One-step Multiplex RT-PCR For The Detection Of Duck Tembusu Virus,duck Plague Virus,Duck Hepatitis A Virus Type1 And Type 3

Duck tembusu virus(DTMUV),Duck plaque virus,Duck hepatitis A virus type 1(DHAV-1)and type 3(DHAV-3)are important infectious viral diseases that results a devastated hit to duck breeding industry.In this work,a multiplex RT-PCR method was developed for simultaneous detection of four viruses,including DTMUV,DPV,DHAV-1 and DHAV-3.The sensitivity,specificity and repeatability of the method were evaluated,and the differential diagnosis of clinical materials was performed.Results are described as follows:1.Establishment of one-step multiplex RT-PCR method for DTMUV,DPV,DHAV-1 and DHAV-3 detectionFour pairs of specific primers were designed according to the genome sequence of DTMUV,DPV,DHAV-1 and DHAV-3 on GenBank.To optimize the conditions of reaction,annealing temperature and primer concentration were tested,and a multiplex RT-PCR method was developed for fragments of 322 bp,804bp,648 bp,and 1314 bp amplification.There is no unwanted amplification when DHBV,MDPV,ILTV,MDV,R.anatipestifer,S.aureus,P.multocida,E.coli,and S.anatis,and RNA from healthy ducklings were used as template.The sensitivity test results showed that the minimum detection limit of DTMUV,DHAV-1,and DHAV-3 was 0.1 pg,and the minimum detection limit of DPV was 1 pg.2.Clinical application of one-step multiplex RT-PCRThe above viruses were selected for the absence of maternal antibodies 7-day-old healthy ducklings challenged with simulated mixed infections,108 samples which collected from artificially-infected ducklings of spleen and Bursa bursa were tested using the one-step multiplex RT-PCR method established in this paper,while a single PCR method was also performed as control.The results show that the coincidence rates of the test results were 98.3%,100%,96.7% and 97.4%,and the negative detection rate of the two detection methods were 87.5%,100%,97.4% and 95.7%.The developed method was used to detect 67 clinical samples from different commercial farms of Guanghan,Shifang,Deyang,Qionglai,Meishan,Mianyang,Leshan Jianyang and Yunnan.The results showed that DHAV-1 and DTMUV positive samples were detected in 16 and 4 samples,respectively.The detection rates were 23.9% and 6.0%,respectively,and no mixed positive samples were detected.The DPV and DHAV-3 test results all showed negative.