Detection Establishment And Application Of SYBR Green Real-Time Polymerase Chain Reaction For Porcine Parvovirus

Porcine parvovirus (PPV) causes reproductive failure in swine, manifested as embryonic resorption, fetal mummification, abortion and stillbirths. The virus is ubiquitous among swine throughout the world and is enzootic in most herds that have been tested. In this research, the PCR method and fluorescence quantitative PCR for the detection of PPV was developed:1. Using PCR assay, a 194 bp region of the porcine parvovirus VP2 gene was cloned to pGEM T vector, and the constructed plasmid was named pGEM-VP2. Serial dilutions of plasmid pGEM-VP2 were used to quantify the virus genomic copy number, and served as standards curve. We developed a SYBR GreenⅠreal-time PCR to detect porcine parvovirus. Sensitivity analysis showed that the developed SYBR Green I real-time PCR could detect 1.0×102 template/μL. The assay exhibited specificity as all negative controls and other porcine pathogens, such as PRRSV, PCV, JEV, PRV, HCV and SIV showed negative results. It is shown the real-time PCR results of 10 suspicious positive samples are positive. However, it was 7 positive by normal PCR. The results suggested that as a result of the sensitivity and specificity of the assay with a rapid and simple procedure, the real-time PCR would be useful for the clinical diagnosis of porcine parvovirus infection.2. HN1 Strain of Porcine Parvovirus Virus was inoculated in PK-15 Cells, some multiplication properties of PPV were studied by using TaqMan fluorescent quantitative real-time PCR. The results showed that after inoculating, the virus began to proliferate at 2 hours, proliferate in the fastest speed from 12 to 60 hours and to the highest rate at 60 hours. The results also found that the virus began to release in 6 hours and reached the highest rate at 72 hours. These results provided basics for vaccines producing and researching the pathology of Porcine Parvovirus Virus.3.To detect porcine parvovirus (PPV) in semen, various DNA extraction techniques have been utilized for PCR, but rarely compared, to determine an optimized extraction protocol. Due to the high content of protein, difficulties can be encountered in obtaining DNA from the seminal cell fraction. This study compared three methods of DNA extractions. All extractions were compared on serially diluted seminal cell fractions and evaluated their ability to extract DNA by real-time PCR. The Trizol method resulting in recovery of the highest amount of DNA, but this reagent was quite expensive and determined not to be used for routine diagnostic testing. When compared the results of the other two methods, the cooking extraction method was more effective than guanidine thiocyanate method. The analysis result indicated that cooking extraction method is a quick, simple, economic and efficient method for DNA extraction and these made it suitable for routine diagnostic testing.