| Classical swine fever and African swine fever are infectious diseases with high contact and high mortality caused by classical swine fever virus and African swine fever virus.Atypical porcine pestivirus can cause congenital tremor of type A-II in piglets.The disease is infectious and can cause death of piglets in severe cases,which is harmful to pig industry.Therefore,it is of great significance to carry out epidemiological detection and clinical rapid diagnosis.At present,the detection methods of ASFV,CSFV and APPV mainly rely on molecular biological methods and serological methods,and it is difficult to distinguish wild virus from vaccine attenuated virus when detecting CSFV.These methods are often single index,time-consuming and laborious,so they are not suitable for the rapid detection of high-throughput samples.In this study,a visual gene chip detection method was established for the differential diagnosis of these three diseases.The research contents are as follows:1.Primer probe design:according to the conserved sequence p72 gene of ASFV,5’UTR sequenceof and NS5B protein gene of CSFV and 5’UTR sequence of APPV,specific primers and gene probes were designed.At the same time,the designed primers were used to amplify the gene fragments of each pathogen nucleic acid,and the positive plasmid was cloned and synthesized as the standard.2.Establishment of visual identification and detection method of CSFV and vaccine attenuated virus by gene chip:designed primers and probes with good specificity,and then hybridized with probe specifically by using biotin labeled target gene fragment.After amplifying the hybridization signal with horseradish peroxidase labeled streptomycin,the results were judged by naked eye observation after color development by TMB chemical precipitation reaction.The reaction conditions were optimized,and the specificity,sensitivity,repeatability and coincidence rate of the chip were evaluated.The results show that the method has good specificity and repeatability.For CSFV wild virus and Vaccine Plasmid standard,the single detection sensitivity can reach 6.98 copies/μL and 6.92×10~1copies/μL,and for the same positive standard,the chip repeatability can reach 100%.177 clinical samples were detected,and the results were compared with the national standard of classical swine fever virus RT-n PCR detection method(GB/T 26875-2018),the overall coincidence rate was 99.43%.3.Establishment of simultaneous identification and detection methods of visual CSFV,ASFV and APPV gene chip:Based on the identification and diagnosis gene chip of CSF wild virus and vaccine,three kinds of synchronous detection methods of pathogen were established by optimizing the amount of primers and reaction conditions.The results showed that the method only detected these three pathogens,but did not detect other 10 kinds of swine viruses,with strong specificity;the sensitivity of single detection for ASFV plasmid standard was 1.92 copies/μL;the single detection sensitivity of APPV plasmid standard was 1.86×10~1copies/μL;the detection sensitivity of mixed detection was 2.43×10~2copies/μL,which was good.For the same sample,the same batch and different batches of chips have the same detection results,which shows that the method has good repeatability.The storage life test results show that the chip can be stored at 4℃for 240 days.219 clinical samples were tested,and compared with the standard test method,the test results were consistent with the results of the standard test method,and the overall coincidence rate reached 99.08%.The results showed that the gene chip detection method established in this study had strong specificity,high sensitivity,and the detection results were intuitive and easy to determine.It could complete the identification and detection of three important swine diseases in 2 hours.It has a good application prospect to carry out the epidemiological monitoring of pig infectious diseases at the grass-roots level and even the prevention and control and purification of pig diseases in China. |
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