Establishment And Application Of Two Rapid Detection Methods For Shrimp Hemocyte Iridescent Virus(SHIV) And Enterocytozoon Hepatopenaei(EHP)

Shrimp hemocyteiridescent virus(SHIV)and Enterocytozoon hepatopenaei(EHP)are the epidemic pathogens in shrimp aquaculture in recent years.Invertebrate iridescent virus,a widespread virus in nature,can live in a variety of aquatic invertebrate animals.SHIV was found in Cherax quadricarinatus and Litopenaeus vannamei,which is a kind of high lethal virus in recent years.The latent virus can stimulate shrimp disease outbreak under some certain conditions.The effective treatment have not been reported for SHIV.The only way to control SHIV is cutting off route of transmission by early detection.EHP will not cause acute death of shrimp,but will cause slow growth,low feed utilization rate and reduced immunity of shrimp.In this study,two kinds of quantitative dection for pathogenic diagnosis technology were set up,which can provide early warning in the initial stage of pathogen infection,even prevent two disease outbreak and reduce loss.It is significant for preventing disease outbreaks.Based on the requirements of laboratory research and field rapid diagnosis,two detection methods,namely,real-time fluorescence quantitative PCR and thermostatic RPA,were established in this study.Real-time fluorescence quantitative PCR detection method of SHIV:a pair of specific primers were designed according to the MCP gene sequence of SHIV,and the SYBR Green I real-time fluorescence quantitative PCR(q PCR)detection method of shrimp iridovirus was established in this study.The results showed that the final primer concentration was 0.3μM,and the linear equation of the standard curve of SHIV-q PCR was C_T=-3.2048X+36.751(X is the logarithm of plasmid copy numbers),and the correlation coefficient R~2=0.9978,indicating that the linear relationship of the standard curve was good.According to the standard curve slope,the reaction amplification efficiency was 105%,which was between the optimal value range(95-110%).The significance analysis results of 8 concentration gradients shows P>0.05,and the variation coefficient within the group ranged from 0.15%to 0.75%.The lower limit of detection sensitivity was up to 10 copies/μL.This method had no amplification for common pathogens of prawn,and had good specificity.RPA of SHIV detection method:according to the MCP gene sequences of SHIV with a pair of specific primers of RPA and probe in this study.Reaction condition was 39℃900 s.A new basic RPA product purification method has been developed,makes the basic RPA agarose electrophoresis effect is similar to the PCR,the sensitivity of 10 copies/μL;At the same time,a real-time fluorescence detection method(real-time RPA)was established to detect SHIV.The standard curve of SHIV-q RPA was C_T=-39.365X+540.48(X is the logarithm of plasmid copy number),the correlation coefficient R~2=0.9945,the coefficient of variation was between 0 and 5.75%,and the lower limit of detection sensitivity was up to 5 copies/μL.The constant temperature RPA established in this experiment can provide a stable,reliable and sensitive diagnosis result in a very short time for the detection of SHIV,and this method can provide technical support for the field rapid diagnosis of SHIV.Real-time quantitative fluorescence PCR detection method of EHP:Based on the q PCR amplification condition for SHIV,a pair of specific primers targeting the SWP gene sequence of EHP was selected,and a SYBR Green I q PCR method for EHP was established.The results showed that 0.3μM was the final primer concentration of this method.The linear equation of the standard curve for EHP-q PCR was C_T=-3.3934X+37.777(X is the logarithm of plasmid copy numbers),and the correlation coefficient R~2=0.9970,indicating that the linear relationship of the standard curve was good.According to the standard curve slope,the reaction amplification efficiency was calculated to be 97.1%,which was between the optimal value range(95-110%).The significance analysis results of 8 concentration gradients were P>0.05,and the variation coefficient within the group ranged from 0.31 to 1.14%.The lower limit of detection sensitivity was up to 5 copies/μL.This method had no amplification for common pathogens of prawn,and had good specificity.Thermostatic RPA of EHP detection methods:according to the SWP gene sequences of EHP with a pair of specific primers of RPA and probe in this study,reaction condition was 39℃900 s.A new basic RPA product purification methods has been developed,makes the basic RPA agarose electrophoresis effect is similar to the PCR,the sensitivity of 100 copies/μL;At the same time,a real-time fluorescence detection method(real-time RPA method)was established to detect EHP.The standard curve of EHP-q RPA was C_T=-45.794x+595.24(X is the logarithm of plasmid copy numbers),the correlation coefficient R~2=0.9939,the coefficient of variation was between 1.29%and 4.10%,and the sensitivity was 10copies/μL.The constant temperature RPA established in this experiment can make a stable,reliable and sensitive diagnosis result in a very short time,and this method can provide technical support for the rapid field diagnosis of EHP.The quantitative method established in this study was used to detect sample,including 59 Macrobrachium rosenbergii,88 Litopenaeus vannamei and 8 M.nipponense,which was collected from Shanghai fengxian,qingpu,jinshan shrimp farms.The results indicated SHIV and EHP infection rates were 86.44%,37.29%in M.rosenbergii,SHIV and EHP infection rates were 89.77%,82.95%in L.vannamei,and SHIV and EHP infection rates were 100.00%,87.50%in M.nipponense.At the same time,the environmental organisms in the breeding pond include tadpoles,small fish,water boatman,crayfish,water striders,and Bellamya sp.The contents of EHP and SHIV were detected.Among them,the content of SHIV in the original crayfish was up to 6.28×10~8 copies/mg.By comparing the nested PCR,real-time thermostatic RPA and real-time fluorescence quantitative PCR,the detection results showed that the detection rate of EHP-q PCR was 94.12%,EHP-q RPA was 94.12%,and the detection rate of EHP in nested PCR was 76.47%.The detection rate of SHIV-q PCR is 100%,that of SHIV-q RPA is 100%,and that of nested PCR is 82.35%.The results of the three methods were consistent for the samples with high pathogen content,while the results of real-time thermostatic RPA and q PCR were consistent for the samples with low pathogen content,when the content of EHP was 76 copies/mg and the content of SHIV was 28 copies/mg,the PCR results were negative but the other methods were positive.The sensitivity of the two methods established in this study has been greatly improved,which is suitable for the early warning of pathogens,and real-time thermostatic RPA has greatly reduced the reaction time,which can provide technical support for the early diagnosis of prawn diseases.