Establishment And Preliminary Application Of RPA-CRISPR-Cas12a Assay For Trichomonas Vaginalis

Trichomonas vaginalis is an important human-animal parasite that is mainly found in the human genitourinary system,but can also infect humans,squirrel monkeys,mice and other animals.Trichomonas vaginalis infection can cause vaginitis,urethritis,perinatal complications and increased risk of HIV infection.Vaginitis caused by Trichomonas vaginalis is the most common sexually transmitted infection（STI）worldwide.According to the World Health Organization（WHO）,there are approximately 276.4 million cases of trichomoniasis each year,accounting for half of all globally transmitted infections that year and approximately 20%of women will develop trichomoniasis during their reproductive years.Therefore,the establishment of a rapid diagnostic method is important for the control of Trichomonas vaginalis.Currently,microscopic observation is used for clinical examination of Trichomonas vaginalis,but the sensitivity is only 44%-68%and requires specialized technicians to operate^[2].To improve the sensitivity and specificity of Trichomonas vaginalis diagnosis,some nucleic acid amplification techniques including PCR and q PCR have also been developed in recent years,and although these methods can amplify the target DNA or RNA exponentially,they require sophisticated and complex instruments and are not suitable for field detection.Thus,there is an urgent need to develop a rapid,sensitive and highly specific visualization Trichomonas vaginalis diagnostic assay.CRISPR-Cas12a technology is a new generation gene editing method that has been rapidly developed in recent years,relying on the DNA enzyme activity of Cas12a,and the CRISPR-Cas system has been applied for rapid detection of viruses,bacteria,fungi,diseases and parasites in a variety of fields.It has been reported that the cleavage activity of CRISPR does not produce detectable signals at low concentrations of target genes（<10 nM）,but single-molecule detection can be achieved under pre-amplified conditions,so amplification of nucleic acids is the key to highly sensitive detection.Recombinase polymerase amplification（RPA）is a novel thermostatic amplification technique for nucleic acids.Recombinant enzymes bound to primers to form DNA-protein complexes were used in the RPA reaction,which does not require a thermal cycling process and allows exponential amplification of template DNA to detectable levels at a constant temperature of 37℃-42℃.In this study,based on RPA isothermal amplification and the trans cleavage activity of CRISPR-Cas12a,we improved the sensitivity of Cas12a by RPA amplification,used the cleavage of CRISPR-Cas12a to compensate for the disadvantages of RPA false positives,and combined with the lateral flow chromatography strips（LFS）biosensor to establish a highly sensitive and on-site detectable method for Trichomonas vaginalis and clinical sample testing was performed.The method was established to provide a new technical tool for the rapid diagnosis of Trichomonas vaginalis.Expression and purification of Cas12a recombinant protein（r Cas12a）and functional validation:The p GEX-4T-1-Cas12a recombinant plasmid was obtained using homologous recombination technique,and the CRISPR-Cas12a recombinant protein（rCas12a）was obtained after induction,prokaryotic expression and purification;a single band of approximately 160 kDa in size was identified by SDS-PAGE and Western Blot;The fluorescent reporter sensor（ssDNA probe）results showed that the T2 experimental group showed a more pronounced fluorescence signal compared to the T2^-/-and Cas12a^-/-controls,and it was determined that the rCas12a obtained in vitro had trans cleavage activity and could cleave ssDNA.Establishment of the Trichomonas vaginalis RPA-CRISPR-Cas12a assay:The target sites were screened within the Trichomonas vaginalis actin gene,and TV2-crRNA was selected as the best target site based on fluorescence intensity;RPA-F5 and RPA-R3 primer pairs were screened as the RPA amplification primer pairs for the target genes.The specificity test showed that the assay did not cross-react with Candida albicans,Mycoplasma hominis,Neisseria gonorrhoeae,Escherichia coli,Cryptosporidium,Giardia,Toxoplasma and human Genome;the sensitivity test showed that the fluorescent reporter sensor was sensitive up to 1 copy/μL or 1Trichomonas vaginalis/m L and the lateral flow chromatography strip was sensitive up to 1 copy/μL or 10 Trichomonas vaginalis/m L.The results can be read using either a portable fluorometer（fluorescent probe）or a lateral flow chromatography strip（biotin probe）.Application of RPA-CRISPR-Cas12a-based Trichomonas vaginalis detection method to clinical samples:30 human vaginal secretions,8 male urine and 8 semen were tested,and the positive rate of secretions was 26.7%（8/30）by fluorescent reporter sensors and lateral flow chromatography strips,while the positive rate was 23.3%（7/30）by nested PCR,and the results of male urine and semen were negative.The PCR-RFLP technique was used to genotype the positive samples and the genotype of Trichomonas vaginalis were identified as E or M.The phylogenetic tree was drawn;after culturing and purifying the clinical samples,a virus-carrying strain of Trichomonas vaginalis was successfully isolated and tested as TVI:MCP.In conclusion,the RPA-CRISPR-Cas12a visualization field test for Trichomonas vaginalis established in this study provides a good technical tool for Trichomonas vaginalis surveillance and has important public health implications.