Multiplex-PCR protocol development for rapid screening of white spot syndrome virus (WSSV) in shrimp

IF 1 Q4 FISHERIES
Sharmin Aktar, M. Parvez, H. Islam, M. Ahsan
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引用次数: 5

Abstract

This study was aimed to develop a faster single step multiplex PCR protocol for the simultaneous detection of white spot syndrome virus (WSSV) with its host (i.e. shrimp) as internal positive control. To do so, four combinations of primer were tested (I. 16S rRNA+Lo F1R1; II. 16S rRNA+Lo F2R2; III. 16S rRNA+Lo F1R2; IV. 16S rRNA+Lo F2R1) which were selected based on two pairs of WSSV specific primer (Lo F1R1 and Lo F2R2) and one pair of shrimp specific primer (16S rRNA). DNA extracted from WSSV infected shrimp were amplified by PCR in a single tube using each of the primer combinations and the thermal cycling conditions as well as reagent compositions were optimized. All the primer combinations yielded their expected band sizes with stronger band resolution intensity that indicated the development of four multiplex PCR protocols. The developed multiplex protocols reduced the chance of cross contamination and these were found to be faster, single step and unique with less effort and resource use. Considering sensitivity and specificity, among the protocols, we suggested the protocols based on 16S rRNA+Lo F1R1 and/or 16S rRNA+Lo F2R2 primer combinations for rapid and routine screening of WSSV in shrimp PL, juvenile and adult.
快速筛选对虾白斑综合征病毒(WSSV)的多重pcr方法的建立
本研究旨在建立一种快速的单步多重PCR方法,用于同时检测白斑综合征病毒(WSSV),其宿主(虾)为内阳性对照。为此,我们测试了四种引物组合(I. 16S rRNA+Lo F1R1;216S rRNA+Lo F2R2;316S rRNA+Lo F1R2;IV. 16S rRNA+Lo F2R1),根据两对WSSV特异性引物(Lo F1R1和Lo F2R2)和一对对虾特异性引物(16S rRNA)选择。利用不同引物组合在单管中扩增WSSV侵染虾的DNA,并对热循环条件和试剂组成进行优化。所有引物组合都获得了预期的条带大小和更强的条带分辨率强度,这表明了四种多重PCR方案的发展。开发的多路协议减少了交叉污染的机会,并且发现这些协议更快,一步单一,独特,更少的努力和资源使用。考虑敏感性和特异性,我们建议采用16S rRNA+Lo F1R1和/或16S rRNA+Lo F2R2引物组合快速常规筛选对虾PL、幼虾和成虾WSSV。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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22
审稿时长
8 weeks
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