摘要
【目的】建立检测胞内劳森菌的SYBR GreenⅠreal-time PCR方法,为猪增生性肠炎的准确诊断奠定基础。【方法】针对胞内劳森菌16SrDNA序列设计引物,扩增16SrDNA,构建pT-LI-16S重组质粒。以pT-LI-16S为模板建立检测胞内劳森菌的SYBR GreenⅠreal-time PCR方法,检测其特异性、敏感性和重复性,并用该方法对51份疑似增生性肠炎病例进行检测。【结果】建立的SYBR GreenⅠreal-time PCR方法特异性强,与大肠杆菌、沙门氏菌、金黄色葡萄球菌和副猪嗜血杆菌等无交叉反应;在标准质粒含量为1.0×10^2-1.0×10^8拷贝/μL时,质粒含量与循环阈值（Ct）之间具有良好的线性关系（R^2=0.992）,最小可检测到10拷贝/μL的重组质粒;重复性检测显示其批内变异系数小于2%。该方法对粪便及小肠组织中胞内劳森菌的检出率分别为46.9%和84.2%,高于普通PCR的检出率（分别为40.7%和78.9%）。【结论】建立的SYBR GreenⅠreal-time PCR方法特异性强、敏感性高、重复性好,能对胞内劳森菌进行快速检测及定量分析,可用于猪增生性肠炎的诊断。
【Objective】The present study established a SYBR GreenⅠreal-time PCR assay for detection of Lawsonia intracellularis.【Method】16SrDNA gene of L.intracellularis,amplified by PCR using specific primers was cloned to construct recombinant plasmid pT-LI-16 S.With the plasmid pT-LI-16 Sas template,the real-time PCR assay for detection of L.intracellularis was established and its specificity,sensitivity and repeatability were evaluated.The method was also applied to 51 clinical samples of porcine proliferate enteritis.【Result】The established assay had no cross reaction with E.coli,Salmonella,Staphylococcus aureus and Haemophilus parasuis.There was a good linear relationship between the template concentration and the Ct value when the standard template concentration was in range of 1.0×10^2-1.0×10^8copies/μL.The detection limit was 10copies/μL recombinant plasmid.The repeatability test indicated that the intra-variations were less than 2%.The positive rates of L.intracellularis in fecal and intestine tissue were 46.9% and 84.2%,respectively,both were higher than the rates（40.7%and 78.9%）tested by con-ventional PCR.【Conclusion】The established real-time PCR assay was specific,sensitive,accurate and suitable for the quick detection and quantity analysis of L.intracellularis.Thus,it can be used to diagnose porcine proliferative enteritis.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2015年第12期29-34,共6页
Journal of Northwest A&F University(Natural Science Edition)
基金
福建省教育厅A类项目(JA11247)
福建省农业科技重点项目(2011N0025)
关键词
胞内劳森菌
荧光定量PCR
猪增生性肠炎
检测方法
Lawsonia intracellularis
real-time PCR
porcine proliferative enteritis
diagnosis
