The Development Of A SYBR Green ? Multiple Real-time Fluorescence Quantitative PCR Assay For Detection Of Porcine Parvovirus,Porcine Circovirus Type 2 And Haemophilus Parasuis

In recent years,with the development of the pig industry,the accompanying diseases have become more and more complex.Porcine parvovirus(PPV)and porcine circovirus type 2(PCV2)often mixed infection in clinical breeding process,and after the infection of PCV2 pig causes immunosuppression,easy to stimulate or other infectious diseases,Haemophilus parasuis(Hps)is a kind of typical "opportunistic pathogen,often found as a secondary pathogen associated with PCV2 lead to mix with the infection.Accurate and rapid diagnosis of these pathogens,so as to provide targeted treatment,is of great significance to the healthy development of the pig industry.Fluorescence quantitative PCR detection technology has the advantages of high sensitivity,strong specificity,rapid,simple and quantitative,and is widely used in the diagnosis of pathogens.This study intends to establish a triple real-time quantitative PCR detection method,and combined with the melting curve analysis,to achieve the rapid diagnosis of PPV,PCV2 and Hps single or mixed infection.To this end,the following research has been done:(a)Fluorescence quantitative PCR detection methods for PPV,PCV2 and Hps were established respectively.Specific primers were designed according to the VP2 gene of PPV,the cap gene of PCV2 and the conserved region of the infB gene of Hps,and plasmid standard was constructed after the amplified fragments were cloned.Diluted with different degrees of standard as a template for SYBR Green ? fluorescence quantitative PCR amplification and making standard curve,three kinds of fluorescence quantitative PCR detection method is established.The sensitivity of this method to the detection of three pathogens is 146 copies/?L,121 copies/?L and 72 copies/?L,respectively.The three pairs of primers do not cross-react with other common pathogens,with good repeatability and specificity,and can be used for the detection of clinicaltissue materials.(b)A dual fluorescence quantitative PCR method was established to detect both PPV and PCV2.Using the PPV and PCV2 specific primers and purification of recombinant plasmid as a template,by adjusting the primer concentration,annealing temperature and other conditions,established the PPV and PCV2 double SYBR Green ? real-time fluorescent quantitative PCR detection method.The sensitivity of this method can reach 167 copies/?L and 140 copies/?L,and the specificity and repeatability are good as the primers have no cross-reaction with other pathogens.The clinical tissue material detection results are consistent with the common PCR,which proves that this method can provide technical support for the rapid differentiation and diagnosis of the mixed infection of these two viruses.(c)A dual fluorescence quantitative PCR method was established for simultaneous detection of PPV and Hps.Using the PPV and Hps specific primers and purification of recombinant plasmid as a template,by adjusting the primer concentration,annealing temperature and other conditions,PPV and Hps double SYBR Green ? real-time fluorescent quantitative PCR detection method,based on the sensitivity,specificity,repeatability of the method and clinical tissue disease material verification,proved that the method can be used for these two kinds of pathogen rapid differential diagnosis.(d)A dual fluorescence quantitative PCR method was established to detect PCV2 and Hps simultaneously.Using the above PCV2 and Hps specific primers and purification of recombinant plasmid as a template,adjust the primer concentration,annealing temperature and other conditions,established the PCV2 with Hps double SYBR Green ? real-time fluorescent quantitative PCR detection method.The sensitivity,specificity,repeatability and clinical tissuematerial of the method were verified,and the results showed that the method could be used for rapid differential diagnosis of the two pathogens.(e)A triple fluorescent quantitative PCR method was established for simultaneous detection of PPV,PCV2 and Hps.Using the PCV2,PPV and Hps of the above three pairs of specific primers and purification of recombinant plasmid as a template,a triple SYBR Green ?real-time fluorescent quantitative PCR detection method was established by adjusting the primer concentration,annealing temperature and other conditions,which can produce three specific Tm peak on the curve in the same fusion.And this method has good repeatability and specificity for having no cross reaction with other pathogens,which can provide technical support for the rapid diagnosis of the three diseases,also immune procedure for these three diseases.