Development Of Real-time RT-PCR And Capture ELISA Tests For North American Type Porcine Reproductive And Respiratory Syndrome

Porcine reproductive and respiratory syndrome (PPRS) is considered one of the important infectious diseases of swine. The disease is associated with reproductive failure in sows and boars and respiratory problems in young pigs. The causative agent, PRRSV is a major pathogen in swine industry of the world. The virus has two different types, the European (EU) type and the North American (US) type, and the North American type is the prevalent type in China.A pair of primers and a probe were designed according to the sequences of the Matrix gene of North American type PPRSV, and a real-time RT-PCR assay was established to detect this virus. By detecting serial dilution of positive control, the TaqMan method was found to 103~104 fold times more sensitive than conventional RT-PCR. To examine the detection limit, the diluted RNA of PRRSV VR-2332 was detected, and the assay was proved to be able to detect 1 TCID50 of the virus. RNA of hog cholera virus, Japanese encephalitis B virus, porcine pseudorabies virus, equine arteritis virus, porcine circovirus virusⅡand MARC145 cell were all detected negative by this assay. To test if the method works for clinical samples, 126 samples were detected and 7 samples were found positive. The results were confirmed by virus isolation. The RNA positive control in the assay was invitro transcribed dsRNA which was stabler than single strand RNA used in other similar test.Based on the monoclonal antibody 1G10, a capture ELISA for detecting serum antibodies against North American PRRSV was established. The concentration of coating McAb was 2.64μg/ml, and the concentration of the roughly extracted PRRSV was 17.51μg/ml. Reference anti-sera for hog cholera virus, porcine parvovirus, brucella suis, Japanese encephalitis B virus and porcine pseudorabies virus were all detected negative by this assay. To determine the sensitivity of the test, sera from 22 artificially challenged pigs were detected showing that this capture ELISA had the same sensitivity (19/22) as IDEXX ELISA kit. The capture ELISA was also used to detect 266 clinical sera together with indirect N-ELISA and IDEXX ELISA kit. The results showed that the positive detectable rate of capture ELISA and indirect N-ELISA is high than IDEXX ELISA kit.