Allele-specific qRT-PCR demonstrates superior detection of single nucleotide polymorphisms as genetic markers for West Nile virus compared to Luminex® and quantitative sequencing

被引：6

作者：

Worwa, Gabriella ^{[1
]}

Andrade, Christy C. ^{[1
]}

Thiemann, Tara C. ^{[1
]}

Park, Bborie ^{[1
]}

Maharaj, Payal D. ^{[2
]}

Anishchenko, Michael ^{[2
]}

Brault, Aaron C. ^{[2
]}

Reisen, William K. ^{[1
]}

机构：

[1] Univ Calif Davis, Sch Vet Med, Dept Pathol Microbiol & Immunol, Ctr Vectorborne Dis, Davis, CA 95616 USA

[2] Ctr Dis Control & Prevent, Div Vector Borne Dis, Ft Collins, CO 80521 USA

来源：

JOURNAL OF VIROLOGICAL METHODS | 2014年 / 195卷

基金：

瑞士国家科学基金会; 美国国家卫生研究院;

关键词：

West Nile virus; Genetic marker; Luminex (R) technology; Quantitative sequencing; qRT-PCR; Fitness competition; VIRULENCE; EVOLUTION; REPLICATION; MUTATIONS; MOSQUITOS; ARBOVIRUS; ISOLATE; CELLS;

D O I：

10.1016/j.jviromet.2013.09.014

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

To enable in vivo and in vitro competitive fitness comparisons among West Nile viruses (WNV), three reference viruses were marked genetically by site-directed mutagenesis with five synonymous nucleotide substitutions in the envelope gene region of the genome. Phenotypic neutrality of the mutants was assessed experimentally by competitive replication in cell culture and genetic stability of the substituted nucleotides was confirmed by direct sequencing. Luminex (R) technology, quantitative sequencing and quantitative RT-PCR (qRT-PCR) were compared in regard to specificity, sensitivity and accuracy for quantitation of wildtype and genetically marked viruses in mixed samples based on RNA obtained from samples of known viral titers. Although Luminex (R) technology and quantitative sequencing provided semi-quantitative or qualitative measurements, a sequence-specific primer extension approach using a specific reverse primer set in singleplex qRT-PCR demonstrated the best quantitation and specificity in the detection of RNA from wildtype and mutant viruses. (C) 2013 Elsevier B.V. All rights reserved.

引用

页码：76 / 85

页数：10