绵羊肺炎支原体安徽株p113基因PCR检测与序列分析
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摘要
旨在应用基于p113基因特异性的PCR方法对绵羊肺炎支原体(Mycoplasma ovipneumoniae,Mo)安徽流行株进行分子生物学鉴定。利用已报道的Mo p113基因引物,采用PCR方法对前期分离的Mo安徽流行株AH-01、AH-02、AH-03和AH-04进行p113基因扩增,并对菌株的p113基因扩增产物进行序列测定及分析。结果表明,利用建立的PCR方法,4株Mo临床分离株均可扩增到大小约为287 bp的特异性目的片段;安徽流行株AH01、AH02、AH03和AH04的p113基因序列两两之间的相似性均在95%以上,与Mo ATCC 29419和Mo贵州流行株GZ-QX1的p113基因序列相似性在88.6%～89.3%。提示基于p113基因的PCR方法可以用于绵羊肺炎支原体的分子生物学鉴定,为开展由Mo引起的绵羊和山羊肺炎的流行病学调查和科学防控提供了新方法。
This study was designed to carry out the molecular identification of clinical isolates of Mycoplasma ovipneumoniae(Mo) prevalent in Anhui Province by using a specific PCR assay targeting at p113 gene. PCR assay was used to amplify the target gene from the four previously isolated Mo strains, AH-01, AH-02, AH-03 and AH-04, using the reported specific primers for p113 gene. Subsequently, the amplified products were sequenced and genetically analyzed. The results showed that the specific PCR products with size of 287 bp were obtained from all of the four clinical isolates; the pairwise sequence similarity of p113 gene of the four Mo isolates were all above 95%, and their sequence similarity with Mo standard strain of ATCC 29419 and Mo clinical strain of GZ-QX1 isolated from Guizou Province ranged from 88.6% to 89.3%. Our results demonstrated that the PCR assay targeting at p113 gene could be used in the molecular identification of Mo clinical isolates, which provided a methodological reference for the epidemiological investigation and scientific control for the respiratory tract infectious diseases in sheep and goat associated with Mo.
This study was designed to carry out the molecular identification of clinical isolates of Mycoplasma ovipneumoniae(Mo) prevalent in Anhui Province by using a specific PCR assay targeting at p113 gene. PCR assay was used to amplify the target gene from the four previously isolated Mo strains, AH-01, AH-02, AH-03 and AH-04, using the reported specific primers for p113 gene. Subsequently, the amplified products were sequenced and genetically analyzed. The results showed that the specific PCR products with size of 287 bp were obtained from all of the four clinical isolates; the pairwise sequence similarity of p113 gene of the four Mo isolates were all above 95%, and their sequence similarity with Mo standard strain of ATCC 29419 and Mo clinical strain of GZ-QX1 isolated from Guizou Province ranged from 88.6% to 89.3%. Our results demonstrated that the PCR assay targeting at p113 gene could be used in the molecular identification of Mo clinical isolates, which provided a methodological reference for the epidemiological investigation and scientific control for the respiratory tract infectious diseases in sheep and goat associated with Mo.
引文
[1]谢秀兰,额尔和花,黎帅,等.宁夏不同绵羊品种中绵羊肺炎支原体的调查[J].中国兽医杂志,2017,53(6):11-13.
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[6]MCAULIFFE L,HATCHELL F M,AYLING R D,et al.Detection of Mycoplasma ovipneumoniae in Pasteurella-vacinnated sheep flocks with respiratory disease in England[J].Veterinary Record,2003,153(22):687-688.
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[9]张建华,黄海碧,王晓晖,等.绵羊肺炎支原体免疫组织化学及间接免疫荧光检测方法的建立[J].中国兽医科学,2017,47(2):172-178.
[10]杨发龙,张焕荣,汤承,等.绵羊肺炎支原体p113基因的序列分析及功能预测[J].华南农业大学学报,2013,34(1):117-121.
[11]张贤宇,杨发龙,冯旭飞,等.绵羊肺炎支原体P113蛋白N端原核表达及其抗原性分析[J].中国兽医杂志,2013,49(7):7-10.
[12]吴燕,袁海文,王琦,等.绵羊肺炎支原体贵州株p113基因的克隆与生物信息学分析[J].西北农业学报,2016,25(3):317-327.
[2]侯宏艳,惠文巧,张丹俊,等.山羊的绵羊肺炎支原体的分离和鉴定[J].安徽农业科学,2015,43(28):159-160.
[3]吴燕,王琦,周碧君,等.绵羊肺炎支原体贵州流行株p113基因序列分析[J].中国兽医学报,2016,36(5):756-762.
[4]储岳峰.我国山羊(接触)传染性胸膜肺炎病原学、流行病学研究及灭活疫苗的研制[D].兰州:中国农业科学院,2011.
[5]吴锦艳,尚佑军,陈妍,等.国内部分地区羊支原体肺炎血清学调查及分析[J].中国预防兽医学报,2017,39(2):156-158.
[6]MCAULIFFE L,HATCHELL F M,AYLING R D,et al.Detection of Mycoplasma ovipneumoniae in Pasteurella-vacinnated sheep flocks with respiratory disease in England[J].Veterinary Record,2003,153(22):687-688.
[7]冯旭飞,张贤宇,王成龙,等.基于p113基因的绵羊肺炎支原体PCR检测方法的建立及应用[J].中国兽医科学,2013,43(11):1157-1161.
[8]侯宏艳,惠文巧,张丹俊,等.绵羊肺炎支原体的培养和PCR鉴定[J].畜牧与饲料科学,2016,37(11):97-98.
[9]张建华,黄海碧,王晓晖,等.绵羊肺炎支原体免疫组织化学及间接免疫荧光检测方法的建立[J].中国兽医科学,2017,47(2):172-178.
[10]杨发龙,张焕荣,汤承,等.绵羊肺炎支原体p113基因的序列分析及功能预测[J].华南农业大学学报,2013,34(1):117-121.
[11]张贤宇,杨发龙,冯旭飞,等.绵羊肺炎支原体P113蛋白N端原核表达及其抗原性分析[J].中国兽医杂志,2013,49(7):7-10.
[12]吴燕,袁海文,王琦,等.绵羊肺炎支原体贵州株p113基因的克隆与生物信息学分析[J].西北农业学报,2016,25(3):317-327.