RT-PCR Detection Of Viral Diarrhea In Neonatal Piglets Of Heilongjiang Province In 2013-2014 And The Establishment Of The RT-LAMP For Detection Of PEDV

Porcine epidemic diarrhea virus(Porcine epidemic diarrhea virus comes, PEDV) is the etiological agent of Pig epidemic diarrhea(Porcine epidemic diarrhea, PED). PEDV is a member of the family Corona virus and can cause the acute and highly contagious enteric disease characterized by severe vomiting, diarrhea and dehydration in piglets from different age groups,. and in neonatal pigs causing up to 100% mortality. Since the winter of 2010, the massive PED outbreaks characterized by high mortality rates in suckling piglets in China. In this study, feces and small intestine of the neonatal piglets that were noted with signs of diarrhea collected form 10 large-scale pig farms in six cities of Heilongjiang province from 2013 to 2014, and the RT-PCR analysis was used to detected the pathogens. The results showed that 56% of the samples were positive for PEDV, 6% of the samples were positive for PEDV and TGEV, 10% of the samples were positive for PRV and 10% of the samples were positive for TGEV. Our investigation provided that PEDV was the main pathogen that caused diarrhea of neonatal piglets in Heilongjiang province. Due to the M gene of PEDV is stable, the M gene genetic evolution analyses was used to detect the mutation of PEDV strains. There were six PEDV-M gene sequences were detected by RT-PCR, the results showed that the nucleotide homology is 98.2%~99% among these genes, and the nucleotide homology between these stains and vaccine strain CV777 was 97.6%~98.2%. Genetic evolutionary tree was build by MEGA 6.0, the result showed that the 6 PEDV-M genes were divided into two series, and most of the genetic distance is close relatively at the same branch. The major structural protein M gene was sequenced and analyzed for the six PEDV strains isolated from Heilongjiang province. The results showed that M gene of the different strains have closed genetic distance with each other. Compared with other Chinese isolates and foreign isolates, the 6 PEDV isolates had a closer genetic relationship with the Korea isolate M1595, CPF259 and Thailand isolates KU06RB08, KU07RB08, but more distant genetic relationship with the vaccine strain CV777. Sequencing and analysis in this study, the regular of genetic evolution has been made clear, It will provide theoretical basis for accurate diagnosis and effective prevention of the disease, and will enrich the data of the molecular epidemiology of PEDV.The study detected PEDV by using RT-LAMP technology. According to published PEDV-N gene sequence on Genbank, a set of primers, which composed of outer primers, inner primers, was designed by using primer Explorer5.0.PEDV was detected by RT-LAMP using the primers, and the reaction conditions were optimized. The experiments result showed that the optimal reaction time and temperature were determined to be 50 min at 61℃. Every component concentration after optimization was Mg2+2.5μL, dNTPs1.5μL and optimum concentration ratio between inner and outer primers was 1:4. RT-LAMP turned out to be more sensitive than conventional PCR. Its sensitivity was 100 times as sensitive as RT-PCR. No cross reaction was observed from the samples and other related viruses. Testing results among groups and within the group were the same.It demonstrated that this detection method was of good stability and repeatability.125 samples were detected by RT-PCR and RT-LAMP respectively in the study. And the test results of the two detection methods were identical. The method of using RT-LAMP method to detect PEDV was successfully established.