The Establishment And Application Of Detection Methods For Giardia Duodenalis Based On RNase P Gene And TPI Gene

Giardia duodenalis is an enteric parasite causing diarrhea, abdominal pain, andadverse reactions and is also associated with epidemic water-borne outbreaks. InChina, dog is commonly in close contact with humans, thus could be serve as a mainhost of giardiasis. It is necessary to develope an effective detection tool for diagnosisof giardiasis. The traditionally used detection methods, such as reactive tissue assaysand immunological diagnostic methods, were in the face of difficulty in samplecollection and treatment, and also need some specialized persons for morphologicaldiscrimination. On the basis of RNase P gene sequence specific to Giardia duodenalis,we designed two pairs of specific primers and established a nested PCR detectionmethod. We tested85canine fecal samples in Changchun, revealing an infection rateof63.5%. Based on TPI gene sequences specific to Giardia, we designed two pairs ofprimers and established a Nested PCR detection method. We used the method to test92fecal samples of dogs in Shenyang,85fecal samples of dogs in Changchun, and60fecal samples of dogs in Harbin, with the infection rate of14.1%in Shenyang,63.5%in Changchun, and32.7%in Harbin. A main TPI genotype (assemblage A) wasdetermined, which has zoonotic potential and is of public health concern. We used thetwo methods to detect Giardia, Trypanosoma evansi, E. tenella, Toxoplasma gondii,Trichomonas vaginalis, and Heartworm. Only Giardia was effectively detected,confirming the specificity of the assay. We also compared single-round PCR methodsand the Nested PCR assays we established to amplify DNAs of Giardia trophozoitesat a range of concentrations of10~0、10~1、10~2、10~3、10~4、10~5、10~6、10~7、10~8、10~9.Nested PCR can detect100of Giardia, showing100-fold more sensitive thansingle-round PCR. Thus, we successfully constructed two specific gene-basedmolecular detection assays for Giardia.