Comparison of RNA extraction and real-time reverse transcription polymerase chain reaction methods for the detection of Porcine reproductive and respiratory syndrome virus in porcine oral fluid specimens
Comparison of RNA extraction and real-time reverse transcription polymerase chain reaction methods for the detection of Porcine reproductive and respiratory syndrome virus in porcine oral fluid specimens
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DOI：
10.1177/104063871102300208
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发表时间：
2011-03-01
影响因子：
1.5
通讯作者：
Zimmerman, Jeffrey J.
中科院分区：
文献类型：
--
作者：
Chittick, Wayne A.;Stensland, Wendy R.;Zimmerman, Jeffrey J.
本研究的目的是评价不同的RNA提取和聚合酶链式反应(PCR)方法检测猪口服液中猪繁殖与呼吸综合征病毒(PRRSV)的方法。提取方案的选择是基于易用性和与高通量、自动化系统的兼容性。结果表明，在检测口服液基质中的PRRSV时，提取方案、PCR方案及其组合之间存在显著差异。一个重要的发现是，聚合酶链式反应被口服液基质中的未知因素部分抑制，并且使用较高浓度的聚合酶链式反应酶抑制作用减弱。结果表明，需要进一步优化猪口服液的PCR检测方法，实验室不应假设优化的血清病毒检测方法将与猪口服液同样有效。
The objective of the current study was to evaluate various RNA extraction and polymerase chain reaction (PCR) protocols for the detection of Porcine reproductive and respiratory syndrome virus (PRRSV) in porcine oral fluids. Extraction protocols were selected based on ease of use and compatibility with high-throughput, automated systems. The results showed marked differences among extraction protocols, PCR protocols, and combinations thereof in detecting PRRSV in the oral fluid matrix. An important finding was that PCR reactions were partially inhibited by unknown factors in the oral fluid matrix and that inhibition was reduced by use of a higher concentration of PCR enzymes. The results suggest that further optimization of PCR assays for porcine oral fluids is needed and that laboratories should not assume that methods optimized for detection of virus in serum will perform equally with porcine oral fluids.