| Giardia canis is a more common zoonotic intestinal parasite protozoan,which spreads worldwide.At present,the classification of Giardia is mainly based on the host,including 8assemblages,namely A-H.Among them,assemblage A and B can infect humans and mammals,assemblage C and D mainly infect canines,aggregate E infects cattle,aggregate F generally infects cats,aggregate G infects murines,aggregate H Mainly infect marine mammals.In clinical situations,traditional immunological methods often use broad-spectrum antibody detection.Broad-spectrum detection has different detection effects for different assemblage.According to the actual clinical situation,false positives and false negatives are often misdiagnosed,and the rate of false and missed detections is high,which makes the actual situation inconsistent with the test expectations.Monoclonal antibodies have strong specificity and can better detect antigens.With the widespread application of monoclonal antibody technology,the specific effect of Giardia immunological detection has been fully enhanced.In this study,the triose phosphate isomerase(TPI)gene and β-giardia protein(BG)gene were used as target genes for genetic identification,so as to determine the main types of canine Giardia in the target area.The positive samples from the canine feces of the first insect were extracted and cultured with Giardia trophozoites.The cultured trophozoites were used to infect the immunized mice,and the corresponding monoclonal antibodies were prepared.The monoclonal antibodies made were based on the sandwich colloidal gold technology.A new rapid immunological diagnosis method for Giardia.1.Identification of the genotype of GiardiaThawing of collected dog feces samples was carried out.DNA in dog feces samples in each region was extracted by phenol extraction.Nested PCR primers were made using triose phosphate isomerase gene and BG(Genotyping at the beta Giardin)gene.The extracted dog fecal genomic DNA was used as a template for nested PCR amplification to determine its positive infection rate.The PCR positive product was sequenced and compared with the sequence of Giardia in Gen Bank to determine its typing.616 samples were collected from Guangzhou,China;ho chi minh city,Vietnam;Kuala Lumpur,Malaysia;Singapore;and Manila,Philippines.Nest PCR primers were designed according to TPI gene and BG gene,and genomic DNA of each region(70-165)samples of dog feces were extracted as templates for nest PCR amplification.PCR products were sequenced and sequenced to determine their typing and infection rate.Of the 616 samples,48 were positive,with a positive rate of 7.79%(48/616).The infection rates were 4.81% in Guangzhou,China,6.90% in ho chi minh city,Vietnam,9.09% in Kuala Lumpur,Malaysia,2.86% in Singapore and 11.52% in Manila,Philippines.In 48 samples,bands were detected at TPI and BG sites,with positive rates of 7.63% and 5.19%,respectively.After sequencing the positive samples for BG and TPI gene loci,it was found that the BG loci contained cluster assemblage B(3.33%)cluster assemblage C 26(86.67%)cluster assemblage D 10%,and the TPI loci contained cluster assemblage B(2.12%)cluster assemblage C(85.1%)cluster assemblage D(12.76%).After statistical analysis and comparison,there was no significant difference between the genotypes of giardia in different regions.2.Giardia trophozoite antigen culture and monoclonal antibody productionTwo different Giardia trophozoites cultured from canine feces samples were prepared with two different Giardia antigens,Giardia excretion and secretion antigens;Giardia soluble antigens,and the prepared antigens were used to infect and immunize mice,and hybridoma cell fusions after screening,seven cell lines that can stably secrete monoclonal antibodies were obtained.Western blotting was used to verify that the prepared monoclonal antibodies successfully recognized Giardia trophozoite excretion and secretion antigens and soluble antigens.Among them,the FG4 strain recognizes bands of 180 k Da and 175 k Da,and has no immune cross-reactivity to antigens such as Ehrlichia canis,Heartworm canine,Schistosoma eggs,etc.The double-antibody sandwich method colloidal gold test paper is established by the prepared monoclonal antibody.After the test paper is made into a finished product,it has good specificity and will not cross-react with the antigens of other insect species;strong sensitivity,and Giardia The detection limit of trophozoite samples is lower than 0.125μg,which has strong repeatability and small gaps between batches.After passing the detection of dog feces samples,the detection ability of the test paper is slightly stronger than that of the commercially available British J&G company’s product and close to the PCR detection result.1.Dogs in the surrounding areas of the South China Sea are infected with Giardia.The infected Giardia is mainly assemblage C,assemblage B and assemblage D exist.2.Antibodies prepared from assemblage C Giardia trophozoites can recognize trophozoite soluble antigens and excretion secretion antigens,and have no cross-reactivity with Ehrlichia canis,Canis heartworm,Schistosoma eggs,and have good specificity3.The colloidal gold test paper established based on the assemblage C antibody can be used for the detection of Giardia faecalis antigen,and has high sensitivity,specificity and stability. |
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