Development and Application of Real-Time PCR for Detection of Subgroup J Avian Leukosis Virus
Development and Application of Real-Time PCR for Detection of Subgroup J Avian Leukosis Virus
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贾亚群禽白血病病毒实时荧光定量PCR检测方法的建立及应用
DOI：
10.1128/jcm.02030-12
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发表时间：
2013-01-01
影响因子：
9.4
通讯作者：
Wang, Xiaomei
中科院分区：
文献类型：
--
作者：
Qin, Liting;Gao, Yulong;Wang, Xiaomei
J亚型禽白血病病毒（ALV-J）是一种禽类逆转录病毒，给家禽业造成严重的经济损失。早期识别和清除脱落病毒的禽类对减少先天性感染和接触性感染的传播非常重要。本研究建立了一种基于taqman的实时PCR方法，利用原病毒DNA快速检测和定量ALV-J。该方法对ALV-J具有较高的特异性。此外，检测限低至10个病毒DNA拷贝。试验间和试验内重复性的变异系数（cv）均小于1%。实时荧光定量PCR检测ALV-J在DF-1细胞中的生长曲线，与50%组织培养感染剂量（TCID50）和p27抗原检测的趋势线相似。对疑似ALV感染的组织标本进行实时PCR、病毒分离和常规PCR检测，阳性率分别为60.1%、41.6%和44.5%。我们的数据表明，实时PCR方法为临床诊断和实验室研究提供了一种敏感、特异性和可重复性的诊断工具，用于ALV-J的鉴定和定量。
Subgroup J avian leukosis virus (ALV-J) is an avian retrovirus that causes severe economic losses in the poultry industry. The early identification and removal of virus-shedding birds are important to reduce the spread of congenital and contact infections. In this study, a TaqMan-based real-time PCR method for the rapid detection and quantification of ALV-J with proviral DNA was developed. This method exhibited a high specificity for ALV-J. Moreover, the detection limit was as low as 10 viral DNA copies. The coefficients of variation (CVs) of both interassay and intra-assay reproducibility were less than 1%. The growth curves of ALV-J in DF-1 cells were measured by real-time PCR, yielding a trend line similar to those determined by 50% tissue culture infective dose (TCID50) and p27 antigen detection. Tissue samples suspected of ALV infection were evaluated using real-time PCR, virus isolation, and routine PCR, and the positivity rates were 60.1%, 41.6% and 44.5%, respectively. Our data indicated that the real-time PCR method provides a sensitive, specific, and reproducible diagnostic tool for the identification and quantification of ALV-J for clinical diagnosis and in laboratory research.