Establishment Of ELISA,Nested PCR,and LAMP Detection Assays For Giardia Duodenalis

Giardiasis is a parasitic zoonotic disease caused by Giardia duodenalis that parasitizes in intestines of humans and animals.The disease is globally distributed and seriously impact human health.G.duodenalis is highly infectious and can cause water-borne outbreaks.The clinical manifestations of human infection with Giardia are mainly diarrhea.At present,the prevention and treatment of giardiasis is getting deeper and deeper.Giardin is a unique cytoskeletal component of Giardia.Currently,giardins are divided into four types: ?-giardin,?-giardin,?-giardin and ?-giardin,Among them,Giardia ?-giardin is a immunogenic Giardia-specific protein.Therefore,this study selected Giardia ?-giardin as antigen to establish a diagnostic method for giardiasis,which should be benificial to prevention and diagnosis of giardiasis.In this study,the prepared ?-giardin recombinant protein expression strain was used for expression and purification,and optimized the conditions.A polyclonal antibody was prepared by immunizing BALB/c mice with the obtained ?-giardin protein.The ?-giardin protein was used as a coating antigen to establish an indirect ELISA assay,and we optimized the conditions of ELISA at the same time.This method was used to test the positive serum of Cryptosporidium,Microsporidium and Trichomonas,and 50 samples of serum were clinically tested.The nested PCR primers were synthesized according to the ?-giardin gene sequence.The DNA of Giardia was extracted as a template,and then the Giardia nested PCR detection method was established.The nested PCR conditions were optimized,and the DNA template containing different Giardia numbers was detected by this method,and the specificity test was carried out with the DNA template of Cryptosporidium,Microsporidium and Trichomonas,and 110 collected fecal samples were tested.According to the ?-giardin gene,the LAMP primers were designed and synthesized,and the extracted Giardia DNA was used as a template.The HNB-LAMP color reaction was used as the result index to establish the Giardia LAMP detection method,and the LAMP reaction program was optimized and utilized.This method detect ed DNA templates containing different Giardia trophozoite numbers.We also conducted specific tests with DNA templates of Cryptosporidium,Microsporidium and Trichomonas,and tested 110 collected fecal samples.The ELISA assay established in this study finally determined that the optimal coating amount of ?-giardin antigen was 200 ng/well;the optimal serum dilution was 1:25600;the negative positive threshold was 0.252.The maximum coefficient of variation for both intra-assay and inter-assay trials was less than 10%,the P/N values of all data are greater than 2.1;and there was no cross-reactivity with Cryptosporidium,Microsporidia and Trichomonas.The positive rate of 50 collected clinical samples was 22%(11/50).The results showed that the ?-giardin protein had strong immunogenicity and good sensitivity and specificity.The established nested PCR detection method can detect Giardia when 100 Giardia trophozoites were contained in the sample,and there was no positive result when detecting Cryptosporidium,Microsporidia and Trichomonas.The positive rate of 110 collected clinical samples was 20.91%(23/110).The results showed that the nested PCR reaction assay established using ?-giardin gene has good sensitivity and specificity.The established LAMP detection method can also have a significant color change when the sample contains 10 Giardia trophozoites at least.When testing the specificity,there was no detectable color change by uses of Cryptosporidium,Microsporidium and Trichomonas DNAs,and the positive rate of 110 collected clinical samples was 23.63%(26/110).The results showed that the LAMP detection method established using ?-giardin gene had good specificity and higher sensitivity compared to nested PCR assay.In addition,the color reaction result was more convenient for visual observation.In this study,three diagnostic methods for giardiasis were established based on ?-giardin gene.All the three methods have good sensitivity and specificity,which provides a new idea for establishment of Giardia immunologic and molecular diagnostic methods.