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1.Zoonotic parasites

1.1A Fluorescent Recombinase Aided AmplificationAssay for Detection of Babesia microt

1.2Rapid Visual Detection of Plasmodium Using Recombinase-Aided Amplification With Lateral Flow DipstickAssay

A Fluorescent Recombinase Aided Amplifica-tion Assay for Detection of Babesia microti

Abstract Babesia microti is one of the most common causative agents of babesiosis.A sensitive and raplddetection is necessary for screening potentially infected individuals.In this study,B. microti cytochrome coxldase subunit l(cox1) was selected as the target gene,multiple primers were designed,and optimized by arecombinase-aided amplification(RAA) assay.The optimal primers and probe were labeled with fluorescein.The sensitivity of fluorescent RAAGRAA) was evaluated using gradient diluents of the cox1 recombinantplasmid and genomic DNA extracted from whole blood of B.microti infected mice.The spedificity offRAA wasassessed by other transfusion transmitted parasites.The analytical sensitivity of the fRAA assay was 10 coplesof recombinant plasmid per reaction and 10 fg/ul B.microti genomic DNA.No cross-re action with any otherblood-transmitted parasites was observed.Our results demonstrated that the fRAA assay would be rapid,sensitive,and specific for the detection of B.microti.

Key words:Babesia microti,recombinase-aided amplification,molecular detection.

Rapid Visual Detection ofPlasmodium Using Recombinase Aided Am-plification With Lateral Flow Dipstick Assay

Background:Malaria is a global public health problem.China has had no case of indigenous malaria since 2016.Hawever,imported cases of malaria remain an issue amang travelers,overseas workers,and foreign traders.Although these cases are always asymptomatic,if they donate blood,there is a great risk of transfusion trans-mitted-malaria(TTM).Therefore,blood banks need a rapid screening tool to detect Pasmodium species.Methods:We designed an assay using recombinase-aided amplification(RAA) and a lateral-flow dipstick(LFD)(RAA-LFD) to detect the 18S ribasomal RNA gene of Plasmodium species.Sensitivity was evaluated using arecombinant plasmid and Plasmodium genomic DNA.Specificity was evaluated using DNA extracted from theblood of patients with malaria ar other infectious parasites.For clinical assessment,blood samples frompatients with malaria and blood donors were evalu ated.Results:The RAA-LFD assay was performed in an incubator block at 37C for 15 min,and the amplicans werevisible to the naked eye an the flaw dipsticks within 3 min.The sensitivity was 1 copy/mL of recombinantplasmid.For genomic DNA from whole blood of malaria patients infected with P.falciparum, P.vivax,P. ovale,and P.malariae,the sensitivity was0.1 pg/mL,10 pg/mL,10-100 pg/mL,and 100pg/mL,respectively.Thesensi-tivity of this assay was 100pg/mL.No cross-reaction withother transfusion transmissible parasites wasdetect-ed.Conclusions:The results demonstrated that this RAA-LFD assay was suitable for reliable field detection ofPlasmodium spedes in low-resource settings with limited laboratory capabilities.

Keywords:malaria,nucleic add detection,plasmodium,recombinase-aided amplification, lateralflow dipstick.