数字农科院2.0

DEV、DHBV与DAdV-3多重PCR检测方法的建立与初步应用

文献类型： 中文期刊

关键词： DEV;;DHBV;;DADV-3

期刊名称：中国预防兽医学报

ISSN： 1008-0589

年卷期： 2025 年

页码：

收录情况： 北大核心（2023版） ; ; CSCD（2025-2026年度） ; ; 科技核心（2024版） ; ; 农林核心（2024版）

摘要： 摘 要：为建立能同时检测鸭瘟病毒(DEV)、鸭乙型肝炎病毒(DHBV)和鸭腺病毒3型(DAdV-3)的多重PCR检测方法,本研究通过比较DEV gE基因、DHBV P基因、DAdV-3 22K基因,选择各基因保守区域,设计并合成3对特异性引物。分别构建3种病毒的重组质粒并鉴定正确后作为质粒标准品,混合后作为模板,分别以温度梯度PCR法确定最适退火温度(Tm),采用单一控制变量法优化各引物浓度,结果显示该方法的最佳退火温度为57 ℃, DHBV、DEV、DAdV-3引物最佳浓度分别为1.5umol/L、0.5 umol/L、0.5 umol/L,初步建立检测这3种病毒的多重PCR方法。以禽流感病毒(AIV)、新城疫病毒(NDV)、鸭肝炎病毒(DHV)、鸭源巴氏杆菌(PM)、鸭疫里默氏杆菌(RA)、鹅细小病毒(GPV)等病原的DNA/cDNA为模板,采用本研究建立的多重PCR方法检测,评估该方法的特异性。结果显示,该方法仅对DHBV、DEV、DAdV-3有目的条带，其他病原均无扩增条带。对同一浓度1:1:1等体积混合的3种重组质粒标准品混合物10倍倍比稀释(10'-10*)后作为模板,采用建立的多重PCR方法检测,评估该方法的敏感性,结果显示,该方法对重组质粒标准品pMD-DEV, pMD-DAdV-3和pMD-DHBV的检测限分别是102拷贝/uL、102拷贝/uL和10拷贝/uL。采用本研究建立的多重PCR检测方法,对同一浓度1:1:1等体积混合的重组质粒标准品混合物分别进行批内及批间重复性试验,结果显示,批内和批间重复性试验的检测结果均一致。对115份来自黑龙江省部分地区以及山东省招远市鸭场腹泻雏鸭粪便样品处理后,采用本研究建立的多重PCR方法与已报道的单一及多重PCR方法检测,结果显示DEV、DAdV-3和DHBV的阳性率分别为3.5%(4/115), 10.4%(12/115)和5.2%(6/115), DEV, DAdV-3和DHBV的混合感染率为0.8%(1/115),与已报道检测方法的检测结果一致。上述结果表明本研究建立的方法特异性强,敏感性高,重复性好,可用于临床样品的检测。本研究首次建立了能够同时快速检测DEV、DHBV和DAdV-3的多重PCR方法,为临床检测上述3种病毒提供了技术支撑。

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