鸭甲型肝炎病毒1型与3型双重TaqMan实时荧光定量PCR检测方法的建立与应用

旨在建立一种针对鸭甲肝病毒1型（DHAV-1）和鸭甲肝病毒3型（DHAV-3）的高效快速、高灵敏度和高特异性的双重TaqMan实时荧光定量PCR （q-PCR）检测方法并应用于临床疑似样品检测。根据DHAV-1和DHAV-3的VP1基因保守区域，分别设计合成了2对特异性引物和探针，在此基础上建立并优化了双重TaqMan实时荧光定量PCR方法。结果显示：优化后标准曲线的相关系数（R²）均在0.999以上，扩增效率（E）在105%~110%，其组内变异系数（i-CV）与组间变异系数（c-CV）均在0.77%以下。运用双重q-PCR方法对不同稀释度的混合质粒与病毒核酸进行检测，结果表明，建立的双重TaqMan q-PCR特异性高；同时检测DHAV-1和DHAV-3的灵敏度均可达10个拷贝，分别是常规PCR方法的1 000倍和100倍。对40份来自苏中、苏北地区的疑似鸭肝炎病毒感染的鸭组织病料进行检测，结果表明，q-PCR方法检出36份阳性病料，阳性率为90%；而常规PCR方法未能检出高Ct值的样品，阳性率为72.5%（29份阳性病料）。建立的双重TaqManq-PCR检测方法为DHAV-1与DHAV-3的临床样品检测提供了高效、灵敏、特异的工具，推动了临床分子流行病学调查及病毒定量分析等研究。

Establishment and Clinical Application of TaqMan Dual Real-time Fluorescent Quantitative PCR for Detection of Duck Hepatitis A Virus Type 1 and 3

This study aims to establish a high-efficiency, rapid, high-sensitivity and high-specificity TaqMan double real-time fluorescence quantification for duck hepatitis virus type 1 (DHAV-1) and duck hepatitis virus type 3 (DHAV-3) PCR (q-PCR) detection method and applied to the detection of clinical suspected samples. According to the conservative regions of the VP1 gene of DHAV-1 and DHAV-3, two pairs of specific primers and probes were designed and synthesized respectively. On this basis, the TaqMan dual real-time fluorescent quantitative PCR method was established and optimized. Results were as follows: The correlation coefficient (R²) of the optimized standard curve is above 0.999, the amplification efficiency (E) is between 105% and 110%, and the coefficient of variation (i-CV) and the coefficient of variation between groups (C-CV) are all below 0.77%. The double q-PCR method was used to detect mixed plasmids and viral nucleic acids of different dilutions. The results showed that the established TaqMan double q-PCR has high specificity; the sensitivity of simultaneous detection of DHAV-1 and DHAV-3 can reach 10 copies, Which are 1 000 times and 100 times higher than conventional PCR methods. Forty tissue samples of duck suspected of duck hepatitis virus infection from central Jiangsu and northern Jiangsu were tested. The results showed that 36 of them were positive by q-PCR method, with a detection rate of 90%; while conventional PCR methods failed to detect samples with high Ct values, and the detection rate was 72.5% (29 positive samples).The establishment of the TaqMan dual q-PCR detection method provides an efficient, sensitive, and specific tool for the detection of clinical samples of DHAV-1 and DHAV-3, and promotes clinical molecular epidemiological investigation and viral quantitative analysis.