Establishment And Application Of Rapid Detection For Mud Crab Dicistrovirus-1

Mud crab (Scylla paramamosain) is an important marine cultured species insoutheast coast of China. However, with the gradual expansion of farming, diseaseproblems continue to arise, resulting in high mortality of mud crab farming. Mud crabdicistrovirus-1(official named Mud Crab virus) which belonged to the familyDicistroviridae，is a novel pathogen isolated from mud crab occurred mass mortality inrecent years and pathogenic to the mud crab. It had caused a widespread economic lossto the crab farming in China. Rapid, accurate and sensitive detection method for thedisease prevention or for further in-depth study of the disease is of great significance.Therefore, this paper established LAMP detection method, Real-time PCR detectionmethod and duplex nested-PCR detection method for MCDV-1detection. And thesemethods were preliminary used in MCDV-1prevalence and infection characteristicsresearch in this study.Above all, a rapid detection of MCDV-1using loop-mediated isothermalamplification (LAMP) is developed. Two pairs of primers targeted to VP2regions of thevirus genes were designed. The optimization of reaction condition, specificity andsensitivity of this method were also taken into account in our study. The results showedthat the optimal LAMP amplification was carried out using0.2μmol/L of F3/B3,1.6μmol/L of FIP/BIP,6mol/L of Mg2+,0.8mol/L of dNTPs,0.8mol/L of betaine, andcompleted in1h at62℃; When visualized by gel electrophoresis, the products of theMCDV-1LAMP assay appeared as a ladder pattern. The MCDV-1LAMP product couldalso be simply detected visually according turbidity, or by adding SYBR Green I to thereaction tube and observing a color change from orange to green. The method couldspecifically amplified MCDV-1template, but was found no cross-reactivity with abalone shriveling syndrome-associated virus (AbSV), acute virus necrobiotic virus(AVNV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), fishnervous necrosis virus (NNV), turbot viral reddish body iridovirus (TRBIV), the whitespot syndrome virus (WSSV) and Mud Crab Reovirus (MCRV). The sensitivity assayrevealed that6copies of viral genome could be detected by this method, which was atleast1000-fold higher than that of conventional PCR for the detection of MCDV-1.Secondly, to establish a real-time PCR for quantitative analysis of MCDV-1gene, apair of specific primers and TaqMan fluorescent probe of the conserved region ofMCDV-1gene were designed. The reaction condition were optimized first. Using thismethod to detect MCDV-1and other seven common aquatic animal virus includingabalone shriveling syndrome-associated virus (AbSV), acute virus necrobiotic virus(AVNV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), fishnervous necrosis virus (NNV), turbot viral reddish body iridovirus (TRBIV), the whitespot syndrome virus (WSSV) and Mud Crab Reovirus (MCRV), only MCDV-1appeared the standard amplification curve. This presented the method is of goodspecificity. Besides, the method is highly sensitive (up to8copies of the viral genes)and reproducible (CV values of intra-and inter-assay were1.11%and1.3%). Thestandard curve linear relationship of copies was excellent within the range8.0×108-8.0(R2=0.992). Then, this real-time PCR was appled to a comparative study on MCDV-1amount on different location of mud crab gill. The results showed that the MCDV-1amount on different location of mud crab gill was range from1010-1011copies/g. Theamount of virus carried have no statistically significant difference between left and rightpart of the gill filaments, or among upper, middle and lower part of the gill filaments, orgill filaments at the top, middle and base. That is to say the MCDV-1evenly distributeat all parts of the gill tissue after MCDV-1infecting the mud crab.Besides, a duplex nested-PCR protocol for the detection of MCRV and MCDV-1inmud crab is developed. Two pairs of primers targeted to conserved regions of the twoviruses genes were designed by Primer Premier5.0. The PCR products weredifferentiated by the size. Data showed the detection limits are101copies viral genomefor the two viruses. The method was found no cross-reactivity with abalone shrivelingsyndrome-associated virus (AbSV), acute virus necrobiotic virus (AVNV), infectioushypodermal and hematopoietic necrosis virus (IHHNV), fish nervous necrosis virus(NNV), turbot viral reddish body iridovirus (TRBIV) and the white spot syndrome virus (WSSV)，indicate the two viruses could be specifically detected. This method was usedin investigating the virus carrying rates of wild mud crab in Yangjiang from May toOctober in2012. The result suggests wild mud crab of Yangjiang area has maintained ahigher rate of virus carrying (44.83-100%) from May to October in2012. The highestrate of MCDV-1carrying appeared months is June, August and September. The highestrate of MCRV carrying appeared months is May, June, August and September. Theseresults reveal the epidemic seasons of two viruses are May-June and August-September.The duplex infection rate of wild mud crab during the epidemic season is between10-100%.In conclusion, results obtained suggest that these rapid detection methods arereliable tools for identification of MCDV-1with high sensitivity, high specificity andhigh efficiency, and could be used for the MCDV-1clinical diagnosis. And these threemethods are suitable for different purposes according to their own characteristics. TheLAMP detection method are both finished the reaction in1hour at a constanttemperature, and easy to judge the test results. It has a wide application prospect inMCDV-1detection at subbase production department or under the wild experimentalconditions. The MCDV-1real-time PCR assay for quantification is accurate and reliable,which can be used in the detection of MCDV-1infection and scientific research. Theestablishment of duplex nested PCR makes it possible to give an early rapid diagnosison the MCDV-1and MCRV at one time. It could be an effective tool for theepidemiological study of MCDV-1and MCRV.