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Volume 43, Number 2, April 2010

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   

 

Corresponding author:

 Department of Nutrition and Health Science, Fooyin University, 151 Chinhsueh Road, Ta-liao City, Kaohsiung County, Taiwan. 

E-mail: citrinin@gmail.com 


 

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.



 

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 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.  



 

Results:

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.



 

Conclusion:

 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.