Development Of Multiplex SYBR Green Ⅰ-Based Real-Time PCR Assay For Detection Of Porcine Pseudorabies, Parvovirus And Circovirus Type 2

Porcine Pseudorabies (PRV), porcine parvovirus (PPV), and porcine circovirus type 2 (PCV-2) are major etiological agents of reproductive failure in swine. PRV manifested as fever, encephalomyelitis and other neurological symtoms in a variety of livestock and wild animals and embryoic resorption, fetal mummification, abortion and stillbirths in sow. PPV infections in pigs may cause fetal death and mummification, still births and other reproductive failures in pregnant sows, and dermatitis and diarrhea in piglets. PCV2 was considered being associated with Postweaning Multisystemic Wasting Syndrome (PMWS) in piglet, reproductive failure, dermatitis and enteritis in sow which was broken out all over the world in recent years.Conventional virus isolation and serogical method are time consuming and are comparatively low in sensitivity. These can not detect exactly and latency infection. Althought, there have been report that TaqMan-based real-time quantitative PCR for detection of PPV, PRV or PCV2. But this mothed is expersive cost, and need especial probe. Therefor, quantitive real-time PCR with a quick, cheap, sensitive, and specific test for detection of PRV, PPV and PCV-2 and duplex or multiplex for the simultaneous detection of two or three viruses would be urgently useful. SYBR Green I is a dye that binds in the minor groove of any double stranded DNA irrespective of the DNA sequence, this avoiding potential failres to detect the amplicon due to sequence variation. However, SYBR Green-based assays are considered less specific than probe-based assays because non-specific amplification and the formation of primers can lead to erroneous results. These unspecific fluorecence signals would not even be detected by probe-based systems. Nevertheless, melting curve analysis (MCA) has been reported as a simple, straightforward way to check SYBR Green-based reactions for artefacts and to ensure reaction specificity. SYBR Green real-time PCR is considered a flexible approach that can be directly applied to any gene without the need to design and synthesize fluorescently labelled target-specific probes.1. Using PCR assay, a 187 bp region of the Pseudorabies virus gH gene was cloned to pGEM T vector, and the constructed plasmid was named pGEM-gH. Serial dilutions of plasmid pGEM-gH were used to quantify the virus genomic copy number, and served as standards curve. We developed a SYBR Green I real-time PCR to detect. Pseudorabies virus sensitivity analysis showed that the developed SYBR Green I real-time PCR could detect 1.26×102 template/μl. The assay exhibited specificity as all negative controls and other porcine pathogens, such as PRRSV, PPV, PCV, CSFV showed negative results. It is shown the real-time PCR results of 15 suspicious positive samples are positive. However, it was 6 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 pseudorabies virus infection.2. 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 Green I real-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.3. According to genome sequences within ORF2 of porcine circovirus type 2 (PCV-2) published in GenBank, A pair of primers were designed. The ORF2 gene which was highly conserved in porcine circovirus type 2 was amplified with traditional PCR, and cloned into T vector which was transformed into E. col i JM109. Following extracting and identifying the positive recombinant plasmid was used as quantitative template to generate standard curve. Sensitivity assay,reproducibility of the assay and specificity assay were determined. The results demonstrated that standard curve established by recombinant plasmid showed a fine linear relationship between threshold cycle and template concentration,melt curve was specificity and the correlation coefficient was 0.9988,the sensitive degree is 1 copies per 25μl,and the quantitative PCR was highly reproducibility and more reproducibility than traditional PCR. A SYBR Green 1 fluorescent quantitative PCR for detecting ORF2 gene of PCV2 was developed for the basis of the early and rapid detection and analysising the infect degree of PCV2 quantitatively.4. In this study, a duplex real-time PCR for simultaneous detection of porcine pavovirus and porcine pseudorabies virus base on SYBR Green was developed and evaluted. The sets of primers were targeted to conserved gH gene of PRV, Non-structional protein (NS1) gene of PPV and used to amplify. The sensitivity, specificity and interference test of duplex real-time PCR assay was analyzed. It was shown that the detection levels of 248/μl copies of PRV, and 160 copies/μl of PPV were achieved, and there was no non-specific amplification and cross-reactivity using cDNA of HCV, PRRSV and JEV. In conclusion, this assay provides an effective etiological diagnostic tool for detecting and quantifying viral loads. The assay may also prove useful for detecting infections, ultimately leading to better disease control on farms.5. In this study, a duplex real-time PCR for simultaneous detection of porcine circovirus and porcine pseudorabies virus base on SYBR Green was developed and evaluted. The sets of primers were targeted to conserved open reading frame-2 (ORF2) gene of PCV type 2, Non-structional protein (NS1) gene of PPV and used to amplify. The sensitivity, specificity and interference test of duplex real-time PCR assay was analyzed. It was shown that the detection levels of 256 copies/μl of PRV, and 168 copies/μl of PPV were achieved, and there was no non-specific amplification and cross-reactivity using cDNA of HCV, PRRSV and JEV. In conclusion, this assay provides an effective etiological diagnostic tool for detecting and quantifying viral loads. The assay may also prove useful for detecting infections, ultimately leading to better disease control on farms.6. In this study, a duplex real-time PCR for simultaneous detection of porcine pavovirus and porcine pseudorabies virus base on SYBR Green was developed and evaluted. The sets of primers were targeted to conserved regions of open reading frame-2 (ORF2) gene of PCV-2, Non-structional protein (NS1) gene of PPV and used to amplify. The sensitivity and specificity test of duplex real-time PCR assay was analyzed. It was shown that the detection levels of 156 copies/μl of PRV, and 175 copies/μl of PPV were achieved, and there was no non-specific amplification and cross-reactivity using cDNA of HCV, PRRSV and JEV. In conclusion, this assay provides an effective etiological diagnostic tool for detecting and quantifying viral loads. The assay may also prove useful for detecting infections, ultimately leading to better disease control on farms.7. A multiplex polymerase chain reaction (PCR) was designed for the simultaneous detection of three viruses involved in reproductive failure in pigs: porcine parvovirus (PPV), porcine pseudorabies virus (PRV), porcine circovirus type 2 (PCV-2). Each of the three pairs of oligonucleotide primers exclusively amplified the targeted fragment of the specific viruses. The sensitiviy of the multiplex PCR using purified plasmid constructs containing the specific viral target fragments was 203,189,173 copies for PPV, PRV, and PCV-2, respectively. When the specificity of the assay using specific PPV, PRV and PCV-2 primers was evaluated by testing the other porcine viruses, no cross-reactions were detected with non-PPV, PRV and PCV-2. This method is a rapid, sensitive and cost-effective diagnostic tool for the routine surveillance of viral infections in pigs.