胞内劳森菌实时荧光RPA方法的建立及初步应用

Development and preliminary application of a real-time recombinase polymerase amplification assay for rapid detection of Lawsonia intracellularis

本研究基于胞内劳森菌(Lawsonia intracellularis)16S rRNA基因保守序列,设计特异性引物和exo探针,建立了一种能够快速检测胞内劳森菌的实时荧光重组酶聚合酶扩增方法(real-time RPA)。进一步对real-time RPA方法的特异性和灵敏性进行了分析,对临床疑似病例的猪粪便和回肠样品进行了检测,并同real-time LAMP和real-time PCR检测结果进行了比较。结果表明,该方法能够在39℃等温条件下,20 min内实现对胞内劳森菌的有效检测;所建立的方法特异性强,仅对L.intracellularis产生特异性扩增,对其他常见引起猪腹泻的病原均无扩增;灵敏性高,以L.intracellularis基因组DNA为模板,检出限为1.4×10^2fg/μL;在60份临床样品中,该方法对L.intracellularis的检出率为66.7%(40/60),同real-time LAMP和real-time PCR方法一致。本研究所建立的real-time RPA方法反应快速、特异性强、灵敏性高,可用于猪场胞内劳森菌的快速检测。

In this study,the specific primers and exo probe were designed based on the 16 S ribosomal RNA gene of Lawsonia intracellularis,a real-time recombinase polymerase amplificatio(real-time RPA)assay was developed for the rapid detection of L.intracellularis.Then the analytical specificity and analytical sensitivity analysis of the real-time RPA assay were performed.Furthermore,the assay was applied in the detection of L.intracellularis in the swine feces and ileum samples,and the results were compared to those of the real-time LAMP and real-time PCR.The results demonstrated that the developed real-time RPA could be performed successfully at 39℃for 20 min.The assay was highly specific for L.intracellularis,as there were no cross-reactions with other tested pathogens,which were commonly identified in the swine with diarrhea.Using the L.intracellularis genomic DNA as the template,the limit of detection of the real-time RPA was 1.4×10^2 fg/μL.The developed assay was further evaluated on 60 clinical samples,and the L.intracellularis positive rate was 66.7%(40/60)in the real-time RPA,which was the same as those of the real-time LAMP and real-time PCR assays.The developed real-time RPA assay was rapid,specific and sensitive,which could be used in the rapid detection of L.intracellularis in pig herds.