Development and evaluation of reverse transcription loop-mediated isothermal amplification assay for the detection of the fathead minnow nidovirus

文献类型： 外文期刊

第一作者： Zhang, Qingli

作者： Zhang, Qingli;Zhang, Qingli;Standish, Isaac;Winters, Andrew D.;Faisal, Mohamed;Winters, Andrew D.;Faisal, Mohamed;Puzach, Corey;Ulferts, Rachel;Ziebuhr, John;Ziebuhr, John

作者机构：

关键词： Fathead minnow nidovirus;Reverse transcription loop-mediated;isothermal amplification;Real-time;Quantitative RT-LAMP;Calcein

期刊名称：JOURNAL OF VIROLOGICAL METHODS （ 影响因子：2.014； 五年影响因子：2.001 ）

ISSN： 0166-0934

年卷期： 2014 年 202 卷

页码：

收录情况： SCI

摘要： Fathead minnow nidovirus (FHMNV) is a serious baitfish-pathogenic virus in North America. Studies to trace the spread of the virus and determine its host range are hampered by the absence of reliable diagnostic assays. In this study, a one-step, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed that targets a region in the FHMNV spike protein gene. The assay was optimized, and the best results were obtained at 8 mM of Mg2+ with an incubation time of 40 min at 63 degrees C in the presence of calcein. The analytical sensitivity of the RT-LAMP method was estimated to be as low as 5 viral copies and was 1000-fold more sensitive than the conventional reverse transcription polymerase chain reaction (RT-PCR) method. The diagnostic sensitivity and specificity of the developed RT-LAMP assay versus the RT-PCR assay was 100% and 95.7%, respectively. A quantitative RT-LAMP of FHMNV with a high correlation coefficient (r(2) = 0.9926) was also developed and the result of quantitation of viral copies in tissue samples of infected fish showed that the viral loads of the infected fish tissue samples reached up to 4.7 Chi 10(10) copies per mg. It is anticipated that the developed RT-LAMP and quantitative RT-LAMP methods will be instrumental for diagnosis and surveillance of FHMNV. (C) 2014 Elsevier B.V. All rights reserved.

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