Development and validation of a rapid detection method for HSV or VZV in intraocular fluid based on a single-tube MIRA-CRISPR/Cas12a system

Objective  To establish and evaluate a novel method for rapid detection of herpes simplex virus (HSV) or varicella-zoster virus (VZV) in intraocular fluid based on single-tube multienzyme isothermal rapid amplification (MIRA) combined with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a trans-cleavage system.

Methods  The conserved gene sequences HSV– UL27 and VZV– ORF28 were selected as detection targets, and standard plasmids were constructed. Specific amplification primers and CRISPR RNA (crRNA) were designed. The MIRA system was established to screen the optimal primer combinations and the single-stranded DNA (ssDNA) reporter probe sequences. The CRISPR/Cas12a detection system was optimized to determine the optimal concentrations and ratios of Cas12a to crRNA, as well as the optimal ssDNA concentration. A single-tube MIRA-CRISPR/Cas12a detection method was developed, and its limit of detection (LOD) and specificity were evaluated. Total 111 samples of nucleic acid extracted from intraocular fluids (aqueous humor or vitreous) were collected at Xuzhou First People’s Hospital from January to December 2023 in Xuzhou First People’s Hospital. These samples were tested to assess the consistency between this method and quantitative real-time PCR (qRT-PCR). This study was approved by the Ethics Committee of Xuzhou First People’s Hospital (No. xyyll[2021]104).

Results  The single-tube MIRA-CRISPR/Cas12a detection method established in this study could be completed within 45 minutes under isothermal conditions at 37 ℃. The method demonstrated high sensitivity and specificity, with LODs of 1 copy/μl for HSV and 10 copies/μl for VZV. The detection of HSV and VZV had no cross-reaction with adenovirus, EB virus, cytomegalovirus, and human herpesvirus types 6, 7, and 8. In the detection of clinical samples, compared with the qRT-PCR, this method demonstrated a sensitivity of 94.12% and specificity of 92.50% for HSV detection, with an overall agreement rate of 92.98%, showing high concordance with qRT-PCR (kappa=0.838). For VZV detection, this method achieved a sensitivity of 94.73% and specificity of 100%, with a total concordance rate of 98.15% (kappa=0.959).

Conclusions  This successfully developed single-tube MIRA-CRISPR/Cas12a nucleic acid detection method can provide rapid, sensitive, and specific detection of HSV and VZV in intraocular fluid with performance comparable to qRT-PCR, and provide a novel approach for laboratory detection of viral eye diseases.