FAstV、FPV和FECoV三重PCR检测方法的建立及应用  

Establishment and application of triple PCR method for FAstV,FPV and FECoV

作　　者：肖连 郭琪 黄坚 杨晓农[1] 李妍 XIAO Lian;GUO Qi;HUANG Jian;YANG Xiao-nong;LI Yan(School of Animal Husbandry and Veterinary Science,Southwest Minzu University,Chengdu 610041,China;Sichuan Medical Device Biomaterials and Products Inspection Center Co.,Ltd,Chengdu 610000,China)

机构地区：[1]西南民族大学畜牧兽医学院,四川成都610041 [2]四川医疗器械生物材料和制品检验中心有限公司,四川成都610000

出　　处：《西南民族大学学报（自然科学版）》2022年第2期135-141,共7页Journal of Southwest Minzu University(Natural Science Edition)

基　　金：四川省科技厅面上项目(2019YFN0133);西南民族大学研究生创新型科研项目(CX2021094)。

摘　　要：猫星状病毒(Feline astrovirus,FAstV)、猫细小病毒(Feline parvovirus,FPV)、猫肠道冠状病毒(Feline enteric coronavirus,FECoV)是临床上引起猫腹泻的重要病原,为建立一种能同时鉴别三种病原的分子检测方法,通过对引物浓度和退火温度的条件进行优化筛选,建立了FAstV/FPV/FECoV的三重PCR方法,并对其特异性、敏感性、重复性和稳定性进行验证.结果表明,该方法可特异性扩增出FAstV(320 bp)、FPV(468 bp)和FECoV(664 bp)的目的片段,而未扩增出其它犬猫相关病原,且重复性好.FAstV、FPV和FECoV的最低检测限分别为2×10^(7)copy/μL(7.1 pg/μL)、4.7×10^(6)copy/μL(2.4 pg/μL)和7×10^(6)copy/μL(5.1 pg/μL).应用建立的三重PCR方法对2019~2020年成都市区采集的207份猫粪便样本(141份腹泻样本,66份临床健康样本)进行检测,结果显示FPV、FAstV和FECoV的检出率分别为37.20%(77/207)、24.15%(50/207)和15.46%(32/207);混合感染以两种病原混合感染为主,其中,FPV/FAstV、FPV/FECoV和FAstV/FECoV的混合感染率分别为9.18%、6.28%和6.28%,检测结果与单一PCR相符.本研究成功建立了FAstV/FPV/FECoV的三重PCR检测方法,可以用于相关病原的临床检测和流行病学调查.Feline Astrovirus(FAstV),Feline Parvovirus(FPV)and Feline Enteric Coronavirus(FECoV)are important pathogens causing diarrhea in cats.In order to establish a molecular detection method which can differentiate the three pathogens in the same PCR system,an FAstV/FPV/FECoV triple PCR method was established with optimized primer concentrations and annealing temperature,and specificity,sensitivity and repeatability were tested.The results showed that the PCR method could only identify FAstV(320 bp),FPV(468 bp)and FECoV(664 bp)genes,while not other canine and feline related pathogens.The detection limits of FAstV,FPV and FECoV were 2×10^(7)copy/μL(7.1 pg/μL),4.7×10^(6)copy/μL(2.4 pg/μL)and 7×10^(6)copy/μL(5.1 pg/μL)respectively.The established triple PCR method was used to detect 207 cat fecal samples collected in Chengdu from 2019 to 2020,including 141 diarrhea samples and 66 clinical health samples.The detection rates of FAstV,FPV and FECoV were 24.15%(50/207),37.20%(77/207)and 15.46%(32/207)respectively,and the co-infection rates of FAstV/FPV,FPV/FECoV and FAstv/FECoV were 9.18%,6.28%and 6.28%respectively.In conclusion,the triple PCR method of FAstV/FPV/FECoV was successfully established,and could be applied for virus detection and epidemiological investigation.

关 键 词：猫星状病毒 猫细小病毒 猫肠道冠状病毒 三重PCR 

分 类 号：S852.65[农业科学—基础兽医学] S829.3[农业科学—兽医学]