Establishment And Application Of Fluorescent Quantitative PCR Methods For Detection Of PRRSV, CSFV And JEV In Boar Semen

Objective:Viruses in semen can easily lead to the spread and prevalence of lots of diseases, which cause tremendous economic losses to the pig industry. Real-time quantitative PCR (RT-qPCR) method has been widely used in disease detection, but there is lack of effective methods for detection of RNA viruses in swine semen. In the present study, to detect the porcine reproductive and respiratory syndrome (PRRSV), classical swine fever virus (CSFV) and Japanese encephalitis virus (JEV), specific quantitative real-time PCR detection methods were established, which would be used to monitor RNA viral infection in field. Semen quality was also analyzed after virus infection.Methods:To meet the requirements of the common PCR cloning and RT-qPCR, three pairs of primers specific to PRRSV, CSFV and JEV were designed using four software as Primer Premier 5.0, Primer Express 3.0, Oligo 7 and DNASTAR were used. Gene sequences of PRRSV, CSFV and JEV were obtained from the GenBank and sequence alignment was performed to identify the conserved region of each virus. Standard plasmids for each virus were constructed, which were used for established the kinetics amplification curve and standard curve for RT-qPCR. After testing the sensitivity, specificity, and reproducibility of the established RT-qPCR system, the three viruses were detected in sperms collected from field. Meanwhile, sperm concentration and their deformity were also counted through sperm smear.Results:1) The effective RT-qPCR systems used to detect the semen PRRSV, CSFV and JEV. For the PRRSV detection, the amplification kinetics curve copies were between 1.824�106/?L and 1.824�101/?L with a good linearity, and the R2 was 0.996, its amplification efficiency was 100.32%. The detection sensitivity could be down to 1.824 � 101 copies/?L. When semen dilutions were detected using the established method, the lowest dilution is 102.0TCID50/mL.2) For the CSFV detection, the amplification kinetics curve copies were between 5.952�106/?L and 5.952�101/?L with a good linearity, and the R2 was 0.998, its amplification efficiency was 104.66%. The detection sensitivity could be down to 101 copies/?L. When semen dilutions were detected using the established method, the lowest dilution is 30 RID/mL.3) For the JEV detection, the amplification kinetics curve copies were between 3.781 �106/?L and 3.781�101/?L with a good linearity, and the R2 was 0.991, its amplification efficiency was 101.66%. The detection sensitivity could be down to 101 copies/?L. When semen dilutions were detected using the established method, the lowest dilution is 1017/mL. For the specificity test, only specific genes for PRRSV, CSFV and JEV could be detected in samples, and no bands for several other swine viruses including PCV2, PRV and PPV were obtained.2) To confirm the established RT-qPCR methods,109 boar semen samples from 15 farms were collected, the results showed that samples from 13 farms (86.67%,13/15). Among them, the positive ratio for PRRSV was 12.8%(14/109, viral load:18.57 copies/?L-294.41 copies/?L); the positive ratio for CSFV was 21.1% (23/109, viral load:from 13.44 copies/?L to 123.39 copies/?L); and positive ratio for JEV was 20.2% (22/109, viral load: 11.20 copies/?L-246.27 copies/?L). The total ratio of combined infection of the three viruses was 2.7%, and ratio for mixed infection of PRRSV and CSFV was 2.7%, CSFV mixed with JEV was 1.8%.Also the size of pig farm and other factors can influence the spread of virus.3) The sperm density and abnormality were further determined in positive semen samples, the data from the non-diluted semen samples showed that the infection of CSFV significantly decreased the sperm number, but no difference was observed for PRRSV-infected semen samples. In positive commercial semen samples (semen was diluted for sale), infection with JEV or with 3 viruses resulted in the decrease of sperm number (p<0.05) with increase of the abnormality ratio.Conclusion:the established RT-qPCR method can be used to monitor the clinic infection of PRRSV, CSFV and JEV in boar semen with high sensitivity and specificity, which also can be used to detect the semen quality and provide technical support for the prediction and control of epidemic diseases.