摘要
目的:建立快速检测致病性小肠结肠炎耶尔森菌的多重PCR方法。方法:利用小肠结肠炎耶尔森菌的ail毒力基因以及该菌属的保守序列16 s rRNA基因,设计针对致病性小肠结肠炎耶尔森菌的PCR引物,进行多重PCR扩增。并对退火温度、Mg2+浓度以及引物浓度等扩增条件进行优化。结果:研究证实本次多重PCR用于检测致病性小肠结肠炎耶尔森菌具有较高的特异性(100%)和敏感性(检测下限为102cfu/ml)。经过多次实验比较,PCR反应的退火温度为62℃,Mg2+浓度为2.5 mmol/L,引物A il和Yer浓度分别为200 nmol/L、100 nmol/L。结论:多重PCR较传统鉴定方法省时省力,对于监测小肠结肠炎耶尔森菌有重要意义。
Objective：To develop a method for detection of pathogenic Yersinia enterocolitica by multiplex PCR. Methods： Characteristic genes of Yersinia enterocolitica such as 16s rRNA and all were chosen as the target genes for multiplex PCR system. Several amplifying conditions such as annealing temperature, the concentration of Mg^2＋ and primers were optimized. Results ： The study demonstrated that high specificity （ 100% ） and sensitivity（ with the detection limit at 10^2 efu/ml） were aehieved for detection of pathogenic Yersinia enterocolitica by multiplex PCR. After trials and errors, the optimized conditions for multiplex PCR Mg^2＋ were determined,with annealing temperature at 62℃, the concentration of Mg , primer Ail and Yer at 2.5 mmol/L,200 nmol/L and 100 nmoL/L, respectively. Conclusion： Being time and labor - saving, the multiplex method has superiority over conventional identification methods and can be effectively employed in surveillance of Yersinia enterocolitica.
出处
《中国卫生检验杂志》
CAS
2009年第4期740-741,760,共3页
Chinese Journal of Health Laboratory Technology
基金
广州市医药卫生科技项目(2006YB129)
广州市卫生局医药卫生科技资助项目(2006-Zdi-11)
