快速荧光定量PCR检测副溶血性弧菌及其毒力基因方法建立与应用效果评价的研究

Rapid fluorescent PCR detection of Vibrio parahaemolyticus and ways of its virulence genes setting up and evaluation of application effect

目的:建立一种快速、特异、灵敏、定量检测副溶血性弧菌及其毒力基因的方法,用于副溶血弧菌性食物中毒的应急检测。方法:根据GenBank中副溶菌(AY-289609)全基因序列上不耐热溶血素基因(tlh)、耐热直接溶血素基因(tdh)、耐热相关溶血素基因(trh)为靶序列,设计引物与Tagman探针并对模板DNA制备方法和PCR反应时间进行改进与优化以提高检测速度,采集食物中毒样本与国家标准方法进行现场平行比对。结果:本方法检测时间仅需30 min。检出限2×101cfu/ml,定量线性范围2×103cfu/ml^2×108cfu/ml,对77份患者粪便增菌前后平行检测结果显示该方法检出率明显高于国家标准方法并具有统计学意义(χ2=58.80,P<0.01;χ2=42.98,P<0.01)。与7种常见肠道细菌均无反应。对6个浓度的副溶血弧菌测定的批内标准差在0.10~0.56之间。结论:该方法可在30 min内完成粪便中副溶血性弧菌的tlh、tdh、trh的检测,具有灵敏度高、特异性好、结果稳定、抗杂菌与药物干扰能力强等优点。非常适合食物中毒的应急检测。

Objective： To establish a rapid,specific,sensitive and quantitative method for detection of Vibrio parahaemolyticus and its virulence genes,for emergency detection of Vibrio parahaemolyticus of food poisoning.Methods：According to GenBank gene sequences of Vibrio parahaemolyticus（AY-289609） of non-thermolabile hemolysin（tlh）,thermostable direct hemolysin（tdh）,thermostable related hemolysin（trh） for the target sequence,design primers and Tagman probe,improve and optimize DNA template preparation method and PCR reaction time to improve the speed,collecting samples of food poisoning,and parallel compared with the national standards.Results：This method takes only 30 minutes,the limit of detection was 2×10^1 cfu/ml;the quantitative linear range of 2×10^3 cfu/ml2×10^8 cfu/ml,parallel detect before and after amplified bacteria of 77 patients′stool,results show that the detection rate higher than the national standard methods and with statistical significance（χ^2=58.80,P〈0.01;χ^2=42.98,P〈0.01）.And non-specific response to seven kinds of common bacterial,the standard deviation between 0.100.56 to detect six kinds of concentration of Vibrio parahaemolyticus.Conclusion：This method can be completed within 30 minutes in the stool of Vibrio parahaemolyticus tlh,tdh,trh detection with high sensitivity and good specificity,resulting in stable,anti-bacteria and drug interference and so on advantages.It is suitable for detection of food poisoning emergency.