Development and application of SYBR Green I real-time PCR assay for the separate detection of subgroup J Avian leukosis virus and multiplex detection of avian leukosis virus subgroups A and B.
Development and application of SYBR Green I real-time PCR assay for the separate detection of subgroup J Avian leukosis virus and multiplex detection of avian leukosis virus subgroups A and B.
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DOI：
10.1186/s12985-015-0291-7
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发表时间：
2015-04-03
期刊：
影响因子：
4.8
通讯作者：
Liao M
中科院分区：
文献类型：
--
作者：
Dai M;Feng M;Liu D;Cao W;Liao M
A、B 和 J 亚组 ALV 是最流行的禽白血病病毒 (ALV)。我们的研究尝试开发两种基于 SYBR Green I 的实时 PCR (RT-PCR) 检测方法，分别用于 ALV J 亚型 (ALV-J) 的特异性检测和 ALV A 亚型和 B 亚型 (ALV-A/B) 的多重检测。这两种检测对 ALV-J 和 ALV-A/B 表现出高度特异性，并且两种检测的灵敏度比常规 PCR 检测至少高 100 倍。病毒培养、常规PCR和实时PCR检测ALV-A株的最低病毒检出限分别为103TCID50单位、102TCID50单位和小于10TCID50单位。此外，测定内和测定间的变异系数均小于5%。对40份临床血浆样本进行实时荧光定量PCR、常规PCR和病毒培养检测，阳性率分别为80%（32/40）、72.5%（29/40）和62.5%（25/40）。当使用ALV-J检测方法对接种ALV-J毒株CHN06和NX0101的鸡各器官组织的病毒载量进行定量时，结果显示，在所有检查的器官组织中都可以检测到ALV-J基因，并且感染后30周时，ALV-J的最高拷贝数主要出现在心脏和肾脏样本中。除肺外，各器官组织中CHN06组的病毒拷贝数均高于NX0101组。基于 SYBR Green I 的实时 RT-PCR 检测为 ALV 检测以及病毒复制和感染研究提供了强大的工具。
Subgroup A, B, and J ALVs are the most prevalent avian leukosis virus (ALV). Our study attempted to develop two SYBR Green I-based real-time PCR (RT-PCR) assays for specific detection of ALV subgroup J (ALV-J) and multiplex detection of ALV subgroups A and B (ALV-A/B), respectively. The two assays showed high specificity for ALV-J and ALV-A/B and the sensitivity of the two assays was at least 100 times higher than that of the routine PCR assay. The minimum virus detection limit of virus culture, routine PCR and real-time PCR for detection of ALV-A strain was 103 TCID50 units, 102 TCID50 units and fewer than 10 TCID50 units, respectively. In addition, the coefficients of variation for intra- and inter-assay were both less than 5%. Forty clinical plasma samples were evaluated by real-time PCR, routine PCR, and virus culture with positive rates of 80% (32/40), 72.5% (29/40) and 62.5% (25/40), respectively. When the assay for detection of ALV-J was used to quantify the viral load of various organ tissues in chicken inoculated by ALV-J strains CHN06 and NX0101, the results exhibited that ALV-J genes could be detected in all organ tissues examined and the highest copies of ALV-J were mainly in heart and kidney samples at 30 weeks post-infection. Except in lung, the virus copies of CHN06 group were higher than that of NX0101 group in various organ tissues. The SYBR Green I-based real-time RT-PCR assay provides a powerful tool for the detection of ALV and study of virus replication and infection.
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影响因子：
3.3
作者：
Li, Delong;Qin, Liting;Gao, Yulong
通讯作者：
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影响因子：
3.8
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影响因子：
1.4
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作者：
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