Establishment And Application Of Multiplex RT-QPCR Method For Detecting Four Porcine Diarrhea Viruses

Porcine epidemic diarrhea virus（PEDV）,Transmissible gastroenteritis virus（TGEV）,Porcine rotavirus（PRo V）and Porcine deltacoronavirus（PDCo V）can all cause diarrhea in pigs.Suckling piglets have acute diarrhea and severe vomiting after infected,moreover,the mortality rate is very high.The symptoms of viral diarrhea which are extremely similar,in addition to distinguish clinically,the multiple infection of porcine diarrhea virus is also common.Therefore,in this study,four porcine diarrhea virus multiplex TaqMan RT-q PCR method were established for clinical differential diagnosis.At present,the magnetic bead method（automation）is commonly used to extract nucleic acids from a large number of porcine-derived samples which in the diarrhea virus detection of large-scale pig farms.However,some samples failed to extract nucleic acid that affected by various factors,resulting in false-negative test results.So,in this study,a TaqMan q PCR detection method for porcine-derived gene was also established in order to monitor the quality of nucleic acid extraction.Besides,the method also has a variety of extended applications:detection of porcine-derived components;as an internal reference in the detection of porcine-derived samples;contamination monitoring of laboratory.The main research contents and results are as follows:（1）Establishment of four porcine diarrhea virus multiplex TaqMan RT-q PCR detection methodFour sets of specific primers and probes were designed based on the conserved sequences of PEDV N,TGEV N,PRo V NSP3 and PDCo V N.Through the optimization of reaction conditions,a quadruple q PCR detection method was established.In terms of sensitivity,the vaccine nucleic acid with known viral load(TCID₅₀/m L)was used as a template for amplification,and the results showed that the lowest detectable viral load was PEDV 10^-1TCID₅₀/m L,TGEV 10^-1TCID₅₀/m L,PDCo V 10^-2TCID₅₀/m L,and PRo V 10^-1TCID₅₀/m L,indicating that the method has favorable sensitivity.In terms of specificity,only the result with positive mixed templates showed specific amplification curve,indicating that they had excellent specificity.The results of the repeatability test showed that the coefficients of variation were all below 3%,indicating that the method had good stability.（2）Application of four porcine diarrhea virus multiplex TaqMan RT-q PCR detection methodUsing the established method,182 diarrhea samples were tested,of which 31were positive for PEDV,1 was positive for PDCo V,and both were negative for TGEV and PRo V.The positive coincidence rate between the self-established method and the commercial kit was 90%,and the self-established method was superior to the purchased commercial kit in terms of sensitivity and accuracy.（3）Establishment of TaqMan real-time quantitative PCR detection method for porcine-derived genePrimers and probe were designed according to the mitochondrial cytochrome B（cytb）gene of porcine-derived cells（PK-15）.By optimizing the reaction system and reaction conditions,a TaqMan q PCR detection method for porcine-derived gene was established.In the sensitivity test,the DNA of PK-15 cells with a known cell number was used as a template for amplification.The results showed that the minimum detectable number of porcine-derived cells was 70/m L,indicating that this method has good sensitivity.In the specificity test,only the result of porcine-derived cell DNA showed a specific amplification curve,indicating that the specificity was better.In the repeatability test,the coefficients of variation were all below 1%,indicating that the established method has excellent repeatability.（4）Application of TaqMan q PCR detection method of porcine-derived geneThe extraction quality of 182 nucleic acids（diarrhea samples）was monitored by using the q PCR detection method of porcine-derived gene.The results showed that169 nucleic acid samples were positive for porcine-derived gene and 13 were negative,and the corresponding diarrhea virus test results were also negative.After re-sampling and nucleic acid extraction,the porcine diarrhea virus was tested again.The re-examination results showed that some of the results of the first detection were inaccurate,and there was actual diarrhea virus infection.In the detection of porcine-derived components,50 feeds and feed ingredients were sampled,and the test results were all negative for porcine-derived gene.