Fast Diagnosis and Quantification for Porcine Circovirus Type 2 (PCV-2) Using Real-Time Polymerase Chain Reaction
Gan-Nan Changa, Jyi-Faa Hwanga, Jing-Tsang Chena, Hau-Yang Tsenb, Jyh-Jye Wangc
Received: March 10, 2009 Revised: May 1, 2009 Accepted: June 11, 2009
Department of Nutrition and Health Science, Fooyin University, 151 Chinhsueh Road, Ta-liao City, Kaohsiung County, Taiwan.
Background and 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 quanti-fication 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.
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 frag-ment was amplified from the 3’ end of the open reading frame-2 of PCV-2 by nested PCR, and its DNA se-quence 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 quantifica-tion 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.
To quantify the PCV-2 levels in field samples, a standard curve (1 × 102–1 × 109 copies/μL) was constructed. PCV-2 concentrations as low as 1 ×102 copies/μL 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 in-fections 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.