Establishment of a dual droplet digital PCR assay for herpes simplex virus type Ⅰ and varicella-zoster virus
Objective To establish a dual droplet digital PCR(ddPCR)assay for herpes simplex virus type Ⅰ(HSV-1)and varicella-zoster virus(VZV).Methods The specific primers and probes were derived based on the conserved regions of HSV-1 and VZV genome.The primer-probe combinations were screened,and the annealing temperatures and primer-probe concentration ratios of the dual-droplet digital PCR reaction were optimized to establish a dual-droplet digital PCR reaction system for HSV-1 and VZV,which was tested for other viruses and validated for clinical samples.The sensitivity,specificity,and reproducibility of the established dual microtiter digital PCR method were analyzed.Results The optimal concentrations of primers and probes for the dual ddPCR detection method of HSV-Ⅰ and VZV were determined to be 800 nmol/L and 250 nmol/L,respectively,with an optimal annealing temperature of 56 ℃.The correlation coefficient(R2)of the standard curve of the dual ddPCR assay was 0.99,showing a clear linear relationship.The method showed high sensitivity,with the lowest detection limit of herpes simplex virus type Ⅰ being 2.97 copies/μL,and for VZV being 2.73 copies/μL.The repeatability was high with a small coefficient of variation and stable detection results;the specificity was excellent,and no cross-reaction was found with herpes simplex virus type Ⅱ,Epstein-Barr virus,Adenovirus,Coxsackievirus(CA6/CA10/CA16),Cytomegalovirus,Human Cytomegalovirus,Human enterovirus 71,Japanese Encephalitis virus,West Nile virus,Measles virus,Mumps virus,and human nucleic acids.Conclusions The dual droplet digital PCR assay for herpes simplex virus type Ⅰ and varicella-zoster virus established in this experiment has strong sensitivity,specificity,and high repeatability,and can provide a solution for rapid quantitative detection of the two viruses in different scenarios.
Herpes simplex virus type Ⅰvaricella-zoster virusdual droplet digital PCRquantitative detection
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