0.01). The bacterial sensitivity and DNA sensitivity were 80 CFU/ml and 100 fg/microl respectively, and the sensitivity to detect Aeromonas hydrophila from stool was 8 x 10(3) CFU/ml, which might be 8 CFU/ml after 8 hours' enrichment. No amplification was observed in the templates of other bacterial.
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.
建立一种用于检测嗜水气单胞菌的TaqMan实时荧光定量PCR方法。
以嗜水气单胞菌主要黏附基因（aha）的保守区设计引物和TaqMan探针。正向引物和反向引物各选取6个浓度梯度，范围为200 - 700 nmol/L，探针选取4个浓度梯度，范围为100 - 400 nmol/L。然后分别采用完全随机设计的方差分析对引物和探针浓度进行优化。以霍乱弧菌45株、副溶血性弧菌20株、河弧菌10株、拟态弧菌4株、创伤弧菌5株、溶藻弧菌1株、弗尼斯弧菌1株、沙门菌5株、志贺菌10株、类志贺邻单胞菌2株作为对照，评价所建立方法的特异性。评价该方法的灵敏度，包括细菌灵敏度和DNA灵敏度。将嗜水气单胞菌人工污染健康人的粪便，评价所建立的TaqMan实时荧光定量PCR系统检测嗜水气单胞菌的能力。
6组引物浓度梯度对应的循环阈值（Ct）值（x±s）分别为：20.69±0.33、20.72±0.21、20.81±0.12、20.74±0.12、20.51±0.16和20.69±0.11，确定正向引物和反向引物浓度为200 nmol/L（F = 1.33，P = 0.28）。4组探针浓度梯度对应的Ct值（x±s）分别为：20.56±0.08、20.82±0.05、20.82±0.11和20.93±0.09，确定探针浓度为100 nmol/L（F = 5.26，P = 0.01）。细菌灵敏度和DNA灵敏度分别为80 CFU/ml和100 fg/μl，从粪便中检测嗜水气单胞菌的灵敏度为8×10³CFU/ml，富集8小时后可能为8 CFU/ml。其他细菌模板未观察到扩增。
靶向嗜水气单胞菌aha基因的TaqMan实时荧光定量PCR方法具有较高的灵敏度和特异性，可用于快速检测纯菌及粪便中的嗜水气单胞菌。
