Molecular Epidemiological Investigation Of PEDV And PDCoV And Evaluation Of Colony Gold Immunochromatographic Test Strips For PEDV

Since 2010,piglet virus diarrheal disease in many provinces in China continue to large-scale outbreaks,often with rapid onset,rapid spread,high morbidity and mortality characteristics,and the main harm to the housing piglets,to the pig industry in China caused Huge economic loss.In order to investigate the situation of swine fever diarrheal disease in Jiangsu province,166 samples of 5 large-scale pig farms in four prefecture-level cities in Jiangsu Province from 2015 to 2016 were analyzed by RT-PCR.PEDV-N and PDCoV-S1 were detected,and some positive genes were sequenced.The results showed that the positive rate of PEDV was 8.43%,the positive rate of PDCoV was 19.88%.But the two pathogens did not occur mixed infection in 5 pig farms.PEDV-N gene sequence analysis showed that the five sequences were not inserted and deleted.Only the point mutation occurred in some positions,which indicated that the PEDV-N gene was relatively conserved during the epidemic.The genetic analysis of N gene showed that the PEDV-N sequences detected in the Lianshui and Longkang pig farms were consistent with the Chinese strain CH-HNLH-2015,CH-HNAY-2015,CH-GDQY-3-2011,CH-SDRZ-2-2011 and CH-BJYQ-2-2011 sequences in the same branch,have close relationship,and the relationship with classical strain CV777 genetic is far.Longkang pig farm strain CHJS/XH-LK10/2015,CHJS/XH-LK37/2015 and the United States strains USA-Texas 128-2013,USA-KS-2013,USA-Minnesotal 88-2014,USA-Kansas 125-2014 have a close relationship,indicating that the occurrence of the infection may be associated with the prevalence of domestic strains and the recent outbreak of the United States PEDV.The nucleotide homology analysis of PDCoV-S1 gene sequence showed that there were no insertions and deletions of genes in the six PDCoV-S1 sequences,and only the mutations appeared in some positions,indicating that the gene was more conserved.The six sequences of this study were 97.7%-97.8%with the S1 sequence of Vietnamese isolates Vietnam/Binh21/2015 and Thai strains of Thailand/S5018/2015;had high homology with the S1 sequence of the China Hong Kong strain HKU15/155,China strain CH/GD01/2015,CHN/JS/2014,CH/SXD1/2015,Korean strain KNU14/04 and USA strain USA/Minnesota140/2015,OH/FD22,USA/Illinois272/2014.The highest homology with Chinese strain CH/GD01/2015,CHN/JS/2014 was 99.4%,followed by Hong Kong strain HKU15/155,which was 99.3%.The genetic evolutionary phylogenetic tree shows that the evolutionary relationship is very close and may originate from the same strain.PEDV detection is mainly dependent on RT-PCR and ELISA and other laboratory methods,although its sensitivity and accuracy is high,but need to rely on equipment,and time-consuming,cumbersome operation.The gold immunochromatography assay(GICA)is an accurate,convenient and rapid method for immunoassay.It is very suitable for grassroots veterinary work units lacking related equipment and equipment.It can also be used in epidemiological investigation.The prevention,control and research of livestock and poultry infectious diseases played an important role.Therefore,the sensitivity,reproducibility,specificity,stability and clinical test of PEDV immunoprecipitation were studied in this study.Experiments show that the test strip specificity is good,stored at 4?for 4 months can still be used normally,with good stability.In the repetitive experiment,the control line of the same batch of test strip and the strip of the test line were consistent.In the sensitivity experiment,the test strip was increased with the dilution ratio of PEDV cell culture medium,and the detection line was gradually weakened.The lowest PEDV cell culture medium TCIDso/O.lml was 3.16,ELISA,real-time fluorescence quantitative PCR and conventional RT-PCR method,the test paper can detect the minimum OD value of 1.418,the minimum concentration of 27.85ppb;can detect the lowest copy number of 1.10×104(1?L in the virus copy number),the highest CT value of 26.74;sensitivity lower than the conventional PCR method.In the test of clinical samples,24 samples of pig manure and intestinal tissue samples were collected by test strip and real-time fluorescence quantitative PCR respectively.Three samples of intestinal tissue samples were positive and the rest were negative;the 24%of the clinical samples of 1 ?L in the virus copy number is lower than the test strip can detect the lowest copy number(1.10×104).Indicating that the sensitivity of the test strip to be further improved.