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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(中国食品药品检定研究院,北京 102629)

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R-33

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Establishment of a fluorescent quantitative PCR detection method for bovine coronavirus and its preliminary application
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(National Institutes for Food and Drug Control, Beijing 102629,China)

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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TaqMan 引物探针,建立基于TaqMan 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TaqMan 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?

    Abstract:

    Objective To investigate the prevalence of bovine coronavirus (BCV) in bovine herds and bovinederivedbioproducts. Methods A fluorescent quantitative PCR (FQ-PCR) method for BCV was developed based on a pairof primers and a TaqMan probe, in accordance with the published sequence of BCV. Results The assay could specificallydetect BCV and had good sensitivity, with a limit of detection of 40 copies. The FQ-PCR method achieved a good linearrelationship within the template concentration range from 103to 107copies/ μL, with a correlation of 0. 999. Theamplification efficiency of the assay was 96. 195%. A total of 64 bovine rectal swabs and 33 bovine-derived bioproducts weresubjected to the FQ-PCR assay, and the positivity rates were 1. 5% and 6%, respectively. The positive sample wasamplified with another pair of primers for the N gene. Conclusions The results of this study demonstrated that the PCRproduct 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.

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王莎莎,王吉,岳秉飞.牛冠状病毒荧光定量PCR 检测方法的建立及初步应用[J].中国比较医学杂志,2019,29(4):69~73.

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  • 收稿日期:2018-09-19
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  • 在线发布日期: 2019-05-10
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