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

doi:10.11843/j.issn.0366-6964.2024.04.030

Abstract

Abstract: 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 Hsp70 gene and to further understand its genetic evolution. In this study, specific primers and probes were designed based on Hsp70 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 Hsp70 genes of the isolated strains were sequenced and analyzed. The results showed that the established qPCR method had a correlation coefficient of 1.00, and the amplification efficiency was 96%, 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 Hsp70 gene of the six Movi isolates were all 1 818 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 Hsp70 gene. Sequence analysis showed that there was high nucleotide homology in the Hsp70 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.

Key words: Mycoplasma ovipneumoniae, Hsp70 gene, TaqMan real-time fluorescent quantitative PCR method, sequence analysis

CLC Number:

S855.3

JIANG Jinxiu, ZHANG Jingpeng, LIN Yusheng, LIU Weiwei, HU Qilin, WAN Chunhe. Establishment of a TaqMan Assay for Mycoplasma ovipneumoniae based on the Hsp70 Gene and Analysis of Its Genetic Evolution[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1684-1695.

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Establishment of a TaqMan Assay for Mycoplasma ovipneumoniae based on the Hsp70 Gene and Analysis of Its Genetic Evolution

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