Development Of An Indirect-ELISA For HRV And HRV Infection Rates Investigation Of Children Diarrhea In Daqing City

Rotavirus is the main pathogen causing acute gastrointestinal infectious diseases in human and animals. Itmainly infects intestinal epithelial cells, causing cell damage, thus leading to diarrhea or even dehydration insevere cases. This further results in water and electrolyte imbalance and threatens life. Rotavirus is widelydistributed in the nature. It can infect various populations. Group A rotavirus is the most common type.Infantile diarrhea caused by infection with rotavirus is the top2disease leading to infantile emergency andmortality in the world. In China,40%-60%infantile diarrhea is caused by infection with rotavirus. Moreover,rotavirus also heavily influences juvenile animals, causing a great loss to animal husbandry. As currently, thereis no specific remedy for rotavirus infection, establishment of a convenient, rapid and sensitive detectionmethod to monitor the situation of rotavirus infection at all times is extremely important to prevent pandemicoutbreak of the infection.In this trial,20stool samples from children with diarrhea were tested for rotavirus. Positive stool sampleswere prepared and inoculated in MA104cells. Cell culture and passage were performed. Since the6rdgeneration, there was CPE change. A viral strain was obtained. Through neutralization test, RT-PCR andsequence analysis, the isolated strain was identified as HRV. Then, differential centrifugation was performed toconcentrate and purify the HRV. With the purified HRV serving as coating antigen, an indirect ELISA methodwas established to detect HRV. The optimal conditions of this method were optimized and determined asfollows: the optimal coating buffer for HRV was50mM pH9.6carbonate (CBS) buffer; the optimal coatingantigen for microplate was10.0μg/mL; the optimal coating condition was12h,4℃; the optimal blockingsolution and applicable concentration was3%gelatin, with blocking time of37℃,1h; the optimal serumdiluent was5%skimmed milk, with optimal dilution strength of1:200and optimal action duration of37℃,30min; the optimal working concentration of the secondary antibody was1:10000, with optimal actionduration of1h,37℃; the optimal substrate color development reagent and stop buffer were TMB and1%SDSrespectively; the optimal reaction time of the substrate was10min,37℃. Through sensitivity test, crossovertest, repeatability test and conformance test, sensitivity, specificity and repeatability of the method wereevaluated. The results showed that the indirect ELISA method established in this trial had high sensitivity, highspecificity and good repeatability. In respect of positive detection rate of HRV, the coincidence rate with virusneutralization test and rotavirus detection kit was94%and92%respectively.Preliminary epidemiological study was performed on infection with rotavirus in children with diarrhea inDaqing between July2011and March2012, using the indirect ELISA diagnostic method established in thistrial. The results showed that during this period, among children with diarrhea in Daqing, the infection rate ofHRV was39.9%at average. According to compare HRV infection rates in the different season, different ageand different regions and so on,As indicated by the results, it was concluded that HRV infection was somehowcorrelated with season, children's age and living environment.