Establishment And Application Of CRISPR/Cas12a Assay For Rapid Detection Of Giardia Duodenalis And Toxoplasma Gondii

Giardia duodenalis is a common intestinal protozoan.Among them,G.duodenalis assemblage A and assemblage B are zoonotic assemblage capable of infecting humans,domestic and wild animals.Toxoplasma gondii has a wide range of hosts and can infect almost all mammals and birds,including humans.Immunocompetent individuals infected with both parasites are usually asymptomatic.Infection in immunocompromised people usually results in severe illness and even death.Both parasites pose a great threat to human health and public health systems.The CRISPR/Cas system,consisting of Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)and CRISPR-associated(Cas)proteins,is an adaptive immune system that exists in most archaea and many bacteria.According to the function of its effector proteins,CRISPR/Cas system is mainly used in gene editing and nucleic acid detection.Notably,type V Cas12 a protein can bind to specific target sequences under the guidance of CRISPR RNA(cr RNA)transcribed by the CRISPR system,and exert its trans-cleavage function to non-specifically cleave any ss DNA.At present,CRISPR/Cas12 a system has been applied to the detection of a variety of pathogen.In this study,the specific target sequences of G.duodenalis assemblage A and assemblage B were screened by alignment of the tpi site sequences of different assemblage,and the corresponding cr RNA were designed.Recombinase Polymerase Amplification(RPA)and CRISPR/Cas12 a system(termed REPORT)were combined to establish rapid detection methods for G.duodenalis assemblage A and assemblage B,respectively.These detection methods do not rely on expensive and large instruments,and the whole reaction process can be carried out in a constant temperature incubator or water bath,and even can be completed by using body temperature.The final test result can be determined by using a cheap blue light meter or portable blue light lamp to observe whether the test product has fluorescence,or can also be determined by observing whether the colloidal gold test strip after adding the reaction product appears the detection line.These detection methods can be used for the rapid detection of G.duodenalis assemblage A and assemblage B in first-line clinical detection and some low-resource areas.The results showed that the rapid detection method based on REPORT for G.duodenalis assemblage A and assemblage B have high specificity and do not have cross-reaction with other Giardia species and other G.duodenalis assemblages.The sensitivity test showed that the G.duodenalis assemblage A detection method based on REPORT could detect the lowest bacterial concentration of 2.04 CFU/m L and 10 trophozoides per gram of feces.The detection method of G.duodenalis assemblage B assay based on REPORT also showed a high sensitivity with a lowest bacterial concentration of 1.1 CFU/m L and 10 cysts per gram of feces.In this study,the target sequence with intraspecific conservation and interspecific specificity was selected by aligning the 529 bp repeat sequences of different genotypes of T.gondii,and the corresponding cr RNA was designed.In this study,the target fragment was amplified by RPA reaction.The Cas12 a protein recognized the target fragment and performed trans-cleavage function under the guidance of cr RNA to nonspecifically cut ss DNA and realize signal conversion.The detection method of T.gondii based on REPORT don’t have cross-reaction with Neospora caninum,Sarcocystis miescheriana,Theileria luwenshuni,Babesia bovis,Aanapalsma phagocytophilum,Cyclospora cayetanensis,Cryptosporidium parvum,G.duodenalis assemblage A,Enterocytozoon bieneusi,and Eimeria spp.of swine,and have high specificity.The sensitivity test results showed that,the detection limit of the detection method of T.gondii based on REPORT were 2.2 copies/μL DNA concentration,5 tachyzoites per m L of blood sample and 3.1 tachyzoites per gram of tissue sample,showing high sensitivity.In this study,the detection methods of G.duodenalis assemblage A and assemblage B based on REPORT were applied to the detection of clinical samples,and were compared with the PCR method and superior to the latter.In this study,the rapid detection method of T.gondii based on the REPORT was applied to the detection of clinical samples,and compared with the PCR method,the results showed 100% consistency.The three rapid detection methods of G.duodenalis assemblage A,assemblage B and T.gondii developed in this study based on the REPORT can be stably used for the detection of clinical samples.