摘要
目的为实验动物微生物检测提供一种简便、灵敏、快速,并且特异性强的小肠结肠炎耶尔森氏菌、假结核耶尔森氏菌、志贺氏菌和空肠弯曲菌多重PCR检测方法。方法根据小肠结肠炎耶尔森氏菌fox基因、假结核耶尔森氏菌inv基因、志贺氏菌ipa H基因及空肠弯曲菌gyr A基因设计并筛选出特异性引物;优化退火温度等条件,分析多重PCR的特异性、敏感性,使用该多重PCR方法检测小鼠和豚鼠样品。结果多重PCR扩增出了预计的PCR产物,即小肠结肠炎耶尔森氏菌(511 bp)、假结核耶尔森氏菌(779 bp)、志贺氏菌(290 bp)和空肠弯曲菌(156 bp)。该方法的灵敏度为1×10-3ng/μL(小肠结肠炎耶尔森氏菌、假结核耶尔森氏菌、志贺氏菌)和1×10-2ng/μL(空肠弯曲菌)。特异性检测未从其他菌中检测出阳性条带。使用该方法从样品中检测出不等的阳性样品数。结论该多重PCR方法对实验动物微生物检测具有很好的应用和开发前景。
Objective To establish a simple,sensitive,rapid,and the specificity multiplex PCR method for Yersinia bacteria, Yersinia pseudotuberculosis, Shigella and Campylobacter jejuni from laboratory animals.Method According to the fox gene of enterocolitis Yersinia and inv of Yersinia pseudotuberculosis,ipa H gene of Shigella and gyr A gene of Campylobacter jejuni,we designed and screened a pair of specific primers. The annealing temperature and other conditions were optimized,then the specificity and sensitivity of The Multiplex PCR were analysis. At last,the multiplex PCR method was use to detect the samples from mouse and guinea pig. Result The multiplex PCR amplified the expected PCR product, namely Yersinia enterocolitis（ 511bp）, Yersinia pseudotuberculosis（ 779 bp）,Shigella（ 290 bp） and Campylobacter jejuni（ 156 bp）. The sensitivity of this method is 1 × 10- 3ng / μL（ Yersinia bacteria,Yersinia pseudotuberculosis,Shigella） and 1 × 10- 2ng / μL（ C. jejuni）. No specific positive was detected from the other samples. Conclusion The multiplex PCR method has good application and development prospects in detection of microorganisms in laboratory animals.
出处
《实验动物科学》
2016年第1期1-6,共6页
Laboratory Animal Science
基金
国家科技支撑计划(No.2015BAI07B02)
