摘要
为建立一种用于检测猪肺炎支原体(Mhp)的套式PCR方法,本研究从GenBank中下载Mhp P36基因序列,选取其保守区域基因片段,设计套式PCR的内外套2对特异性引物,经过反应条件优化,建立了Mhp套式PCR检测方法。用该方法对Mhp 168株、鸡毒支原体、猪鼻支原体、猪链球菌、胸膜肺炎放线杆菌、副猪嗜血杆菌、猪圆环病毒2型、猪伪狂犬病毒进行特异性检测。结果显示,除了Mhp 168株能扩增出外套793 bp和内套448 bp的目的条带,其余病原均未扩增出相应的片段,特异性较强;该方法对Mhp的最低检测限为10拷贝/μL,敏感性是常规PCR方法的100倍。对50份含Mhp的培养基经本研究建立的套式PCR、常规PCR、Mhp分离培养方法的阳性检出率分别为88%、82%、88%,套式PCR方法与常规PCR的总体符合率为94%,与Mhp分离培养方法的符合率为100%。本实验建立的套式PCR方法,为Mhp的流行病学调查提供了检测手段。
In order to develop a nested PCR method for the detection of Mycoplasma hyopneumoniae(Mhp),two pairs of specific primers for nested PCR were designed referring to the conserved region gene fragment of Mhp P36 gene deposited in GenBank,and the reaction conditions were optimized.Mhp of 168 isolates,Mycoplasma Galliscepticum,Mycoplasma hyorhinitis,Streptococcus suis,Actinobacillus pleuropneumoniae,Haemophilus parasuis,Porcine circovirus type 2,and Pseudorabies virus were detected using this method.The results showed that the method could amplify the 793 bp and the 448 bp target gene of Mhp specifically by using the two pairs of specific primers,respectively,and had no cross reaction with any of others.The sensitivity of the nested PCR was 10 copies/μL,which was more sensitive of 100 times than that of conventional PCR kit.50 samples of culture medium DNA containing Mhp were tested by using the developed nested PCR,conventional PCR and Mycoplasma isolation and culture methods,the positive detection rates of 88%,82%and 88%,respectively.The total coincidence rate of nested PCR and conventional PCR was 94%,and that of Mycoplasma isolation and culture method was 100%.The nested PCR method developed in this study can be used for the epidemiological investigation of Mhp.
作者
栾璐
刁小龙
刘云迎
张家铭
王飞
何海蓉
LUAN Lu;DIAO Xiao-long;LIU Yun-ying;ZHANG jia-ming;WANG Fei;HE Hai-rong(Key Laboratory of Safety Assessment of Agricultural Products,Livestock and poultry Inputs of the Ministry of Agriculture,Zhongchong Sino Biotech Taizhou Co.,Ltd.,Taizhou 225300,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2020年第5期469-473,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
兽用生物制品及检测试剂应用示范及平台建设(2017YFF0208606)
关键词
猪肺炎支原体
套式PCR
应用
Mycoplasma hyopneumoniae
nested PCR
application
