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鸭瘟病毒微滴数字PCR方法的建立与优化

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为快速、准确检测鸭瘟病毒(duck enteritis virus,DEV),根据GenBank中DEV UL6 基因序列,设计了微滴数字PCR(ddPCR)的引物和探针,通过条件优化,建立了检测DEV的ddPCR方法,并对该方法的灵敏度、特异性和重复性进行了验证.结果显示:在57.1℃退火温度下,当引物浓度为1.50 µmol/L,探针浓度为0.45 µmol/L时,病毒拷贝数浓度达到最大,为332 copies/µL;该方法的最低检测限为0.7 copies/µL,且对非鸭瘟病原均仅扩增出阴性微滴,无交叉反应;组内和组间重复试验变异系数均小于 10%.结果表明,本研究建立的DEV ddPCR方法具有灵敏度高、特异性强、重复性好和稳定性高的优点,可用于低DEV含量样品的检测及其感染的早期诊断和流行病学调查.
Establishment and Optimization of Droplet Digital PCR for Duck Enteritis Virus
In order to quickly and accurately detect duck enteritis virus(DEV),primers and probes for droplet digital PCR(ddPCR)were designed based on DEV UL6 gene sequence registered in GenBank,and a ddPCR method for detection of DEV was established after optimization of reaction conditions,followed by the verification of its sensitivity,specificity and reproducibility.The results showed that the virus concentration reached to the maximum(332 copies/µL)when the concentrations of primers and probes were 1.50 µmol/L and 0.45 µmol/L,respectively,at an annealing temperature of 57.1℃;the lowest detection limit was 0.7 copies/µL,and it couldn't generate positive microtitres for non-DEV pathogens,without cross-reactivity;the variable coefficients of both the intra-and inter-group repeated tests were less than 10%.In conclusion,the DEV ddPCR method established in this study was characterized by the advantages of high sensitivity,strong specificity,good reproducibility and high stability,and could be used for the detection of samples with low DEV content and early diagnosis and epidemiological investigation of the disease.

DEVddPCRmethod establishmentoptimization

邱艳红、廖维连、蒋文明、杨得胜、宋迟、洪瑾芳、刘道泉

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福州市动物疫病预防控制中心,福建福州 350025

将乐县动物疫病预防控制中心,福建将乐 365001

中国动物卫生与流行病学中心,山东青岛 266032

福建省动物疫病预防控制中心,福建福州 350003

永泰县金蛋发展有限公司,福建永泰 350714

永泰县畜牧水产服务中心,福建永泰 350700

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鸭瘟病毒 微滴数字PCR 方法建立 优化

2024

中国动物检疫
中国动物卫生与流行病学中心

中国动物检疫

影响因子:0.437
ISSN:1005-944X
年,卷(期):2024.41(10)