| Echinococcosis,also known as hydatidosis,is a zoonotic parasitic disease with global public health significance,which can cause a serious disease burden and affect public health safety and livestock development and can lead to chronic human diseases and cause serious medical,social and economic burden.Therefore,early and accurate detection and differentiation,and real-time monitoring of the infection status of various hosts are of great significance for the prevention and control of the disease In this study,based on the needs for the sensitivity,specificity,and operability of the detection of Echinococus and the diagnosis of echinococcosis,and according to the principle of recombinase-aided isothermal amplification,the isothermal amplification technology for detecting Echinococcus granulosus G1 and E.multilocularis were established.The following three parts of study specifically described how to develop novel tools in order to provide useful options for the detection of Echinococcus infections:Part Ⅰ Establishment and preliminary application evaluation of the recombinase-aided nucleic acid isothermal amplification technique for identifying Echinococcus granulosus G1In this section,we use the mitochondrial gene sequence of E.granulosus G1(EgG1)(GenBank No.AF297617)as the target sequence.And according to the principle of RAA reaction,6 pairs of primers were designed and synthesized.After screening to obtain the best primers,the RAA reaction system was optimized from three aspects:temperature,primers,and magnesium acetat.The sensitivity of RAA amplification tests were performed with genomic DNA at different dilution concentrations and pMD19-T(Simple)cloned plasmids containing different copies of the target gene fragments in different dilutions.And the genomic DNA of Taenia saginata,T.asiatica,T.multiceps,Dipylidium caninum,Toxocara canis,Trichuris trichiura Linnaeus,Giardia lamblia,Fasciola hepatica,Paragonimus westermani,F.gigantica and Clonorchis sinensis was used as template for RAA amplification to evaluate the specificity.At the same time,we used the PCR reaction as parallel control.In addition,different samples which infected with EgG1,including animal tissue samples(10),simulated field-positive dog feces samples(3),and field-positive dog feces samples(5)were tested separately to verify the reliability and practicability of the established RAA detection technologyThe RAA method established in this study can specifically amplify the target gene fragment of EgG1 within 40 minutes.The reaction system of RAA was established with the optimal temperature of 37℃.When the primer concentration was 10μmol/L,the optimal volume of primer was 2μL(the reaction concentration was 0.4μmol/L);and when the magnesium acetate concentration was 280 mmol/L,the optimal volume of magnesium acetate of 2.5μL(the reaction concentration was 14 mmol/L).In terms of sensitivity,the RAA method can detect a minimum of 10 pg of genomic DNA and 104 copies of plasmids;as for specificity,amplification results using other parasite genomic DNA as templates were negative.The test results of infected EgG1 samples were all positive,and were consistent with the PCR test results.Part Ⅱ Establishment and preliminary application evaluation of the recombinase-aided nucleic acid isothermal amplification technique for identifying Echinococcus multilocularisIn this section,we use the mitochondrial gene sequence of E.multilocularis(Em)(GenBank No.AB018440)as the target sequence.And according to the principle of RAA reaction,5 pairs of primers were designed and synthesized.After screening to obtain the best primers,the RAA reaction system was optimized from three aspects temperature,primers,and magnesium acetat.The sensitivity of RAA amplification tests were performed with genomic DNA at different dilution concentrations and pMD19-T(Simple)cloned plasmids containing different copies of the target gene fragments in different dilutions.And the genomic DNAs of other parasite were used as template for RAA amplification to evaluate the specificity.At the same time,we used the PCR reaction as parallel control.In addition,different samples which infected with Em,including animal tissue samples(9),simulated field-positive dog feces samples(3),and field-positive dog feces samples(2)were tested separately to verify the reliability and practicability of the established RAA detection technology.The RAA method established in this study can specifically amplify the target gene fragment of Em within 40 minutes.The optimization reaction system of RAA was established with the optimal amplification temperature of 37℃.When the primer concentration was 10μmol/L,the optimal volume of primer was 2μL(the reaction concentration was 0.4μmol/L);and when the magnesium acetate concentration was 280 mmol/L,the optimal volume of magnesium acetate of 2.5μL(the reaction concentration was 14 mmol/L).In terms of sensitivity,the RAA method can detect a minimum of 10 pg of genomic DNA and 104 copies of plasmids;in terms of specificity,amplification results using other parasite genomic DNA as templates were negative.The test results of infected Em samples were all positive,and were consistent with the PCR test results.Part Ⅲ Establishment and preliminary application evaluation of a multiplex recombinase-aided nucleic acid isothermal amplification technique for identifying Echinococcus granulosus G1 and Echinococcus multilocularisThis section uses the primers designed in the first and second parts(patent application number:201911164337.5)to establish a multiplex recombinase-aided isothermal amplification(mRAA)that can specifically amplify the target gene of EgG1 and Em within 40 minutes.The mRAA reaction system was optimized from three aspects:temperature,primers,and magnesium acetat.The sensitivity of mRAA amplification tests were performed with genomic DNA at different dilution concentrations and pMD19-T(Simple)cloned plasmids containing different copies of the target gene fragments in different dilutions.And the genomic DNAs of other parasite were used as template for mRAA amplification to evaluate the specificity.At the same time,we used the mPCR reaction as parallel control.In addition,some different samples which infected with EgG1 or Em,including animal tissue samples(no.=19,10 for EgG1 and 9 for Em),simulated field-positive dog feces samples(no.=3,3 samples mixed with EgG1 and Em),and field-positive dog feces samples(no.=7,5 for EgG1 and 2 for Em)were tested separately to verify the reliability and practicability of the established RAA detection technology.The optimal temperature of this study was 37℃.When the primer concentration was 10 μmol/L,the optimal primer amount of the upstream and downstream was 1μL for EgG1(the reaction concentration was 0.2μmol/L)and 1.5μL for Em(the reaction concentration was 0.3 μmol/L);and when the concentration of magnesium acetate was 280 mol/L,the amount of magnesium acetate was 2.5μL(the reaction concentration was 14mmol/L).The minimum detectable concentration of genomic DNA was 0.1 ng and the copy number of recombinant plasmids was 104 by mRAA method.The genomic DNAs of other parasites used in this study did not show any positive amplificants by this method.The samples infected with EgG1 or Em were all detected by mRAA as well,and the results were consistent with multiplex PCRThe RAA and mRAA detection methods were established in this study,which are faster than conventional PCR and perform well in sensitivity and specificity.These methods have good value in the identification of EgGl and Em and in the diagnosis of echinococcosis,and provides a new sensitive and specific tool for rapid detection.And these new methods may provide efficient and fast technical support for the detection of echinococcosis,which is conducive to the on-site prevention and control. |
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