Establishment And Preliminary Application Of A Colloidal Gold Immunochromatographic Assay And LFD-RPA Assay For The Detection Of Pentatrichomonas Hominis Infection In Dogs

Pentatrichomonas hominis（P.hominis）is a trichomonas protozoan that parasitizes the cecum and colon of humans and other mammals and is transmitted mainly by the fecal-oral route.In the past,it was considered a non-pathogenic commensal parasite of the intestinal tract or a conditional pathogenic protozoan.However,in recent years,P.hominis infection has been found to cause diarrhea in both animals and humans and is a potentially zoonotic protozoan.Moreover,molecular epidemiological investigations have found that P.hominis infection is closely associated with gastrointestinal cancers.Humans and many animals can be infected with the same P.hominis,such as the P.hominis CC1 genotype,can infect humans and dogs.As companion animals,dogs are close to humans,making them at risk for transmitting P.hominis infection to humans.Therefore,it is of great public health importance to strengthen the detection of P.hominis infection in dogs.However,the current detection methods for P.hominis infection are mainly fecal examination and PCR.The fecal examination is difficult to distinguish similar parasites,low detection rate,and is easy to cause misdiagnosis and missed diagnosis.PCR has high requirements on operators and requires specific instruments,so it is not suitable for clinical field detection.The colloidal gold chromatographic strip in immunological detection and the RPA technology in molecular biological detection have the advantages of being fast and convenient,suitable for the clinical field.However,in the detection methods of P.hominis,there are no relevant reports on immunological detection such as colloidal gold chromatography strip and RPA technology.Therefore,this study first screened candidate antigens for immunological diagnosis,and established an indirect ELISA assay,a Colloidal gold immunochromatographic assay,and an LFD-RPA assay for clinical scene detection,in order to provide a new direction for the detection of P.hominis infection.Screening of candidate antigens for detection of P.hominis infection in dogs The excretory-secretory antigens（ESAs）of P.hominis was collected,and the western blot analysis revealed that the sera of dogs infected with P.hominis could respond with the P.hominis ESAs.Mass spectrometry analysis of the bands in response to infected dog serum resulted in a total of three candidate diagnostic proteins for P.hominis.Among them,glyceraldehyde-3-phosphate dehydrogenase（GAPDH）scored the highest and was therefore selected to construct the prokaryotic expression plasmid p ET-32a-GAPDH,which was expressed and purified in E.coli to obtain GAPDH.Indirect immunofluorescence assays revealed that GAPDH protein was present in the cytoplasm,surface,and flagellum of P.hominis.Western blot assays revealed that GAPDH has good reactogenicity and the potential to detect P.hominis.Establishment and preliminary application of a Colloidal gold immunochromatographic assay for P.hominis infection in dogs An Indirect ELISA assay for P.hominis infection was established using GAPDH protein as the detection antigen.After optimizing the conditions,it was found that the optimal antigen coating dose was 0.25μg/well,the optimal sample dilution was 1:10,the optimal blocking agent was 5%skim milk powder,the optimal dilution concentration of HRP-labeled rabbit anti-dog Ig G was 1:3000,and the optimal reaction time of the substrate was 15 min,and the sensitivity of the method could reach 1:160.Based on the indirect ELISA method,a Colloidal gold immunochromatographic assay for P.hominis infection was established.After optimizing the conditions,it was found that the optimal p H value was 2μL of 0.2 mol/L K₂CO₃ added to each milliliter of colloidal gold solution,the optimal SPA protein labeling dose was 6μg per milliliter of colloidal gold solution,the optimal coating concentration for the quality control line（C）was 0.0625mg/m L,the optimal coating concentration for the detection line（T）was 0.5 mg/m L.The sensitivity of the test strip could reach 1:80,and the shelf-life test found that the test strip could be stored at 4℃for 80 days and at both room temperature and 37℃for60 days.After detection,both methods were found to be non-cross-reactive with Giardia duodenalis-infected dog sera and Tritrichomonas foetus-infected dog serum,with good specificity,reproducibility,and stability.The established Colloidal gold immunochromatographic assay were applied to detect feces and serum of 20 dogs,respectively,and compared with the pathogenic method,resulting in four positive samples detected by all three methods,with a 100%compliance rate.The established indirect ELISA and Colloidal gold immunochromatographic assays were applied to detect 95 dog serum samples in Changchun,and the results showed that 11 positive samples were detected by both methods,with a positivity rate of 11.58%.Establishment and preliminary application of an LFD-RPA assay for P.hominis infection in dogs The highly conserved SPO11-1 gene was used as the target gene to screen the best primers and probes for the establishment of an LFD-RPA assay for P.hominis infection in dogs,and compared with the established nested PCR method.The method was found to be non-cross-reactive with the genomes of Giardia duodenalis,Tritrichomonas muris,Tritrichomonas foetus,Isospora,and Toxocara canis,indicating good specificity.The minimum detection limit of the method was 10²copies/μL.The established LFD-RPA and Colloidal gold immunochromatographic assay were applied to detect the feces and serum of 20 dogs,respectively,and compared with the pathogenic method,resulting in four positive samples detected by all three methods,with a 100%compliance rate.Using the established LFD-RPA,7 positive samples were detected in 72 canine fecal samples from a stray animal rescue station in Changchun,with a positivity rate of 9.72%,which was consistent with the results of the established nested PCR method.In summary,this study established an indirect ELISA and Colloidal gold immunochromatographic assay for the detection of P.hominis infection in dogs using the screened GAPDH protein as the antigen,and an LFD-RPA assay for the detection of P.hominis infection using the SPO11-1 gene as the target gene,both methods showed good specificity and sensitivity when applied to clinical samples in a different scene,which can provide technical support for the control of P.hominis.