A nanoparticle-assisted PCR assay to improve the sensitivity for rapid detection and differentiation of wild-type pseudorabies virus and gene-deleted vaccine strains
A nanoparticle-assisted PCR assay to improve the sensitivity for rapid detection and differentiation of wild-type pseudorabies virus and gene-deleted vaccine strains
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DOI：
10.1016/j.jviromet.2013.07.018
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发表时间：
2013-11-01
影响因子：
3.1
通讯作者：
Cui, Shangjin
中科院分区：
文献类型：
--
作者：
Ma, Xingjie;Cui, Yuchao;Cui, Shangjin
纳米颗粒辅助聚合酶链式反应是一种新的DNA快速扩增方法，因其简便、快速、特异等优点而被广泛应用于病毒检测。建立了一种检测和区分伪狂犬病病毒野生型和基因缺失型伪狂犬病病毒(PRV)的方法。从PRV的保守区筛选出3对特异性扩增片段，分别扩增出431bp(GB)、316bp(GE)和202bp(GG)的特异性扩增片段。用含有特定基因片段的纯化质粒构建的这种方法的灵敏度比传统的PCR高100-1000倍。PRV纳米聚合酶链式反应不能扩增猪细小病毒、猪圆环病毒2型、猪繁殖与呼吸综合征病毒、猪病毒或非洲猪瘟病毒，但与缺失基因的PRV-Bartha-K61扩增出3条预期大小的条带和2条预期大小的条带。从中国所在的7个省采集的110份临床标本中，用纳米聚合酶链式反应和病毒分离方法检测，分别有53%和48%的标本检出野生型伪狂犬病毒。(C)2013爱思唯尔B.V.保留所有权利。
Nanoparticle-assisted polymerase chain reaction (nanoPCR) is a novel method for the rapid amplification of DNA and has been adopted for the detection of virus because of its simplicity, rapidity, and specificity. A nanoPCR assay was developed to detect and differentiate wild-type and gene-deleted pseudorabies virus (PRV). Three pairs of primers for nanoPCR developed in this study were selected from conserved regions of PRV, producing specific amplicons of 431 bp (gB), 316 bp (gE), and 202 bp (gG). The sensitivity of this assay using purified plasmid constructs containing the specific gene fragments was 100-1000 fold higher than conventional PCR. The PRV nanoPCR assay did not amplify porcine parvovirus, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, porcine teschovirus, or African swine fever virus but produced three bands of expected size with PRV and two bands of expected size with the gene-deleted PRV-Bartha-K61. Of 110 clinical samples collected from seven provinces in China, 53% and 48% were positive for wild-type PRV according to the nanoPCR assay and virus isolation, respectively. (C) 2013 Elsevier B.V. All rights reserved.