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Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction
Published on: November 1, 2018
Fast diagnosis and quantification for porcine circovirus type 2 (PCV-2) using real-time polymerase chain reaction
Gan-Nan Chang1, Jyi-Faa Hwang, Jing-Tsang Chen
1Department of Biotechnology, Tajen University, Pingtung, Taiwan.
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 x 10(2) -1 x 10(9) copies/microL) was constructed. PCV-2 concentrations as low as 1 x 10(2) copies/microL 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.
