胞内劳森菌的纳米PCR检测方法的建立
The nano-PCR assay establishment for detection of Lawsonia intracellularis
白云 1郑英帅 2左奕 1孙继国1
作者信息
- 1. 河北农业大学动物医学院,河北保定071001
- 2. 天津市动物卫生监督所,天津300211
- 折叠
摘要
为建立一种猪增生性肠炎(PPE)的快速诊断方法,本研究根据GenBank中已登录的胞内劳森菌(LI)基因组16S rDNA基因序列,设计1对引物,以疑似PPE的病料样品,通过PK-15细胞培养,分离到一株LI,建立了LI的纳米金PCR检测方法.结果表明该方法能够扩增得到与实验设计相符的202 bp (LI)的特异性片段;与大肠杆菌、沙门氏菌、流行性腹泻病毒、猪传染性胃肠炎病毒、猪轮状病毒无交叉反应,特异性良好.与普通PCR法进行比较,该方法检测灵敏度比普通PCR高100倍.经临床检测,从50份病料样品中检出5份阳性样品,并且发现回肠粘膜和粪便中LI的检出率明显高于组织中LI的检出率.该方法可以用于LI的临床快速检测与流行病学调查.本研究为进一步研究LI的人工培养和实验室诊断PPE奠定了基础.
Abstract
The objective of the present study was to develop a nanogold-assisted PCR assay for detecting Lawsonia intracellularis.One pair of specific primers were designed according to the 16S rDNA sequence of L.intracellularis available in the GenBank by primer 5.0.The sensitivity and specialty of the method was optimized.One specific fragment of 202 bp was amplified from genomic DNA of L.intracellularis by the nanogold-assisted PCR,but no any amplification was found from the related pathogens,such as E.coli,Salmonella,porcine epidemic diarrhea virus,Transmissible gastroenteritis virus,porcine rotavirus.In addition,the nano-PCR was 100 time sensitive than that of traditional PCR.The method provides a fast and reliable way for applification in clinical detection of the L.intracellularis infection and diagnosis of porcine proliferative enteropenthy in pigs.
关键词
胞内劳森菌/猪增生性肠炎/分离/检测/纳米金PCRKey words
Lawsonia intracellularis/porcine proliferative enteropenthy/detection/Nano-PCR引用本文复制引用
出版年
2016
NETL
NSTL
维普
万方数据