A duplex nested-PCR assay for detection of mud crab reovirus and mud crab dicistrovirus-1

Abstract

Recent outbreaks of "sleeping disease" have caused mass mortality in cultured mud crab and widespread economic loss in southeast China.Two novel pathogens(Mud Crab Reovirus,MCRV and Mud Crab Dicistrovirus,MCDV-1) have been isolated from crab exhibiting "sleeping symptoms".Thus,there is an urgent need for the accurate and early detection of this pathogen to facilitate disease control.We developed a duplex nested-PCR protocol for the detection of MCRV and MCDV-1 in Scylla paramamosain.Two pairs of primers targeting the conserved regions of the two viruses genes were designed using Primer Premier 5.0.The outer primers for MCRV were ReoF(5′-GCAAATTGAACTACTACTACTTA-3′) and ReoR(5′-GATTCCTATTGTCAACTATCTCA-3′),and the inner primers were ReoNF(5′-ACTCATAGAGCAGTCATGGG-3′) and ReoNR(5′-ATATCGTCAGAATGTC GTTC-3′).The outer primers for MCDV-1 were DicF(5′-GCACTGGGTACTCTTCCTG-3′) and DicR(5′-AC ACCTACCAAAGCCCTAC-3′),and the inner primers were DicNF(5′5′-GGATACTATGGATGATGTTTC-3′) and DicNR(5′-ACAAAATACCAGATAAAGCAA-3′).The PCR products were differentiated by size.The first PCR was carried out using a PCR mix consisting of 1 μL DNA,1 μL MCRV outer primer(ReoF/ReoR,0.5 μmol/L),0.4 μL MCDV-1 outer primer(DicF/DicR,0.2 μmol/L),and dH2O(to a final volume of 20 μL).The thermal cycle was: 94℃ for 3 min,followed by 30 cycles of 94℃ for 30 s,55℃ for 30 s,and 72℃ for 1 min,followed by a final extension at 72℃ for 10 min.The nested PCR was carried out using the same mix with 0.2 μL template DNA(the product of the first PCR),0.8 μL MCRV inner primer(ReoNF/ReoNR,0.4 μmol/L),and 0.8 μL MCDV-1 inner primer(DicNF/DicNR,0.4 μmol/L).The conditions were the same as in the first PCR,except the annealing temperature was 53℃.We evaluated the specificity and sensitivity of the duplex nested-PCR.The assay did not exhibit any cross-reactivity with abalone shriveling syndrome-associated virus(AbSV),acute virus necrobiotic virus(AVNV),infectious hypodermal and hematopoietic necrosis virus(IHHNV),fish nervous necrosis virus(NNV),turbot viral reddish body iridovirus(TRBIV),or white spot syndrome virus(WSSV).We used a standard recombinant plasmid for each virus in the sensitivity assay and MCRV/MCDV-1 served as the position control.The detection limit was 10 1 copies of the viral genome for the two viruses.Our results suggest that this technique is a rapid,reliable,and cost effective tool for identification of crab virus with high sensitivity,high specificity.The assay could also be applied as a rapid diagnostic tool in clinical samples in which MCRV or MCDV-1 infection is suspected and differential diagnosis is required.