Fast Diagnosis and Quantification for Porcine Circovirus Type 2 (PCV-2) Using Real-Time Polymerase Chain Reaction
| Autor: | Hau-Yang Tsen, Jing-Tsang Chen, Gan-Nan Chang, Jyh-Jye Wang, Jyi-Faa Hwang |
|---|---|
| Jazyk: | angličtina |
| Předmět: |
Microbiology (medical)
Circovirus Time Factors Swine Polymerase Chain Reaction complex mixtures law.invention Porcine Postweaning Multisystemic Wasting Syndrome law Immunology and Microbiology(all) Genotype Immunology and Allergy Animals Transition Temperature Multiplex Polymerase chain reaction DNA Primers General Immunology and Microbiology biology General Medicine Viral Load biology.organism_classification Virology Molecular biology circovirus typing Standard curve Porcine circovirus Real-time polymerase chain reaction Infectious Diseases DNA Viral multiplex real-time PCR Recombinant DNA Nested polymerase chain reaction porcine circovirus type 2 |
| Zdroj: | Journal of Microbiology, Immunology and Infection. (2):85-92 |
| ISSN: | 1684-1182 |
| DOI: | 10.1016/S1684-1182(10)60014-X |
| Popis: | Background/Purpose The postweaning multisystemic wasting syndrome, caused by the porcine circovirus type 2 (PCV-2), is a major disease that poses a significant threat to the global swine industry. The purpose of this study was to establish a real-time polymerase chain reaction (PCR) method for the quantification of PCV-2 and to enable the rapid differentiation of porcine circoviruses type 1 and 2 (PCV-1 and PCV-2). Such a method would significantly speed up the process of clinical diagnosis, and could also be used to study the pathogenic mechanisms of diseases associated with PCV-2. Methods Multiplex real-time PCR, together with LightCycler PCR data analysis software, was used for the quantification of PCV-2, and for the rapid differentiation of PCV-1 and PCV-2. A 263-bp DNA fragment was amplified from the 3′ end of the open reading frame-2 of PCV-2 by nested PCR, and its DNA sequence was verified as having 100% identity with a PCV-2 standard (NCBI accession number: AF055394). The 263-bp DNA fragment was cloned into the pGEM-T easy vector, and the recombinant plasmid was serially diluted and quantified using real-time PCR. A standard curve was then constructed for quantification of the PCV-2 levels in field samples. The differentiation of PCV-1 and PCV-2 was carried out by analyzing the melting temperatures of the genotype-specific PCR products. Results To quantify the PCV-2 levels in field samples, a standard curve (1 × 10 2 −1 × 10 9 copies/μL) was constructed. PCV-2 concentrations as low as 1 × 10 2 copies/mL could be detected in specimens taken from the lymph nodes or infected tissues in samples of PCV-2-infected pigs. The diagnosis of PCV-1 and PCV-2 infections and the quantification of the viral load in the field samples could be completed within 45 minutes after extracting the viral DNA using a commercial extraction kit. Conclusion This study demonstrate that real-time PCR is a clinically feasible method for the accurate quantification of PCV-2, and for the rapid differentiation of PCV-1 and PCV-2. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|