The development and evaluation of cross-priming amplification for the detection of avian reovirus
The development and evaluation of cross-priming amplification for the detection of avian reovirus
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DOI：
10.1111/jam.12705
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发表时间：
2015-02-01
影响因子：
4
通讯作者：
Gawel, A.
中科院分区：
文献类型：
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作者：
Wozniakowski, G.;Niczyporuk, J. S.;Gawel, A.
目的根据禽呼肠孤病毒（Avian Reovirus，ARV）S1133株的sigma NS基因序列，设计5条特异性引物，建立检测ARV的交叉引物扩增（cross-priming amplification，CPA）方法，并对CPA方法进行初步评价。确定孵育温度和引物浓度。最佳水浴条件为613 ℃水浴45 min。不需要逆转录阶段。在UV光照射下，阳性样品的结果记录为明亮的绿色荧光，阴性对照和样品则无此荧光。此外，在分析期间进行的凝胶电泳显示存在由发夹状CPA产物形成的梯状图案。开发的CPA方法进行了比较，逆转录聚合酶链反应（RT-PCR）和实时RT-PCR。用标准S1133菌株的7个稀释液估算CPA的灵敏度，其灵敏度可达0.05log（10）TCID（50）ml（-1）。RT-PCR的灵敏度达到25 log（10）TCID（50）ml（-1），比CPA低1000倍，而实时RT-PCR的灵敏度达到15 log（10）TCID（50）ml（-1）。对从野外标本中提取的32个RNA的分析显示，在4个（125%）样品中存在ARV sigma NS片段。有趣的是，阳性样品来自感染马立克氏病（MD）或禽腺病毒（FadV）的鸡群。由于RT-PCR的敏感性较低，无法检测到ARV。然而，进行的实时RT-PCR证实了CPA study.ConclusionsCPA是一个非常敏感和快速的方法，它允许使用简单的实验室设备ARV检测。意义和影响的StudyThis是第一次报告的应用程序的CPA方法检测ARV，使用简单的实验室设备。
AimsThe aim of this study was to develop and evaluate cross-priming amplification (CPA) for the detection of avian reovirus (ARV).Methods and ResultsFive specific primers were designed, on the basis of the sigma NS sequence of the S1133 ARV strain. Incubation temperature and primer concentrations were determined. The optimal incubation conditions in a water bath were 613 degrees C for 45min. No reverse transcription stage was required. The results were recorded under UV light illumination as a bright, greenish fluorescence in positive samples, and through the lack of this in negative controls and samples. Additionally, the gel electrophoresis performed during analysis showed the presence of ladder-like patterns, formed by hairpin-like CPA products. The developed CPA method was compared to reverse-transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR. Sensitivity of CPA was estimated using seven dilutions of standard S1133 strain and reached 005log(10) TCID(50)ml(-1). RT-PCR sensitivity reached 25log(10) TCID(50)ml(-1) and was 1000 times lower than for CPA, whereas real-time RT-PCR sensitivity reached 15log(10) TCID(50)ml(-1). Analysis of 32 RNAs extracted from field specimens showed the presence of an ARV sigma NS fragment in 4 (125%) samples. Interestingly, the positive samples originated from flocks affected by Marek's disease (MD) or fowl adenovirus (FadV). RT-PCR was unable to detect ARV, due to its lower sensitivity. However, the real-time RT-PCR that was conducted confirmed the CPA study.ConclusionsCPA is a very sensitive and rapid method, which allows ARV detection using simple laboratory equipment.Significance and Impact of the StudyThis is the first report on the application of the CPA method for detection of ARV, using simple laboratory equipment.