嗜水气单胞菌TaqMan实时PCR检测方法的建立

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

目的建立嗜水气单胞菌TaqMan实时PCR实验室检测方法。方法根据嗜水气单胞菌主要黏附素基因（aeromonas hydrophila major adhesion gene，aha）的保守序列设计引物和TaqMan探针。引物选取200～700nmol／L6个浓度梯度，探针选取100～400nmol／L4个浓度梯度，以完全随机设计资料的方差分析分别优化引物和探针的浓度。以45株霍乱弧菌、20株副溶血弧菌、10株河流弧菌、4株拟态弧菌、5株创伤弧菌、1株溶藻弧菌、1株弗尼斯弧菌、5株沙门菌属细菌、10株志贺菌属细菌和2株类志贸邻单胞菌为对照，评价该方法的特异性。对所建立的方法进行菌液灵敏度检测和DNA灵敏度检测，并将嗜水气单胞菌人工污染健康人的粪便，实验室内评价该方法从粪便中检测嗜水气单胞菌的能力。结果6组引物浓度所得的循环阈值（cycle threshold，Ct）分别为（x^- ±s）：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，选择上下游引物的浓度为200nmol／L（F=1．33，P=0．28），4组探针浓度所得的Ct值分别为（x^- ±s）：20．56±0．08、20．82±0．05、20．82±0．11和20．93±0．09，选择探针的浓度为100nmol／L（F=5．26，P=0．01）。该方法DNA的检测下限为100fg／μl，纯菌液的检测下限为80CFU／ml，粪便中嗜水气单胞菌的检测下限为8×10^3CFU／ml，人工粪便标本增菌8h后的检测下限为8CFU／ml。该方法对其他细菌的染色体无扩增。结论以aha基因为目标检测片段建立的嗜水气单胞菌实时PCR方法灵敏度高、特异度强，可用于纯菌和粪便标本中嗜水气单胞菌的快速检测。

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 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 parahemolyticus, 10 strains Vibrio fluvialis,4 strains Vibrio mimicus,5 strains Vibrio vulnificus, 1 strain Vibrio alginoayticus, 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. 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 = 0.01 ）. 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 ×10^3 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.