Novel multiplex PCR assay using locked nucleic acid (LNA)-based universal primers for the simultaneous detection of five swine viruses
| Autor: | Chang-Xu Song, Qiu Yang, Xiao-Bo Gao, Xiao-Lu Yu, Ru Chen |
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| Rok vydání: | 2015 |
| Předmět: |
0301 basic medicine
Circovirus Swine 030106 microbiology Oligonucleotides Biology Sensitivity and Specificity 03 medical and health sciences Plasmid Limit of Detection Virology Multiplex polymerase chain reaction Animals Porcine respiratory and reproductive syndrome virus Locked nucleic acid DNA Primers Detection limit Swine Diseases Oligonucleotide biology.organism_classification Molecular biology Herpesvirus 1 Suid 030104 developmental biology Classical Swine Fever Virus Virus Diseases Viruses Nucleic acid Primer (molecular biology) Multiplex Polymerase Chain Reaction |
| Zdroj: | Journal of virological methods. 228 |
| ISSN: | 1879-0984 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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