| Based on the PCR and real-time fluorescence quantitative PCR (FQ-PCR) principles, single and triplex Two-step Temperature and Taqman FQ-PCR methods were established, for rapid detection of Porcine parvovirus (PPV), Pseudorabies virus (PRV) and Porcine circocirus type2(PCV-2). The LAMP method was used for development of a new detection technique of PCV-2, with advantages of simple, high sensitivity and specificity. The established methods would facilitate lab detection of these pathogens. This study consists of three parts:Part1:Establishment of single and triplex Two-Step Temperature PCR assay.Conserved regions were selected after aligned genome sequences based on GenBank database. Three pairs of high Tm tempercture and specificity primers were designed based on the conserved domains of the VP2gene of PPV, the gH gene of PRV and the ORF2gene of PCV-2. The size of the amplified products were142bp for PPV,300bp for PRV and469bp for PCV-2. Single and multiplex Two-Step Temperature PCR methods were established after optimization of reaction mixture and conditions, and sensitivity, specificity and repeatability were verified. The total process of triplex PCR could be finished within66minutes, which saved about70%of the time than the conventional single PCR and at least20%than conventional multiplex PCR. The sensitivity of the methods could be down to200copies of the positive plasmid, and there was no cross-reaction with other common swine pathogen (Haemophilus parasuis, Streptococcus, CSFV, PRRSV, Escherichia coli and JEV). The methods also have good repeatability and stability.Part2:Establishment of single and triplex FQ-PCR assay.According to the principle of TaqMan(?) Real-time fluorescence quantitative PCR assay, one pair of primers and one probe for each of PPV, PRV and PCV-2were designed with related software, the probe reporter dye were JOE (PPV), TAMARA (PRV) and FAM(PCV-2), respectively. Dynamic amplification curve and standard curve of Single and triplex Taqman(?) FQ-PCR were established after optimization of reaction mixture and conditions, and the sensitivity, specificity and repeatability of the methods were verified. The total detection process took less than70min, the sensitivity could be down to10copies of the positive plasmid, and there was no cross-reaction with other common swine pathogen. In this study, the CV%of intra and inter-assay repeat testing were both less than3%, the repeatability of the method was good. The amplification efficiency E>95%, the correlation coefficient R2>0.99, indicating a high degree of accuracy in forming the standard curves.Part3:Establishment of the LAMP method for detection of PCV-2.According to the principle of the LAMP technique, four specific primers within the ORF2gene were designed by the Primer Explorer3.0software online. The LAMP for detection of PCV-2was established after optimization of reaction mixture and conditions, and verification of the sensitivity, specificity and repeatability. The sensitivity could be down to0.5pg of PCV-2genomic DNA, and there was no cross-reaction with other common swine pathogen. Positive LAMP products could be verified by restriction enzyme digestion. The results could be visual by addition of SYBR Green I fluorescence dye.205clinical samples were tested by the triplex PCR methods, and compared with the conventional single PCR method. The results showed that the coincidence rate of single and triplex Two-Temperature PCR methods for detection of PPV, PRV and PCV-2were all more than97%with the conventional single PCR method. The detection rate of single and triple FQ-PCR assay for the relevant pathogen was higher than the conventional single PCR method:PPV was0.38%higher, PRV was0.97%higher, PCV-2was1.96%higher. All30positive PCV-2DNA which has been identified by the standard detection method were amplified by the LAMP method, and there was no amplification of negative control, indicating the reliability of the method. |
PDF Full Text Request