基于Hsp70基因的绵羊肺炎支原体TaqMan检测方法的建立及其遗传演化分析

Establishment of a TaqMan Assay for Mycoplasma ovipneumoniae based on the Hsp 70 Gene and Analysis of Its Genetic Evolution

热休克蛋白70 (heat shock protein 70,Hsp70)是绵羊肺炎支原体(Mycoplasma ovipneumoniae, Movi)的重要膜蛋白,是机体内高度保守的生物分子,但在种间差异大,可作为分子生物学检测的候选靶区。为建立基于Hsp70基因的Movi通用型TaqMan实时荧光定量PCR(qPCR)检测方法,并进一步了解其遗传变异情况,本研究基于GenBank中Movi的Hsp70基因特征,设计特异性的引物及探针,建立了基于Hsp70基因的绵羊肺炎支原体qPCR检测方法。应用建立的检测方法对88份山羊鼻拭子样品及43份疑似羊支原体性肺炎(Mycoplasmal pneumonia of sheep and goats, MPSG)病料进行检测。将检测结果为Movi阳性的肺组织样品进行分离鉴定,并对分离株Hsp70基因进行序列分析。结果显示,建立的qPCR检测方法其相关系数为1.00,扩增效率为96.0%,斜率为-3.411,Y轴截距为37.29。特异性强,与丝状支原体山羊亚种(Mycoplasma mycoides subsp.capri, Mmc)、山羊支原体山羊肺炎亚种(Mycoplasma capricolum subsp.capripneumoniae, Mccp)、莱氏无胆甾原体(Acholeplasmalaidlawii, AL)、无乳支原体(Mycoplasma agalactiae, Maga)、伪结核棒状杆菌(Corynebacterium pseudotuberculosis, CP)、羊口疮病毒(orf virus, ORFV)、牛支原体(Mycoplasma bovis, Mb)等牛羊常见病原均无交叉反应;敏感性高,最低检测限为5.72 copies·μL^(-1);重复性优,组内变异系数和组间变异系数均小于1.00%。6株Movi分离株Hsp70基因全长均为1 818 bp,与其它Movi参考株核苷酸和氨基酸同源性分别为96.0%~99.4%和98.0%~100.0%;进一步分析发现,山羊源Movi均比绵羊源多1个N-糖基化位点。遗传演化分析表明,其均处于ClusterⅠA亚分支(均为山羊源)。综上,本研究建立了特异性强、敏感性高、重复性优的基于Hsp70基因的Movi的qPCR检测方法。序列分析发现,不同来源Movi的Hsp70基因核苷酸同源性高;遗传演化分析证实,Movi福建株与山羊源分离株遗传关系较近。本研究不仅为Movi临床诊断提供了技术支持,更为进一步了解Movi遗传演化规律提供参考。

Heat shock protein 70(Hsp70)is an important membrane protein of Mycoplasma ovipneumoniae(Movi).It is highly conserved in the body but varies greatly in species and can be used as a candidate target gene for molecular biological detection.The purpose of this research is to establish a universal TaqMan real-time fluorescence quantitative PCR(qPCR)assay for the detection of Movi based on the Hsp 70 gene and to further understand its genetic evolution.In this study,specific primers and probes were designed based on Hsp 70 genes retrieved from GenBank to establish a qPCR method for the screening of Movi infection,after which eighty-eight goat nasal swabs and 43 lung samples of suspected Mycoplasmal pneumonia of sheep and goats(MPSG)were tested by the established qPCR method.Lung samples that tested positive for Movi were then used for pathogen isolation and identification,and the Hsp 70 genes of the isolated strains were sequenced and analyzed.The results showed that the established qPCR method had a correlation coefficient of 1.00,with a 96.0%and the amplification efficiency was 96%,a-3.411 slope and a 37.29 Y-intercept slope was-3.411,Y-axis intercept was 37.29.The method was specific,and there was no cross reaction with other pathogens,such as Mycoplasma mycoides subsp.capri(Mmc),Mycoplasma capricolum subsp.capripneumoniae(Mccp),Acholeplasmalaidlawii(AL),Mycoplasma agalactiae(Maga),Corynebacterium pseudotuberculosis(CP),Orf virus(ORFV),or Mycoplasma bovis(Mb).The method was sensitive,with a minimum detection limit of 5.72 copies.μL^(-1).The method is reproducible,with intragroup and intergroup coefficients of variation both less than 1.00%.The length of the Hsp 70 gene of the six Movi isolates were all 1818 bp,and there were 96.0%~99.4%and 98.0%~100.0%homology of the nucleotide and amino acid,respectively,between the six Movi isolates with other Movi reference strains.Further analysis showed that goat-origin Movi had 1 more N-glycosylation site than sheep-origin Movi.The genetic analysis showed that they were both in the ClusterⅠA subgroup(goat-origin).In conclusion,this study established a specific,sensitive and reproducible qPCR method for the detection of Movi based on the Hsp 70 gene.Sequence analysis showed that there was high nucleotide homology in the Hsp 70 gene of Movi from different sources.Phylogenetic analysis confirmed that the Movi Fujian-origin strains had a close genetic relationship with the goat-origin strains.This study not only provides technical support for the clinical diagnosis of Movi but also provides reference for further understanding the genetic evolution of Movi.