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

摘要/Abstract

摘要： 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。

关键词: 绵羊肺炎支原体, Hsp70基因, TaqMan实时荧光定量PCR方法, 序列分析

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

中图分类号:

S855.3

江锦秀, 张靖鹏, 林裕胜, 刘维巍, 胡奇林, 万春和. 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[J]. 畜牧兽医学报, 2024, 55(4): 1684-1695.

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.

参考文献

[1] ZHAO J Y, DU Y Z, SONG Y P, et al. Investigation of the prevalence of Mycoplasma ovipneumoniae in southern Xinjiang, China[J]. J Vet Res, 2021, 65(2):155-160.
[2] GAETA N C, DE SÁ GUIMARÃES A M, TIMENETSKY J, et al. The first Mycoplasma ovipneumoniae recovered from a sheep with respiratory disease in Brazil-draft genome and genomic analysis[J]. Vet Res Commun, 2022, 46(4):1311-1318.
[3] SPAAN R S, EPPS C W, CROWHURST R, et al. Impact of Mycoplasma ovipneumoniae on juvenile bighorn sheep (Ovis canadensis) survival in the northern basin and range ecosystem[J]. Peer J, 2021, 9:e10710.
[4] BESSER T E, CASSIRER E F, POTTER K A, et al. Association of Mycoplasma ovipneumoniae Infection with population-limiting respiratory disease in free-ranging rocky mountain bighorn sheep (Ovis canadensis canadensis)[J]. J Clin Microbiol, 2008, 46(2):423-430.
[5] HANDELAND K, TENGS T, KOKOTOVIC B, et al. Mycoplasma ovipneumoniae-a primary cause of severe pneumonia epizootics in the Norwegian muskox (Ovibos moschatus) population[J]. PLoS One, 2014, 9(9):e106116.
[6] HIGHLAND M A, HERNDON D R, BENDER S C, et al. Mycoplasma ovipneumoniae in wildlife species beyond subfamily Caprinae[J]. Emerg Infect Dis, 2018, 24(12):2384-2386.
[7] MANLOVE K, BRANAN M, BAKER K, et al. Risk factors and productivity losses associated with Mycoplasma ovipneumoniae infection in United States domestic sheep operations[J]. Prev Vet Med, 2019, 168:30-38.
[8] JA? M, AMBROSET C, TRICOT A, et al. Population structure and antimicrobial susceptibility of Mycoplasma ovipneumoniae isolates in France[J]. Vet Microbiol, 2020, 248:108828.
[9] LINDSTRÖM L, TAUNI F A, VARGMAR K. Bronchopneumonia in Swedish lambs:a study of pathological changes and bacteriological agents[J]. Acta Vet Scand, 2018, 60(1):54.
[10] DEENEY A S, COLLINS R, RIDLEY A M. Identification of Mycoplasma species and related organisms from ruminants in England and wales during 2005-2019[J]. BMC Vet Res, 2021, 17(1):325.
[11] CHEN C, QIAO J, MENG Q L, et al. Serological and molecular survey of sheep infected with Mycoplasma ovipneumoniae in Xinjiang, China[J]. Trop Anim Health Prod, 2015, 47(8):1641-1647.
[12] XU J, CHU Y F, ZHAO P, et al. Investigation of gene polymorphism on Mycoplasma ovipneumoniae isolates from some areas of China[J]. Acta Veterinaria et Zootechnica Sinica, 2011, 42(9):1295-1301. (in Chinese)
许健, 储岳峰, 赵萍, 等. 中国部分地区绵羊肺炎支原体的基因多态性分析[J]. 畜牧兽医学报, 2011, 42(9):1295-1301.
[13] XU J, CHU Y F, GAO P C, et al. Molecular typing and immunoblotting of 17 Mycoplasma ovipneumoniae isolates from China[J]. Acta Microbiologica Sinica, 2011, 51(10):1421-1426. (in Chinese)
许健, 储岳峰, 高鹏程, 等. 我国17株绵羊肺炎支原体分子分型及其菌体蛋白免疫印迹[J]. 微生物学报, 2011, 51(10):1421-1426.
[14] MAKSIMOVIĆ Z, RIFATBEGOVIĆ M, LORIA G R, et al. Mycoplasma ovipneumoniae:a most variable pathogen[J]. Pathogens, 2022, 11(12):1477.
[15] LI M, MA C J, LIU X M, et al. Molecular cloning of HSP70 in Mycoplasma ovipneumoniae and comparison with that of other mycoplasmas[J]. Genet Mol Res, 2011, 10(2):834-848.
[16] JIANG F, HE J Y, NAVARRO-ALVAREZ N, et al. Elongation factor Tu and heat shock protein 70 are membrane-associated proteins from Mycoplasma ovipneumoniae capable of inducing strong immune response in mice[J]. PLoS One, 2017, 11(8):e0161170.
[17] TIWARI S, THAKUR R, SHANKAR J. Role of heat-shock proteins in cellular function and in the biology of fungi[J]. Biotechnol Res Int, 2015, 2015:132635.
[18] MULTHOFF G, POCKLEY A G, STREFFER C, et al. Dual role of heat shock proteins (HSPs) in anti-tumor immunity[J]. Curr Mol Med, 2012, 12(9):1174-1182.
[19] MULTHOFF G, POCKLEY A G, SCHMID T E, et al. The role of heat shock protein 70(Hsp70) in radiation-induced immunomodulation[J]. Cancer Lett, 2015, 368(2):179-184.
[20] LIN Y S, JIANG J X, ZHANG J P, et al. Establishment of a TaqMan real-time fluorescent quantitative PCR assay for detection of Mycoplasma ovipneumoniae[J]. Chinese Journal of Preventive Veterinary Medicine, 2018, 40(4):316-320. (in Chinese)
林裕胜, 江锦秀, 张靖鹏, 等. 绵羊肺炎支原体TaqMan荧光定量PCR检测方法的建立[J]. 中国预防兽医学报, 2018, 40(4):316-320.
[21] HUANG J, YIN Z J, YUE H, et al. Development and application of quantitative real-time PCR for the detection of Mycoplasma ovipneumoniae[J]. Chinese Veterinary Science, 2016, 46(10):1270-1276. (in Chinese)
黄坚, 尹正军, 岳华, 等. 绵羊肺炎支原体荧光定量PCR检测方法的建立与应用[J]. 中国兽医科学, 2016, 46(10):1270-1276.
[22] CHENG Z T, ZHANG S X, WANG H, et al. Establishment of real-time fluorescent quantitative PCR assay for detection of Mycoplasma ovipneumoniae[J]. Acta Agriculturae Boreali-Occidentalis Sinica, 2013, 22(2):7-12. (in Chinese)
程振涛, 张双翔, 王慧, 等. 绵羊肺炎支原体实时荧光定量PCR检测方法的建立[J]. 西北农业学报, 2013, 22(2):7-12.
[23] DESHUILLERS P L, SANTOS A P, DO NASCIMENTO N C, et al. Complete genome sequence of Mycoplasma ovis strain Michigan, a hemoplasma of sheep with two distinct 16S rRNA genes[J]. Genome Announc, 2014, 2(1):e01235-13.
[24] NOLL L W, HIGHLAND M A, HAMILL V A, et al. Development of a real-time PCR assay for detection and differentiation of Mycoplasma ovipneumoniae and a novel respiratory-associated Mycoplasma species in domestic sheep and goats[J]. Transbound Emerg Dis, 2022, 69(5):e1460-e1468.
[25] KAMLA V, HENRICH B, HADDING U. Phylogeny based on elongation factor Tu reflects the phenotypic features of mycoplasmas better than that based on 16S rRNA[J]. Gene, 1996, 171(1):83-87.
[26] SÖDERLUND R, BÖLSKE G, HOLST B S, et al. Development and evaluation of a real-time polymerase chain reaction method for the detection of Mycoplasma felis[J]. J Vet Diagn Invest, 2011, 23(5):890-893.
[27] ZHANG B, HAN X, YUE H, et al. Molecular characterization of the heat shock protein 70 gene in Mycoplasma ovipneumoniae[J]. Vet J, 2013, 198:299-301.
[28] MCAULIFFE L, HATCHELL F M, AYLING R D, et al. Detection of Mycoplasma ovipneumoniae in Pasteurella-vaccinated sheep flocks with respiratory disease in England[J]. Vet Rec, 2003, 153(22):687-688.
[29] YANG F L, HAN X, TANG C, et al. A Hsp70 gene-based PCR for detection of Mycoplasma ovipneumoniae[J]. J Anim Vet Adv, 2013, 12(19):1495-1497.
[30] JIANG J X, LIN Y S, YOU W, et al. A molecular epidemiological study on Mycoplasma pneumonia of sheep and goats in Fujian[J]. Fujian Journal of Agricultural Sciences, 2017, 32(1):12-16. (in Chinese)
江锦秀, 林裕胜, 游伟, 等. 福建省羊支原体性肺炎分子流行病学研究[J]. 福建农业学报, 2017, 32(1):12-16.
[31] MAKSIMOVIĆ Z, DE LA FE C, AMORES J, et al. Comparison of phenotypic and genotypic profiles among caprine and ovine Mycoplasma ovipneumoniae strains[J]. Vet Rec, 2017, 180(7):180.
[32] GUO P, LI C F, JU Y, et al. N-glycosylation modification of heat shock protein gp96 affects its immunological function[J]. Chinese Journal of Biotechnology, 2021, 37(11):4036-4046. (in Chinese)
郭鹏, 李长菲, 鞠莹, 等. N-糖基化修饰对热休克蛋白gp96免疫学功能的影响[J]. 生物工程学报, 2021, 37(11):4036-4046.
[33] ZHANG Z Z, WANG X, ZAI J J, et al. Roles of N-glycosylation in immunity of prME and NS1 gene of JEV[J]. Chinese Journal of Virology, 2012, 28(3):213-218. (in Chinese)
张子重, 汪雪, 宰俊杰, 等. N-糖基化对乙型脑炎病毒prME和NS1基因免疫效果影响的研究[J]. 病毒学报, 2012, 28(3):213-218.
[34] BANKS J, SPEIDEL E S, MOORE E, et al. Changes in the haemagglutinin and the neuraminidase genes prior to the emergence of highly pathogenic H7N1 avian influenza viruses in Italy[J]. Arch Virol, 2001, 146(5):963-973.
[35] GU M, LI Q H, GAO R Y, et al. The T160A hemagglutinin substitution affects not only receptor binding property but also transmissibility of H5N1 clade 2.3.4 avian influenza virus in guinea pigs[J]. Vet Res, 2017, 48(1):7.