Establishment and application of a one-pot lyophilized CRISPR system for detecting CMV in liver transplant recipients
10.3760/cma.j.cn114452-20250702-00388
- VernacularTitle:一锅法干粉化CRISPR体系检测肝移植受者巨细胞病毒方法的建立与验证
- Author:
Junheng ZHANG
1
;
Jingsong XU
1
;
Yu LIU
1
;
Haiqian HUANG
1
;
Min LI
1
;
Hua WANG
1
Author Information
1. 上海交通大学医学院附属仁济医院检验科，上海 200127
- Publication Type:Journal Article
- Keywords:
Clustered regularly interspaced short palindromic repeats;
Liver transplantation;
Cytomegalovirus;
Point-of-care testing
- From:
Chinese Journal of Laboratory Medicine
2025;48(10):1317-1322
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish a one-pot lyophilized detection system based on recombinase polymerase amplification (RPA) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas13a) technology for the rapid diagnosis of cytomegalovirus (CMV) infection in liver transplantation recipients.Methods:This study is a methodology study. CRISPR RNA (crRNA) and RPA primers were designed targeting the CMV gene sequence. Optimal RPA primer sets were screened to establish the RPA-CRISPR/Cas13a-based CMV detection system. The limit of detection (LOD) was evaluated using gradient-diluted CMV plasmid standards. Cross-reactivity was assessed using genomic DNA from common opportunistic viruses in organ transplant recipients. Lyophilized reagents were validated with CMV-negative and positive samples. P-values were computed using two-sample t-tests for pairwise comparisons and one-way ANOVA for multi-group analyses to assess fluorescence value differences. Subsequently, lyophilized reagents were employed to detect 22 plasma samples from liver transplantation recipients collected at Renji Hospital, Shanghai Jiao Tong University School of Medicine, from June 3, 2024, to May 31, 2025. The test results were then compared with those obtained using quantitative real-time polymerase chain reaction (qPCR). Consistency between the two methods was evaluated using the Kappa coefficient calculated by Kappa test.Result:The established RPA-CRISPR/Cas13a system achieved a detection sensitivity of 1 copy/reaction and exhibited no cross-reactivity with other common opportunistic viruses in organ transplantation. Lyophilized RPA-CRISPR/Cas13a reagents demonstrated performance equivalent to non-lyophilized reagents. Concordance between lyophilized reagent detection and qPCR results for 22 clinical samples was 100% (22/22).Conclusion:A lyophilized CMV detection method based on RPA-CRISPR/Cas13a technology was successfully developed and validated for convenient diagnosing CMV infection in liver transplant recipients.
