文献类型： 外文期刊

作者： Chen, Ru ¹ ; Gao, Xiao-Bo ² ; Yu, Xiao-Lu ³ ; Song, Chang-Xu ⁴ ; Qiu, Yang ¹ ;

作者机构： 1.Guangdong Entry Exit Inspect & Quarantine Bur, Ctr Tech, 66 Huacheng Dadao Ave, Guangzhou 510623, Guangdong, Peoples R China

2.Natl Res Inst Family Planning, Dept Genet, Beijing 100081, Peoples R China

3.Sun Yat Sen Univ, Sch Life Sci, Guangzhou 510675, Guangdong, Peoples R China

4.Guangdong Acad Agr Sci, Vet Med Inst, Guangzhou 510640, Guangdong, Peoples R China

关键词： Locked nucleic acid (LNA);Universal primer;Multiplex PCR (mPCR);Swine virus

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

ISSN：

年卷期：

页码：

收录情况： SCI

摘要： A novel multiplex PCR assay using non-homologous oligonucleotides with locked nucleic acid (LNA) modifications as universal primers was developed and validated for the simultaneous detection of five swine viruses. The assay utilizes five virus-specific primer pairs modified at the 5' end through the addition of the universal primer sequence. In the reaction, small amounts of target templates with the 5' tail were generated and subsequently amplified through the extension of a LNA universal primer set. To validate the specificity of this assay, 27 viral target strains and 12 non-target pathogens were tested. The lower limit of detection of viral nucleic acids was 1.1-1.9 pg per reaction or 11-32 pg in a five-plex viral nucleic acid mixture. The LNA mPCR assay displayed higher analytical sensitivity and efficiency for the detection of plasmid standards compared with the conventional assay, which uses standard primers without the 5' tail. A total of 207 field samples were tested using both assays. The LNA mPCR assay provided numerically higher detection rates for all pathogens in independent samples. Moreover, the LNA mPCR assay had significantly higher detection rates in independent samples compared with the conventional assay. (C) 2015 Published by Elsevier B.V.