摘要
目的利用荧光定量PCR技术,研制适合各层次实验室使用的,快速、准确地检测鉴定食品中副溶血弧菌污染的定性、定量方法。方法根据副溶血弧菌gyrase基因序列,设计并合成特异性引物和荧光标记探针,将扩增后的特异片断制成阳性模板,绘制标准曲线。以副溶血弧菌菌株8株及同源、非同源参考菌株24株,做特异性检测;将阳性污染菌按梯度加入阴性样品为模拟样本,做敏感性检测。同时使用PE7000型定量PCR仪和国产DA620微量荧光检测仪检测。结果该方法有良好的特异性:8株副溶血弧菌结果均为阳性,而其它24株菌株均为阴性(其中8株是弧菌科,16株分别来自不同的菌属);该方法有良好的敏感性:阳性模板浓度与定量曲线循环阈值之间相关系数达到0.9871。在纯培养的条件下,其检测低限为10cfuml。对32个模拟样本直接进行定量检测,则检测低限在102cfug;经过24小时增菌培养,检测低限可达2cfug。结论该方法特异性强、敏感性高,操作简便快速,能避免常规PCR方法易污染的缺陷,在国产仪器上测试同样取得较好的敏感性和特异性,便于基层实验室使用,具有很好的推广应用前景。
Objective\ To establish a rapid and accurate qualitative and quantitative method to detect Vibrio Parahaemolyticus in food. Methods Primers and Taqman probe were designed according to the sequence of gyrase gene of Vibrio Parahaemolyticus. The PCR fragment was cloned into PTM vector and was used as positive template for establishing the criterion curve. The simulated samples, made from negative food samples with Vibrio Parahaemolyticus positive strain, were used to evaluate the sensitivity of the PCR reaction. 8 Vibrio Parahaemolyticus standard strains and other 24 negative strains ~8 strains were Vibrionaceae other than Vibrio Parahaemolyticus, the rest 16 strains were none-Vibrionaceae) were treated in the same way to evaluate the specificity. All samples were detected with PE7000 sequence detection system and DA620 fluorescene detection system. Results FQ-PCR method had good specificity and high sensitivity. All 8 strains of Vibrio Parahaemolyticus tested showed positive results while all other 24 strains were negative ~8 strains were Vibrionaceae, 16 strain were from different). The correlation was 0.9871 between the concentration of positive template and the quantitative curve circular threshold. The threshold for detecting Vibrio Parahaemolyticus in pure culture is 10cfu/ml, and the threshold is as low as 2cfu/ml with 16 simulative samples after these samples were incubated for a period of time. By direct quantitative detection for uncultured 16 food samples, the threshold is 100cfu/g. Conclusion FQ-PCR method has high sensitivity and specificity, and it is a handy and rapid detection method. Comparing with regular PCR method, it is not easily contaminated in operation, and can achieve high sensitivity and specificity with domestic instruments. FQ-PCR method has potential and applied value for detection of pathogenic bacteria in food.
出处
《卫生研究》
CAS
CSCD
北大核心
2005年第4期457-460,共4页
Journal of Hygiene Research
基金
广东省科技攻关基金资助项目(No.2002C1041002)
关键词
荧光定量PCR副溶血弧菌
定量检测试剂盒
fluorescent-quantitative PCR, Vibrio Parahaemolyticus, quantitative detection kit
