猪δ冠状病毒实时荧光RT-PCR检测方法的建立与应用
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S852.65

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上海市自然科学基金项目(04713)


Establishment and Application of a Real-time Fluorescent RT-PCR Assay for Porcine Deltacoronavirus
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    摘要:

    为灵敏、快速检测猪δ冠状病毒(porcine deltacoronavirus,PDCoV),根据PDCoV M基因保守区域设计引物和探针,建立了一种PDCoV实时荧光RT-PCR检测方法,并评价了该方法的特异性、灵敏性和重复性。结果显示,该方法的拷贝数对数值与Ct值在1.3×103~1.3×107 copies/μL拷贝数浓度范围内呈现良好的线性关系,R2 = 0.994;最低检出限为13 copies/μL,且无非特异性扩增;批内和批间重复性试验变异系数(CV)为0.24%~2.15%,均小于3%。利用该方法和普通RT-PCR对42份临床样品同时进行检测,发现本方法比普通RT-PCR灵敏性更高,二者符合率为95.2%。以上结果表明,本试验建立的实时荧光RT-PCR方法灵敏、特异、可靠,可用于PDCoV的临床检测和流行病学调查。

    Abstract:

    In order to sensitively and rapidly detect porcine deltacoronavirus(PDCoV),probe and a pair of primers were designed based on the conserved region of PDCoV M gene to establish a real-time fluorescent RT-PCR assay for PDCoV,followed by evaluation on its specificity,sensitivity and repeatability. The results showed that a good linear relationship was observed between the logarithmic value of copy number and Ct value from 1.3×103 to 1.3×107 copies/μL,R2=0.994;the lowest detection limit was 13 copies/μL without any nonspecific amplification;the coefficient of variation(CV)of intra and inter batch repeatability tests were from 0.24% to 2.15%,both of which were less than 3%. 42 clinical samples were detected by the established method and general RT-PCR assay,it was found that the former was more sensitive and reliable than the latter,the coincidence rate of both methods was 95.2%. In conclusion,the established method could be used for clinical diagnosis and epidemiological investigation on PDCoV with the help of its good specificity,reliability and sensitivity.

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苑建军,杨德全,鞠厚斌,庄建萍,葛菲菲,杨显超,李鑫,沈海潇.猪δ冠状病毒实时荧光RT-PCR检测方法的建立与应用[J].《中国动物检疫》编辑部,2022,39(11):115-120.

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  • 在线发布日期: 2022-11-03
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