Establishment of a visualized detection method of Sendai virus by reverse transcription loop-mediated isothermal amplification
Received:February 02, 2016  
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DOI:10.3969/j.issn.1005-4847.2016.03.015
KeyWord:Sendai Virus;RT-LAMP;Real time;Rapid detection
                 
AuthorInstitution
周洁 上海实验动物研究中心, 上海
赵丽娟 上海实验动物研究中心, 上海 ;扬州大学兽医学院, 扬州
陶凌云 上海实验动物研究中心, 上海
倪丽菊 上海实验动物研究中心, 上海
高诚 上海实验动物研究中心, 上海
陈洪岩 中国农业科学院哈尔滨兽医研究所实验动物中心, 哈尔滨
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Abstract:
      Objective To establish a simple and sensitive detection method of Sendai virus (SeV) by reverse transcription loop-mediated isothermal amplification (RT-LAMP) technique.Methods According to the published GenBank sequences (DQ219803.1), six pairs of primers were designed targeting the conserved region of SeV. The amplification products were detected with a LAMP real-time Turbidimeter.(LA-302). Through optimizing the LAMP primers and reaction conditions, a rapid and specific detection method of SeV was established.Meanwhile, the amplified products were colored by fluorescence detection reagent after completion of the reaction, so that the amplification could be visualized and detected by naked eyes. Then, methodological evaluation of the RT-LAMP was tested. Results The method of RT-LAMP showed a highly efficient amplification for SeV viral target gene which was performed at 63℃ for 60 min with the LAMP real-time Turbidimeter (LA-302).The detection limit was 2.1 TCID50, 100 times higher than that of RT-PCR, and no cross-reaction with other RNA and DNA viruses of mice was observed. The results of SeV LAMP reaction was visualized and the tube could be directly observed by naked eyes with the addition of fluorescence detection reagent. The results were consistent with the results detected by real-time tubidimeter.92 clinical samples were detected byRT-LAMP, RT-PCR and indirect ELISA, and the coincidence rate was 100%.Conclusions This established SeV RT-LAMP detection method is fast, specific, highly sensitive,easy to perform under simple conditions, and is suitable for rapid detection of Sendai viirus.
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