Establishment Of CRISPR-Cas12a-based Varicella-zoster Virus Detection Method And Its Application

Varicella-zoster virus(VZV)infects more than 90%of the world’s population.Primary and secondary infections of VZV can cause two different clinical symptoms,with primary infections occurring mainly in childhood and usually presenting as a generalized rash(chickenpox).VZV reactivation causes a wide spectrum of diseases,and in addition to postherpetic neuralgia(PHN),VZV reactivation may lead to a variety of neurological and other systemic complications.Herpes zoster is usually diagnosed by the characteristic unilateral distribution of clustered small blisters.However,some atypical rashes,such as generalized herpes zoster and herpes zoster without rash,are not available,and in these cases pathogenic diagnostic methods are essential for accurate diagnosis and prompt treatment.Common diagnostic methods for VZV include viral culture and direct fluorescent antibody assay(DFA),polymerase chain reaction(PCR),and serologic testing.However,these methods inevitably require a trade-off between sensitivity,specificity,simplicity and rapidity.Therefore,it is of great clinical value to establish an assay that is simple to perform,requires low personnel and equipment,and can be used for clinical diagnosis in the field.Therefore,we developed a lateral flow assay(CQ-LFA)based on CRISPR/Cas12a and quantum dot nanobeads(QDNBs)to detect VZV by targeting the ORF31 gene of herpes zoster virus.The results of our study are as follows:(1)Recombinase-aided amplification(RAA)primers targeting the conserved sequence of ORF31 encoding the VZV g B protein,were successfully designed.According to the RAA primer design principle,two pairs of primers were designed,and the best pair of primers was selected by agarose gel electrophoresis,which could successfully amplify the target with high amplification efficiency.(2)Successful design of cr RNA targeting VZV RAA amplicons and applicable to Lb Cas12a.Based on the PAM(TTTN)sequence required for Lb Cas12a activation,we searched for TTTN sequence on the sequence of RAA amplicons and designed the cr RNA.The experimental results showed that the designed cr RNA could successfully match with the target and activate Lb Cas12a cleavage activity.(3)Lateral flow assay labeled with quantum dot nanobeads(QDNBs)were successfully constructed.The QDNBs were conjugates with anti-FAM antibody,and the biotin-ss DNA-FAM reporter probe was added and loaded onto the test strips for detection,with streptavidin embeded in the test(T)line and goat anti-mouse Ig G antibody encapsulated in the control(C)line.As a result,a clear fluorescent signal appeared in the T line,indicating the successful construction of quantum dot nanobeads(QDNBs)-labeled Lateral flow assay strips.(4)Successfully built and optimized CQ-LFA detection platform.We optimized the isothermal amplification time,the concentration of reporter probes and the ratio of QDNBs to anti-FAM antibodies,respectively.The optimal amplification time of 30 min,the optimal reporter probe concentration of 10 n M,and the optimal ratio of anti-FAM antibody to QDNBs of 80μg/mg were selected.(5)The established assay was tested and demonstrated to have good specificity and sensitivity.Blister samples caused by herpes simplex virus,Trichophyton rubrum,and Trichophyton mentagrophytes were tested with our assay and all results were negative.The standard plasmids constructed based on VZV isothermal amplification products were 0.2-10~5 copies/μL,and the results showed that our method could detect as low as0.2 copies/μL.(6)The established assay was tested and demonstrated to be applicable to the clinical detection of VZV.By identifying 86 clinical blister samples,our method yielded 49positive and 37 negative results,which were in 100%agreement with the q PCR results.It was superior to clinical diagnosis in terms of specificity.In summary,we established an lateral flow assay(CQ-LFA)based on CRISPR/Cas12a and quantum dot nanobeads(QDNBs).Our method can specifically identifies VZV from a small amount of herpes fluid without the need for nucleic acid extraction and allows for subsequent detection by rapid release of varicella-zoster virus nucleic acid,reducing sample processing time.From sample processing to results can be done in less than an hour.Therefore,this method has great potential for clinical VZV detection,especially in areas with limited medical resources.