Establishment And Application Of Three Visual Nucleic Acid Detection Methods For Feline Herpesvirus

Feline rhinotracheitis is an acute,highly contactable upper respiratory disease in which the causative agent,feline herpesvirus type 1（FHV-1）,primarily infects cats and felines,with a mortality rate of up to 50%in young cats.Like other members of the herpesvirus family,FHV-1can be latent in the host trigeminal nerve,optic nerve,and submandibular lymph nodes,causing persistent,latent infection and resulting in lifelong virulence in infected cats.Once the body’s resistance decreases,there is a risk of reactivation and outgrowth of the virus.At the same time,FHV-1 often causes mixed infections with other viruses,such as feline calicivirus（FCV）,further increasing mortality and seriously endangering the health of cats and felines.Currently,the main detection methods for FHV-1 in China are PCR or fluorescent quantitative PCR methods,which have great advantages in terms of technical maturity,but the reliance on sophisticated instruments and professional personnel,as well as high costs,make them still limited in clinical application.Therefore,there is a need to establish simple,rapid,and applicable FHV-1 nucleic acid detection methods for pet hospitals or primary units in order to assist vets in rapidly developing effective treatment plans.When compared to other nucleic acid amplification techniques,recombinase-aided amplification（RAA）has the advantages of short detection time,no need for precision instruments,and diverse result reading.In this study,several circulating strains of FHV-1 were selected and used Meg Align software to compare and analyze them,selected the highly conserved TK gene of FHV-1 as the target gene,and used the RAA isothermal amplification technique to amplify exponentially.The targets were combined with immunochromatographic test paper,a home portable glucose meter（PGM）,and CRISPR/Cas12a technology,three visual nucleic acid detection methods targeting FHV-1 TK gene were established..The main findings are as follows:1.Establishment and evaluation of the FHV-1 RAA-VF assay.A RAA-VF nucleic acid visualization assay targeting the FHV-1 TK gene was established by combining recombinase-aided amplification（RAA）with immunochromatographic test paper.The specific primer pairs labeled with FITC and biotin were designed and synthesized,and the double-labeled amplification products could be obtained by RAA amplification.Visual interpretation of double-labeled amplification products was realized by using nucleic acid test paper fixed with FITC antibody and streptavidin.The sensitivity of this method is better than that of conventional PCR methods,as it can detect FHV-1 at as low as 14.2 copies/μL recombinant plasmid and 20TCID₅₀/m L in 20 min at 42°C.And the method has no cross-reactivity with FCV,FPV,FCo V,CAV,or CPIV and good specificity.At the same time,the FHV-1 RAA-VF assay does not require special instruments or equipment and is easy to operate.To achieve rapid visualization detection of FHV-1,it only takes about 5 minutes to read the results from the closed disposable nucleic acid visualization test paper device.2.Establishment and evaluation of the FHV-1 RAA-PGM assay.The FHV-1 RAA nucleic acid visualization detection method（RAA-PGM）based on a home blood glucose meter（PGM）was established using RAA technology and biosensing technology.Using the principle that sucrose converting enzyme can convert sucrose to glucose,a probe coupled with sucrose converting enzyme at the 5’end and complementary to the upstream primer sequence was introduced after the RAA reaction,and the remaining amount of the upstream primer not involved in the reaction was detected in reverse after the addition of the substrate sucrose to indirectly achieve the quantitative detection of FHV-1 DNA.The method can detect as few as14.2 copies/μL of recombinant plasmid at a constant temperature of 42°C,which is more sensitive than the common PCR method.The results of the specificity experiments also showed that the method had no cross-reactivity with FCV,FPV,FCo V,CAV or CPIV.Since PGM replaced the precision power supply equipment,it has not only reduced the cost of detection but also realized the visualization and semi-quantitative detection of FHV-1.3.Establishment and evaluation of the FHV-1 RAA-CRISPR/Cas12a assay.A rapid visualization assay for FHV-1 RAA-CRISPR/Cas12a nucleic acid was established by combining RAA isothermal amplification technology with the CRISPR/Cas12a system.Firstly,cr RNA specifically recognizing FHV-1 was designed and screened,and the rapid detection of FHV-1nucleic acid was achieved by using Cas12a,which can bind RAA amplification products under cr RNA guidance and activate its trans cleavage activity,to cut FAM-BHQ-labeled ss DNA to release fluorescence.The method requires only about 40 min at a constant temperature of 42°C to detect recombinant plasmids down to 1.42 copies/μL.There is no cross reaction with FCV,FPV,FCo V,CAV,CPIV nucleic acid,and has good specificity.The method does not require opening the cap during the reaction,avoids aerosol contamination,and generates a fluorescent signal visible to the naked eye for rapid visualization of FHV-1.In conclusion,this study targeted the FHV-1 TK gene and provided three simple,rapid,and sensitive nucleic acid visualization methods based on the RAA amplification technique combined with three different resultant signal reading methods for rapid screening of cats suspected of being infected with FHV-1,as well as effective technical support for feline infectious rhinobronchitis in primary laboratories,veterinary hospitals,and even for achieving self-testing adequacy.