1. Zoonotic parasitic diseases

1.1 A Fluorescent Recombinase Aided Amplification Assay for Detection of Babesia microti

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

1.3 Establishment of a method for detecting Plasmodium using recombinase-mediated isothermal amplification technology

1.4 Application of real-time fluorescence recombinase-mediated nucleic acid amplification in the rapid detection of Plasmodium

1.5 Establishment and preliminary evaluation of a recombinase-mediated isothermal amplification fluorescence detection method for Schistosoma japonicum

1.6 Establishment of a recombinase-mediated isothermal amplification detection method for specific gene fragments of Schistosoma japonicum

1.7 Rapid detection of Schistosoma japonicum gene fragments by combining recombinase-mediated isothermal nucleic acid amplification and fluorescent probes

1.8 Strengthening the research and application of molecular diagnostic methods to promote precise schistosomiasis prevention and control in China

1.9 Rapid detection of Schistosoma japonicum-infected Oncomelania hupensis by recombinase-mediated isothermal nucleic acid amplification fluorescence method

1.10 Establishment and evaluation of a recombinase-mediated isothermal nucleic acid amplification method specific for Cryptosporidium

1.11 Establishment and preliminary evaluation of a recombinase-mediated isothermal nucleic acid amplification method specific for Clonorchis sinensis

1.12 Sending the "plague god" again: the latest research progress on appropriate technologies for eliminating schistosomiasis

1.13 Establishment and preliminary application of a recombinase-mediated isothermal nucleic acid amplification technique for the detection of Echinococcus multilocularis

1.14 Establishment and preliminary application evaluation of a recombinase-mediated isothermal nucleic acid amplification technique for the detection of Echinococcus granulosus

1.15 Establishment of a nucleic acid detection method for Gnathostoma spinigerum based on recombinase-mediated isothermal amplification technology

1.16 Establishment of a nucleic acid detection method for Echinococcus granulosus based on recombinase-mediated isothermal amplification technology

1.17 Establishment of a gene detection method for Schistosoma mansoni based on recombinase-mediated isothermal nucleic acid amplification reaction

1.18 New isothermal amplification technology promotes the improvement of rapid on-site detection capacity for parasitic diseases

1.19 Establishment and evaluation of a recombinase-mediated isothermal nucleic acid amplification method specific for Giardia lamblia

1.20 Establishment of a nucleic acid detection method for Leishmania based on fluorescence recombinase-mediated isothermal amplification technology

1.21 Establishment of a nucleic acid dipstick detection method for specific gene fragments of Schistosoma japonicum based on recombinase-mediated isothermal nucleic acid amplification technology

1.22 Establishment of a rapid detection method for recombinase-mediated isothermal nucleic acid amplification of Apodemus agrarius Babesia

1.23 Evaluation of the effectiveness of recombinase-mediated isothermal nucleic acid amplification fluorescence method for detecting Schistosoma japonicum-infected Oncomelania hupensis

1.24 Establishment and evaluation of a method for detecting Plasmodium using recombinase-mediated isothermal amplification technology

1.25 Study on the application of recombinase-mediated isothermal nucleic acid amplification fluorescence method for the detection of Schistosoma japonicum-infected Oncomelania hupensis in the dry season

A Fluorescent Recombinase Aided Amplification Assay for Detection of Babesia microti

Abstract Babesia microti is one of the most common causative agents of babesiosis. A sensitive and rapid detection is necessary for screening potentially infected individuals. In this study, B. microti cytochrome coxidase subunit I (cox1) was selected as the target gene, multiple primers were designed, and optimized by a recombinase-aided amplification (RAA) assay. The optimal primers and probe were labeled with fluorescein. The sensitivity of fluorescent RAA (fRAA) was evaluated using gradient diluents of the cox1 recombinant plasmid and genomic DNA extracted from whole blood of B. microti infected mice. The specificity of fRAA was assessed by other transfusion transmitted parasites. The analytical sensitivity of the fRAA assay was 10 copies of recombinant plasmid per reaction and 10 fg/μl B. microti genomic DNA. No cross-reaction with any other blood-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 of Plasmodium Using Recombinase Aided Amplification With Lateral Flow Dipstick Assay

Background: Malaria is a global public health problem. China has had no case of indigenous malaria since 2016. However, imported cases of malaria remain an issue among travelers, overseas workers, and foreign traders. Although these cases are always asymptomatic, if they donate blood, there is a great risk of transfusion-transmitted malaria (TTM). Therefore, blood banks need a rapid screening tool to detect Plasmodium species. Methods: We designed an assay using recombinase-aided amplification (RAA) and a lateral-flow dipstick (LFD) (RAA-LFD) to detect the 18S ribosomal RNA gene of Plasmodium species. Sensitivity was evaluated using a recombinant plasmid and Plasmodium genomic DNA. Specificity was evaluated using DNA extracted from the blood of patients with malaria or other infectious parasites. For clinical assessment, blood samples from patients with malaria and blood donors were evaluated. Results: The RAA-LFD assay was performed in an incubator block at 37°C for 15 min, and the amplicons were visible to the naked eye on the flow dipsticks within 3 min. The sensitivity was 1 copy/mL of recombinant plasmid. For genomic DNA from whole blood of malaria patients infected with P. falciparum, P. vivax, P. ovale, and P. malariae, the sensitivity was 0.1 pg/mL, 10 pg/mL, 10-100 pg/mL, and 100 pg/mL, respectively. The sensitivity of this assay was 100 pg/mL. No cross-reaction with other transfusion transmissible parasites was detected. Conclusions: The results demonstrated that this RAA-LFD assay was suitable for reliable field detection of Plasmodium species in low-resource settings with limited laboratory capabilities.

Keywords: malaria, nucleic acid detection, Plasmodium, recombinase-aided amplification, lateral flow dipstick.

Establishment of a method for detecting Plasmodium using recombinase-mediated isothermal amplification technology

Abstract: This study utilizes the recombinase-aided isothermal amplification (RAA) technology. Targeting the Plasmodium 18SrDNA sequence, universal primers for detecting Plasmodium were designed, screened, and their specificity was tested. Four primer sets with good amplification effects were selected. The entire reaction process is carried out at 37℃, with a short amplification time (40min) and good specificity. A RAA method for detecting Plasmodium has been successfully established, and this method is suitable for the rapid detection of Plasmodium at import and export ports.

Keywords: Recombinase-aided amplification; Plasmodium; Molecular detection; Malaria.

Establishment and preliminary evaluation of a recombinase-aided isothermal amplification fluorescence detection method for Schistosoma japonicum

Abstract: Objective: To establish a rapid nucleic acid detection method for Schistosoma japonicum based on recombinase-aided isothermal amplification (RAA) technology and to evaluate its detection effect. Methods: Schistosoma japonicum, Schistosoma mekongi, and Schistosoma spindale cercariae were isolated from crab samples, and genomic DNA was extracted for molecular identification. The mitochondrial cytochrome c oxidase subunit I gene (cox1) sequence of Schistosoma japonicum was used as the target sequence to design, prepare, and screen primers and probes. The fluorescence RAA detection method was verified using genomic DNA from Schistosoma japonicum cercariae collected in Jiyuan City, Henan Province and Yiyang County, Luoyang City. Different concentrations of recombinant plasmids containing the Schistosoma japonicum cox1 gene sequence and genomic DNA from Schistosoma japonicum cercariae were used as templates for fluorescence RAA amplification to evaluate its detection sensitivity. The established fluorescence RAA method was used to detect the genomic DNA of Schistosoma mekongi, Schistosoma spindale, Clonorchis sinensis, and Schistosoma japonicum, to evaluate its detection specificity. Results: Schistosoma japonicum, Schistosoma mekongi, and Schistosoma spindale cercariae were isolated from crab samples. Molecular identification and phylogenetic analysis confirmed their homology with the standard strain gene sequences of Schistosoma in GenBank. A fluorescence RAA detection method for Schistosoma japonicum was successfully established. It can amplify the genomic DNA of Schistosoma japonicum cercariae collected in Jiyuan City, Henan Province and Yiyang County, Luoyang City within 5 min, while the negative control showed no amplification. Using recombinant plasmids as templates, the lowest detection limit of the fluorescence RAA method was 10 copies/L of recombinant plasmid, and all showed positive amplification within 5 min. Using genomic DNA as a template, the lowest detectable template DNA concentration was 10 pg/μL, and all showed positive amplification within 10-15 min. The fluorescence RAA method showed negative results for the detection of genomic DNA of Schistosoma mekongi, Schistosoma spindale, Schistosoma japonicum, and Clonorchis sinensis. Conclusion: A rapid, sensitive, and specific nucleic acid detection method for Schistosoma japonicum based on fluorescence RAA technology has been successfully established, which has potential application value in rapid on-site detection and species identification of Schistosoma japonicum in endemic areas.

Keywords: Schistosoma japonicum; Recombinase-aided isothermal amplification; Nucleic acid detection; Detection effect.