牛冠状病毒荧光定量PCR检测方法的建立及初步应用
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摘要
目的建立特异灵敏的牛冠状病毒(bovine coronavirus,BCV)荧光定量PCR检测方法,并对牛源性样本进行筛查。方法根据Genbank中登录的BCV的N基因序列设计Taq Man引物探针,建立基于Taq Man探针法的BCV PCR检测方法,对收集到的牛源样本进行BCV筛查,并对部分阳性样本的PCR产物进行测序鉴定。结果建立了能够特异检测BCV的Taq Man探针法荧光定量PCR检测方法,其标准曲线相关系数Slope为-3.417,相关系数r2为0.999,,扩增效率Eff为96.195%。该方法检测BCV的敏感度为40 copies。使用该方法检测64份牛肛拭子样本检出率为1.5%,检测33份牛源生物制品检出率为6%,部分阳性样本的PCR产物经测序比对为BCV核酸序列,说明BCV在国内牛群中流行,牛源相关生物制品中有感染BCV的潜在风险。结论经测序验证,建立的方法能够灵敏的检测样本中的BCV核酸,应加强牛源相关生物制品中BCV的监测,避免人感染BCV的潜在风险。
Objective To investigate the prevalence of bovine coronavirus(BCV) in bovine herds and bovinederived bioproducts.Methods A fluorescent quantitative PCR(FQ-PCR) method for BCV was developed based on a pair of primers and a Taq Man probe,in accordance with the published sequence of BCV.Results The assay could specifically detect BCV and had good sensitivity,with a limit of detection of 40 copies.The FQ-PCR method achieved a good linear relationship within the template concentration range from 103 to 107 copies/μL,with a correlation of 0.999.The amplification efficiency of the assay was 96.195%.A total of 64 bovine rectal swabs and 33 bovine-derived bioproducts were subjected to the FQ-PCR assay,and the positivity rates were 1.5% and 6%,respectively.The positive sample was amplified with another pair of primers for the N gene.Conclusions The results of this study demonstrated that the PCR product had 99% similarity to BCV,suggesting the existence of a BCV epidemic in bovine herds and the potential risk of BCV contamination in bovine-derived bioproducts.
Objective To investigate the prevalence of bovine coronavirus(BCV) in bovine herds and bovinederived bioproducts.Methods A fluorescent quantitative PCR(FQ-PCR) method for BCV was developed based on a pair of primers and a Taq Man probe,in accordance with the published sequence of BCV.Results The assay could specifically detect BCV and had good sensitivity,with a limit of detection of 40 copies.The FQ-PCR method achieved a good linear relationship within the template concentration range from 103 to 107 copies/μL,with a correlation of 0.999.The amplification efficiency of the assay was 96.195%.A total of 64 bovine rectal swabs and 33 bovine-derived bioproducts were subjected to the FQ-PCR assay,and the positivity rates were 1.5% and 6%,respectively.The positive sample was amplified with another pair of primers for the N gene.Conclusions The results of this study demonstrated that the PCR product had 99% similarity to BCV,suggesting the existence of a BCV epidemic in bovine herds and the potential risk of BCV contamination in bovine-derived bioproducts.
引文
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[11]Kim JH,Jang JH,Yoon SW,et al.Detection of bovine coronavirus in nasal swab of non-captive wild water deer,Korea[J].Transbound Emerg Dis.,2018,65(3):627-631.
[2]耿合员,谭文杰.新近发现的冠状病毒研究进展[J].病毒学报,2013,29(1):65-70.
[3]沈付娆,杨建乐,赵贵民,等.牛冠状病毒SYBR GreenⅠ实时荧光定量PCR检测方法的建立及初步应用[J].中国兽医杂志,2016,(6):22-24.
[4]孙留霞,朱平军,李文华,等.牛冠状病毒检测方法的研究进展[J].上海畜牧兽医通讯,2014(5):20-22.
[5]薛麒,吴思捷,王锡乐,等.实验用牛微生物学质量控制标准的研究[J].实验动物科学.2017,(04):55-58.
[6]Gomez DE,Arroyo LG,Poljak Z,et al.Detection of bovine coronavirus in healthy and diarrheic dairy calves[J].J Vet Intern Med,2017,31(6):1884-1891.
[7]Toftaker I,Holmoy I,Nodtvedt A,et al.A cohort study of the effect of winter dysentery on herd-level milk production[J].JDairy Sci.,2017,100(8):6483-6493.
[8]Zhang XM,Herbst W,Kousoulas KG,et al.Biological and genetic characterization of a hemagglutinating coronavirus isolated from a diarrhoeic child[J].J Med Virol.,1994,44(2):152-161.
[9]Oma VS,Klem T,Travén M,et al.Temporary carriage of bovine coronavirus and bovine respiratory syncytial virus by fomites and human nasal mucosa after exposure to infected calves[J].BMCVet Res.,2018,14(1):14-22.
[10]Wolff MH,Sattar SA,Adegbunrin O,et al.Environmental survival and microbicide inactivation of coronaviruses.[M].Coronaviruses with Special Emphasis on First Insights Concerning SARS.Birkhuser Basel,2005:38343-38350.
[11]Kim JH,Jang JH,Yoon SW,et al.Detection of bovine coronavirus in nasal swab of non-captive wild water deer,Korea[J].Transbound Emerg Dis.,2018,65(3):627-631.