Application of CRISPR-based one-pot droplet assay for BK virus nucleic acid detection
10.3760/cma.j.cn114452-20240823-00470
- VernacularTitle:基于成簇规律间隔短回文重复序列液滴一锅法的BK病毒核酸检测及应用
- Author:
Yu LIU
1
;
Jingsong XU
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;
Microfluidic droplet;
BK virus;
Nucleic acid detection
- From:
Chinese Journal of Laboratory Medicine
2025;48(6):743-749
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish a method for quantitative detection of BK virus (BKV) nucleic acid using microfluidic droplet one-pot Recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats, RPA-CRISPR/Cas13a.Methods:A methodology establishment study. Plasma or urine samples were collected from 50 post renal transplant patients and 40 healthy individuals in Renji Hospital, Shanghai Jiao Tong University School of Medicine, from May 16 to August 31, 2024, and the viral genomic DNA was extracted. In the renal transplant group, there were 28 males and 22 females aged (46.1±11.6) years; in the healthy control group, there were 21 males and 19 females aged (45.6±11.3) years. Three crRNAs were designed for screening the optimal crRNA in CRISPR/Cas13a reaction taking PCR product as templet. Two pairs of RPA primers targeting BKV sequences were designed and selected an optimal primer set from four combinations for RPA-CRISPR/Cas13a reaction. Microfluidic droplet generation chip was designed and fabricated for rapid quantitative detection of BKV nucleic acids, which was combined with a droplet reader. BKV plasmid standards were utilized to assess the detection sensitivity of the one-pot assay. The CRISPR droplet one-pot assay and qPCR were utilized to detect the BKV load in the above samples, respectively. The methodological consistency was then analyzed, and Kappa coefficients of the results were calculated using the Kappa test, and the correlation between the two methods was evaluated using linear regression analysis. Results:The RPA-CRISPR/Cas13a reaction system was combined with a droplet chip to establish a CRISPR microfluidic droplet one-pot assay for BKV detection. The sensitivity of the CRISPR microfluidic droplet one-pot assay for detecting BKV was 10 copies/ml, which surpassed that of qPCR (100 copies/ml) without cross-reaction with JC virus. Methodological consistency analysis demonstrated that the Kappa coefficient of the two methods was 0.96, and the coefficient of determination R 2 was 0.95. Conclusion:In this study, a novel BKV detection technology based on the one-pot CRISPR droplet assay was successfully established.
