Bead-based suspension array for simultaneous differential detection of five major swine viruses
| Autor: | Chang-Xu Song, Xiao-Lu Yu, Cun-Yi Xue, Ru Chen, Xiao-Bo Gao, Yong-Chang Cao, Li Yan |
|---|---|
| Rok vydání: | 2014 |
| Předmět: |
Circovirus
Swine Real-Time Polymerase Chain Reaction Applied Microbiology and Biotechnology Sensitivity and Specificity Specimen Handling Classical Swine Fever chemistry.chemical_compound Plasmid Multiplex polymerase chain reaction Animals Multiplex Porcine respiratory and reproductive syndrome virus DNA Primers Detection limit Swine Diseases biology Parvovirus Reproducibility of Results General Medicine Parvovirus Porcine biology.organism_classification Virology Molecular biology Reverse transcription polymerase chain reaction Real-time polymerase chain reaction chemistry Classical Swine Fever Virus DNA Viral RNA Viral Multiplex Polymerase Chain Reaction DNA Biotechnology |
| Zdroj: | Applied microbiology and biotechnology. 99(2) |
| ISSN: | 1432-0614 |
| Popis: | A novel multiplex detection array based on Luminex xMAP technology was developed and validated for simultaneous detection of five major viruses causing swine reproductive diseases. By combining one-step asymmetric multiplex reverse transcription polymerase chain reaction (RT-PCR) with xMAP bead-based hybridization and flow cytometry analysis, the resulting multiplex assay was capable of detecting single and mixed infections of PRRSV, PCV-2, PRV, CSFV, and PPV in a single reaction. The assay accurately detected and differentiated 23 viral strains used in this study. The low detection limit was determined as 2.2–22 copies/μL (corresponding to 0.5–6.8 fg/μL DNA template) on plasmid constructs containing viral fragments. The intra-assay and inter-assay variances (CV%) were low that ranged from 2.5 to 5.4 % and 4.1 to 7.6 %, respectively. The assay was applied to test field samples and detected single and mixed viral infections. The detection rate was higher than that of uniplex conventional PCR and RT-PCR methods. The detection of PRRSV by the bead-based multiplex assay was comparable with a commercially available real time RT-PCR kit. The test procedure on purified DNA or RNA samples could be completed within 2 h. In conclusion, the bead-based suspension array presented here proved to be a high-throughput practical tool that provided highly specific and sensitive identification of single and multiple infections of five major viruses in pigs and boar semen. |
| Databáze: | OpenAIRE |
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