鸡传染性喉气管炎病毒SYBR Green Ⅰ实时荧光定量PCR检测方法的建立与应用

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中文摘要：鸡传染性喉气管炎病毒(Infectious laryngotracheitis virus，ILTV)以往多感染蛋鸡，近年来在蛋种鸡、商品肉鸡中连续多发，表明该病毒感染谱有扩大风险，因此有必要加强对ILTV的监测。为建立高效、灵敏的ILTV检测方法，本研究以ILTV的TK基因为靶基因设计引物，扩增并构建pMD18-T-TK质粒标准品，建立SYBR Green Ⅰ实时荧光定量PCR检测方法和标准曲线，对其特异性、敏感性和重复性进行验证，并对临床疑似病例进行检测。结果发现，该检测方法特异性良好，与其他常见症状相似病原无交叉反应;最低检测浓度为1。55 拷贝/μL，灵敏度是普通PCR方法的 10 倍;重复性好，批内、批间变异系数在 0。79%～1。83%之间。表明本研究建立的ILTV实时荧光定量PCR方法特异性强、灵敏度高，可为ILTV的临床诊断和流行病学研究等提供有效的检测方法。

外文标题：Establishment and Application of SYBR Green Ⅰ Real-Time Fluorescent Quantitative PCR Method for Chicken Infectious Laryngotracheitis Virus

外文摘要：The chicken infectious laryngotracheitis virus(ILTV)has been prevalent in layer in the past,but it has been continuously isolated in breeders and broilers in recent years,indicating an expanding risk of virus infection spectrum.Therefore,it is necessary to strengthen the monitoring of ILTV.To establish an effi-cient and sensitive ILTV detection method,a pair of specific primers were designed based on the TK gene of ILTV,and then the gene was amplified and the pMD18-T-TK plasmid standard was constructed.A SYBR Green Ⅰ real-time fluorescent quantitative PCR method and standard curve were established.The specificity,sensitivity,and repeatability of the method were tested,and the ILTV suspected cases were detected.The re-sults showed that the method had good specificity with no cross reactivity with other pathogens exhibiting simi-lar symptoms.The minimum detection concentration was 1.55 copies/μL,and the sensitivity of this method was 10 times that of conventional PCR.The repeatability of the method was good with the coefficient of varia-tionsfor intra-and inter batch were from 0.79%to 1.83%.It indicated that the real-time fluorescent quantita-tive PCR method for ILTV detection established in this study had strong specificity and high sensitivity,which could provide an effective detection method for clinical diagnosis and epidemiological researches of ILTV.

外文关键词：

Chicken infectious laryngotracheitis virusTK geneReal-time fluorescent quantitative PCRSpecificitySensitivityRepeatability

作者：

张玉霞、董雯雯、袁小远、孟凯、徐怀英

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作者单位：

山东省农业科学院家禽研究所,山东济南 250100

关键词：

鸡喉气管炎病毒 TK基因实时荧光定量PCR 特异性敏感性重复性

基金：

山东省农业科学院农业科技创新工程项目山东省农业科学院农业科技创新工程项目山东省重点研发计划项目山东省自然科学基金项目

项目编号：

CXGC2023A10CXGC2023A222022LZGC014ZR2021MC060

出版年：

2024

DOI：

10.14083/j.issn.1001-4942.2024.06.017

山东农业科学

山东省农业科学院,山东农学会,山东农业大学

山东农业科学

CSTPCD北大核心

影响因子：0.578

ISSN：1001-4942

年,卷(期)：2024.56(6)