Establishment And Application Of LAMP And RAA-LFD Methods For Detection Of Toxoplasma Gondii

Objective:To understand the infection status and infection characteristics of Toxoplasma gondii infected in newborns and pregnant women in Wuhu area.Furthermore to established the rapid detection methods of Loop mediated isothermal amplification(LAMP)and Recombinase-Aid Amplification-Lateral flow dipstick(RAA-LFD)for Toxoplasma gondii.The sensitivity and specificity of the established LAMP and RAA-LFD methods were evaluated by further detection of clinical samples,in order to provide some experimental basis for the rapid diagnosis of clinical toxoplasmosis.Methods:According to the 529 bp gene repeat sequence of Toxoplasma gondii,the best LAMP primers and RAA-LFD primers and probes were designed and screened;the reaction system and reaction conditions were optimized;the 529 bp gene-positive recombinant plasmid of Toxoplasma gondii was used as the template gradient sensitivity test after dilution;the genomic DNA of human blood,Plasmodium vivax,Ascaris lumbricoides,Trichinella spiralis,Schistosoma japonicum,Digramma interrupta,Taenia solium,Digramma interrupta and Entamoeba coli were used for specificity detection.Finally,the established LAMP and RAA-LFD methods were used to further detect clinical blood samples,and their detection rates and consistency were compared with Electrochemiluminescence(ECL)and quantitative fluorescence PCR(qPCR).Results:(1)The positive rates of T.gondii IgM and T.gondii IgG in newborns in Wuhu area were 0.06%(2/3371)and 13.02%(439/3371),respectively.There was no significant difference in the positive rates of T.gondii IgG among newborns of different genders(P>0.05).There were significantly difference between the positive rate of TOX-IgG in premature infants compared with the normal newborns controls(P<0.05).The positive rate of T.gondii IgM and T.gondii IgG in pregnant women was 0.14%(2/1443)and 5.96%(86/1443),respectively.There was significant difference in the positive rates of T.gondii IgG in pregnant women at different age groups(P<0.05).(2)The 529 bp gene of T.gondii could be detected within 30 minutes at the optimum reaction temperature of 67℃ for the LAMP method,and the established detection limit of plasmid was 1.0×10-5ng/μL.There was no cross-reaction with human blood,Plasmodium vivax,Ascaris lumbricoides,Trichinella spiralis,Schistosoma japonicum,Digramma interrupta,Taenia solium,Digramma interrupta and Entamoeba coli.(3)The optimal combination of primers and probes for the 529 bp gene of T.gondii had the best effect when the primer dosage was 240pmol/L,the reaction amplification time was 30min at 39℃ for the RAA-LFD method.The established detection limit of plasmid was 1.0×105ng/μL.There was no cross-reaction with human blood,Plasmodium vivax,Ascaris lumbricoides,Trichinella spiralis,Schistosoma japonicum,Digramma interrupta,Taenia solium,Digramma interrupta and Entamoeba coli.(4)The positive rates of ECL(IgM),qPCR,LAMP and RAA-LFD in the detection of clinical samples were 1.12%,1.50%,1.50%,and 1.50%,respectively.The detection rates of qPCR,LAMP and RAA-LFD were consistent.By comparing the consistency of the four detection methods,it was found that the coincidence rate of ECL,qPCR,LAMP and RAA-LFD methods was 85.77%(229/267).Conclusion:Newborns and pregnant women in Wuhu area were infected with Toxoplasma,and premature infants were related to T.gondii infection.The established LAMP and RAA-LFD methods for the detection of T.gondii are simple,rapid,sensitive and specific,and have a high coincidence rate with the detection results of ECL and qPCR methods.Therefore,the established LAMP and RAA-LFD methods in this study are suitable for the rapid detection of T.gondii in clinical blood samples.