Establishment And Application Of CRISPR-Cas12a And Cas13a Nucleic Acid Detection Methods For Feline Herpesvirus Type 1(FHV-1) And Feline Calicivirus(FCV)

Feline herpesvirus I（FHV-1）and feline calicivirus（FCV）are two major viral pathogens causing upper respiratory tract disease（RTD）in cats.FHV mainly causes rhinotracheitis,also known as feline viral rhinotracheitis virus,while FCV often causes stomatitis,gingivitis and localized lesions of tongue.The prevalence of FHV-1 in cold seasons（spring and winter）is higher than that in warm seasons（summer and Autumn）,while the prevalence of FCV is not affected by the weather.These two pathogens have high prevalence,and the detection and isolation rate can be as high as 84%.Both viruses have strong vitality,which are posing a great threat to the health of the cat group.The purpose of this study was to establish a rapid detection method of feline herpesvirus type I（FHV-1）and feline calicivirus（FCV）based on RPA-Cas12a and RPA-Cas13a system and to carry out clinical validation.According to the analysis and comparison results of FCV and FHV-1 genome sequences,cr RNA and RPA amplification primers were designed with FHV-1-TK gene sequence and FCV ORF1 gene sequence as targets respectively.The method established in this study was compared with SYBR Green RT q PCR to evaluate the effectiveness of FHV-Cas12a and FCV-Cas13a methods from the aspects of RPA primer screening,sensitivity,specificity and clinical sample detection.Furthermore,based on the single disease detection method,the double RPA-CAS12A/Cas13a system with fluorescence and LFD detection method was established to realize the simultaneous detection of FHV-1 and FCV.The results of this study are as follows:1.Detection target screening and primer design of FHV-1 and FCV.Download the sequence of FHV-1 TK and FCV reference strain from NCBI,and conduct multiple sequence alignment of the whole gene and target region sequence.The results of phylogenetic tree showed that FHV-1 gene sequence was highly conserved,while FCV showed rich genetic diversity.The homology of TK genes of 12 FHV-1 reference strains was 99.89%,and the homology of fragment genes in 18 FCV ORF1 was 95.8%.RPA-FHV-F/R and RPA-FCV-F/R primers,FCV cr RNA and FHV-1 cr RNA sequences were designed in the locking region.After screening,RPA-FHV-F2/R1 and RPA-FCV-F3/R were selected as the optimal primer pairs,and the detection limit of the target gene reached single copy number,showing good sensitivity and specificity,which provided basic materials for the establishment of nucleic acid detection methods for FHV-1and FCV.2.Establishment and application of nucleic acid detection methods for FHV-1-Cas12a and FCV-Cas13a.The fluorescence intensity was monitored by fluorescence quantitative PCR reaction detection system,and verified by chemiluminescence imaging system and flow chromatography test strip.The method established in this study was compared with SYBR Green fluorescence quantitative PCR to evaluate the effectiveness of FHV-1 RPA-Cas12a and FCV RPA-Cas13a methods from the aspects of sensitivity,specificity and clinical sample detection.The results show that the established FHV-1RPA-Cas12a method and FCV RPA-Cas13a method can accurately detect the target gene,have no cross reaction with other cat related pathogens,have good specificity,and have higher sensitivity than SYBR Green fluorescence quantitative PCR method,and can detect the plasmid template with single copy number.The minimum detection limit of FHV-1RPA-Cas12a detection method is 2.35×10^-1 copies/μL,the minimum detection limit of RPA-Cas13a detection method is 5.5×10⁰copies/μL。Finally,20 and 56 clinical samples with clinical symptoms were detected by two methods,and FHV-1 was detected in the first batch of samples（20）.The detection rate of SYBR Green q PCR method was 25%（5/20）,and the detection rate of FHV-1 by FHV-1-Cas12a method was 35%（7/20）.The second batch of samples（56 samples）were detected by FCV.The detection rate of SYBR Green RT q PCR was 42.8%（24/56）,and that of FCV-Cas13a was 67.8%（38/56）.The detection rate of pathogens was higher than that of SYBR Green q PCR.The above results show that the established detection methods of FHV-1-Cas12a and FCV-Cas13a can be used as tools for rapid clinical diagnosis of FHV-1 and FCV.3.Double detection of FHV-1 and FCV by RPA-CRISPR-Cas12a/13a.In the reaction system,Cy5 and FAM probes were combined respectively,and the chemical excitation light was used for multiple color imaging to output the detection results.At the same time,the side flow chromatography test strip was used for verification.The sensitivity and specificity of this method are consistent with that of single detection method.The detection rate of FHV-1 was 33.9%（19/56）;The detection rate of FCV was37.5%（21/56）;The double positive detection rate of FHV-1 and FCV was 12.5%（7/56）,which was higher than that of fluorescence quantitative PCR.Finally,the one-step detection of RPA and cas12a/cas13a is preliminarily tried,and the detection can be completed under the conditions of 40°C-42°C and 60 min.The FHV-1-Cas12a and FCV-Cas13a single nucleic acid detection methods established in this study have the characteristics of high sensitivity and short detection time.The equipment involved is relatively simple.There is no need for complex denaturation,annealing and extension steps to amplify the target.The pathogen detection can be realized under the constant temperature condition of 37°C.Then,based on the single detection system,the double detection method of RPA-Cas12a/Cas13a is established,which is combined with the flow chromatography test strip to realize the simultaneous detection of FHV-1 and FCV and the visualization of the results.The detection method based on CRISPR/Cas system established in this study does not rely on special equipment,the reaction process is fast and convenient,and the detection rate is high.It has application value for the on-site rapid diagnosis of corresponding pathogens in clinic.