应用荧光定量PCR方法检测伪狂犬病病毒弱毒株在猪体组织中的分布

Detection of in-tissue distribution of an attenuated pseudorabies virus strain in piglets using a real-time quantitative PCR method

为了解伪狂犬病病毒(PRV)TK/PK/gE三基因缺失弱毒株在猪体组织中的分布情况,针对PRV gB基因保守区设计并合成1对特异性引物,建立并优化了一种可快速、定量检测PRV的SYBR-GreenⅠ荧光定量PCR方法。该检测方法特异性强且敏感度高,最低检测浓度为10^(1)拷贝/μL。PRV TK/PK/gE三基因缺失弱毒株免疫仔猪后可少量存在于大脑、小脑、肝脏、脾脏、肺脏、扁桃体和颌下淋巴结中,部分仔猪鼻甲骨病毒含量较高。应用本研究建立的荧光定量PCR方法阳性检出率为79.2%,而常规PCR方法阳性检出率仅为45.8%。结果表明,本研究建立的荧光定量PCR方法为了解弱毒株在猪体的分布提供了快速、敏感的检测手段。PRV TK/PK/gE三基因缺失弱毒株在猪组织中的分布情况为进一步揭示其组织嗜性、安全性和免疫机制提供了数据。

To explore the distribution of a pseudorabies virus(PRV) TK/PK/gE deletion strain in inoculated piglets, a pair of primers specific to the gB gene were designed and synthesized, and a SYBR-Green Ⅰ real-time quantitative PCR method was developed. The established method seemed to be of high sensitivity and strong specificity. Its lowest detection limit was as low as 10^(1) copies/mL. The PRV TK/PK/gE deletion strain might be detected in small amounts in the brain, cerebellum, liver, spleen, lung, tonsils, and submandibular lymph nodes of the inoculated piglets, and a higher viral load in the turbinate in some inoculated piglets. Comparison of the detection results between the SYBR-Green Ⅰ real-time quantitative PCR and conventional PCR showed that the positive rates of these two methods were 79.2% and 45.8%, respectively. In conclusion, the SYBR-Green Ⅰ real-time quantitative PCR method established in this study could be used in rapid and accurate detection of the PRV TK/PK/gE deletion strain. The in-tissue distribution of the PRV TK/PK/gE deletion strain in pigs would provide informative data for further revealing the tissue tropism, safety and immune mechanism of the PRV TK/PK/gE deletion strain.