Development Of HRM And Multi-ARMS-qPCR Method For Detection Of Hookworms From Cats And Dogs

Ancylostoma ceylanicum,A.canincum and A.tubaeforme are widely distributed parasites all over the world,which can infect humans,dogs,cats and many other animals and cause hookworm disease with anemia and diarrhea as the main symptoms.This study treated the hookworm from stray dogs and cats in South China as research object to establish HRM and Multi-ARMS-qPCR method for detection of hookworms from cats and dogs based on ITS1 sequence,in order to provide new technical supportment for molecular epidemiological survey,risk assessment of zoonosis and curative effect assessment of hookworm disease.Firstly,a new method for detection of dog-derived and cat-derived hookworm included Ancylostoma ceylanicum,A.canincum and A.tubaeforme in South China was developed by HRM assay based on ITS1.The result showed that the primers HRM-F and HRM-R were able to differentiate among three hookworms.A series of experiments on the stability,sensitivity and accuracy of the HRM method were also conducted.Results showed that the melting curves of intra-and inter-assay reproducibility were almost coincided.The lowest detection concentration was about 5.69×10~-44 ng/μL.The HRM detection results from18 canine and feline hookworm samples were in complete accordance with their sequencing results.The results indicated that the HRM method is rapid,stable,sensitive,accurate,and suitable for clinical detection and molecular epidemiological survey of dog-derived and cat-derived hookworms.Secondly,we compared clinical detection effect of dog-derived and cat-derived hookworm by HRM with that by PCR-RFLP,where the universal primer AF and AR were used to amplify the ITS1 sequence of 98 canine and feline samples,and the PCR products were digested by EcoRII and RsaI restriction endonuclease.Results showed that all positive fecal samples were detected to be positive by using the HRM method,they were identified as A.ceylanicum(n=6),A.caninum(n=15),A.tubaeforme(n=4)and mixed infections of A.ceylanicum and A.caninum(n=17).While only thirty-nine positive fecal samples were detected by using PCR-RFLP,including A.ceylanicum(n=4),A.caninum(n=18),A.tubaeforme(n=4)and mixed infections of A.ceylanicum and A.caninum(n=13).So,it is concluded that HRM method has a better clinical detection effect.Thirdly,for the purpose of establishing ARMS-qPCR molecular detection method of dog-derived and cat-derived hookworm,three sets of primers(ARMS-Cey/F,ARMS-Tub/F and ARMS-Can/F)were designed according to three SNPs(ITS253,ITS150 and ITS78)to detect three hookworms by combining ARMS with qPCR.Results showed that three sets of primers were able to detect A.ceylanicum,A.tubaeforme and A.caninum,respectively.Among the three pairs of primers,the specificity of ARMS-Cey and ARMS-Tub were good,but the specificity of ARMS-Can primer was poor,which needed to be optimized by annealing temperature.the specificity of ARMS-Can in Multi-ARMS-qPCR was good.Results indicate that the ARMS-qPCR method for three hookworms was established basically,which will be applied on the research of subsequent Multi-ARMS-qPCR detection method.Finally,a Multi-ARMS-qPCR method for detecting simultaneously A.ceylanicum,A.tubaeforme and A.caninum was established based on ARMS-qPCR.Results showed that three specific melt peaks were generated by Multi-ARMS-qPCR,and three specific bands(268 bp,170 bp and 94 bp)were produced by PCR.The linear correlation coefficients of the three hookworms were 0.994,0.999 and 0.999,respectively;the efficiency were 94%,102%and 97%,respectively;the lowest quantitative detection was 104 eggs/g,2 eggs/g and 1 egg/g,respectively.The intra-and inter-assay reproducibility were good.Seventeen out of fifty feline and canine fecal samples were positive,including A.caninum(n=8),A.tubaeforme(n=2),mixed infections of A.ceylanicum and A.caninum(n=5)and mixed infections of caninum and A.tubaeforme(n=2).Among the 10 samples with single infection,three samples were severe infection(C≥100),five samples were moderate infection(10≤C<100),two samples were mild infection(C<10).It is concluded that Multi-ARMS-PCR method is specific,stable and capable of quantitative analysis,which can remedy the deficiency of the traditional detection method,and provide a new technical means for the risk assessment of zoonotic ancylostomiasis and the curative effect evaluation of hookworm disease.