开发基于 CRISPR/Cas12a 的核酸检测平台并进行临床评估，用于诊断角膜炎

IF 3.2 Q1 OPHTHALMOLOGY Ophthalmology science Pub Date : 2024-03-28 DOI:10.1016/j.xops.2024.100522

Hanith Raj Deivarajan MSc , Vignesh Elamurugan MBBS , Padmapriya Sivashanmugam MSc , Jaishree Pandian PhD , Karvannan Sevugamurthi MSc , Gunasekaran Rameshkumar MSc , Swagata Ghosh PhD , Daipayan Banerjee PhD , Anitha Venugopal MBBS , Anju Jose MBBS, DNB , Ram Rammohan PhD , Anita Raghavan FRCOphth , Revathi Rajaraman MBBS , Dharmalingam Kuppamuthu PhD , Lalitha Prajna MBBS , Venkatesh N. Prajna FRCOphth , Siddharth Narendran MBBS

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引用次数: 0

摘要

目的本研究旨在开发一种快速准确的基于聚类规则间隔短回文重复序列（CRISPR）/Cas12a的分子诊断测定（使用CRISPR快速鉴定真菌病，RID-MyC测定）来检测真菌核酸，并将其与现有的传统真菌学方法进行比较，以诊断真菌性角膜炎（FK）。设计本研究是一项开发和验证研究，重点是创建和评估 RID-MyC 检测法，将其作为 FK 的一种新型诊断方法。方法 RID-MyC 检测法利用重组酶聚合酶扩增，以 18S 核糖体 RNA 基因为目标进行等温扩增，然后进行 CRISPR/Cas12a 反应。主要结果测量主要结果测量侧重于 RID-MyC 检测真菌核酸的分析灵敏度和特异性。结果RID-MyC测定对4种常见真菌的检测限为13.3至16.6个基因组拷贝。在微生物性角膜炎患者中，RID-MyC 检测法与显微镜检查（kappa = 0.714）显示出很大的一致性，与培养（kappa = 0.399）显示出相当的一致性。该检测法对 FK 诊断的灵敏度为 93.27%（95% 置信区间 [CI]，86.62%-97.25%），特异度为 89.47%（95% 置信区间 [CI]，66.86%-98.70%），中位诊断时间为 50 分钟（范围为 35-124 分钟）。RID-MyC测定利用CRISPR-Cas12a技术，对FK的诊断准确率很高，其在护理点使用的潜力可以加快和提高真菌诊断的准确性，为解决目前的诊断难题提供了一个前景广阔的解决方案。

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Development and Clinical Evaluation of a CRISPR/Cas12a-Based Nucleic Acid Detection Platform for the Diagnosis of Keratomycoses

Objective

The objective of this study was to develop a rapid and accurate clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-based molecular diagnostic assay (Rapid Identification of Mycoses using CRISPR, RID-MyC assay) to detect fungal nucleic acids and to compare it with existing conventional mycologic methods for the diagnosis of fungal keratitis (FK).

Design

This study was structured as a development and validation study focusing on the creation and assessment of the RID-MyC assay as a novel diagnostic modality for FK.

Subjects

Participants comprised 142 individuals presenting with suspected microbial keratitis at 3 tertiary care institutions in South India.

Methods

The RID-MyC assay utilized recombinase polymerase amplification targeting the 18S ribosomal RNA gene for isothermal amplification, followed by a CRISPR/Cas12a reaction. This was benchmarked against microscopy, culture, and polymerase chain reaction for the diagnosis of FK.

Main Outcome Measures

The primary outcome measures focused on the analytical sensitivity and specificity of the RID-MyC assay in detecting fungal nucleic acids. Secondary outcomes measured the assay's diagnostic sensitivity and specificity for FK, including its concordance with conventional diagnostic methods.

Results

The RID-MyC assay exhibited a detection limit ranging from 13.3 to 16.6 genomic copies across 4 common fungal species. In patients with microbial keratitis, the RID-MyC assay showed substantial agreement with microscopy (kappa = 0.714) and fair agreement with culture (kappa = 0.399). The assay demonstrated a sensitivity of 93.27% (95% confidence interval [CI], 86.62%–97.25%) and a specificity of 89.47% (95% CI, 66.86%–98.70%) for FK diagnosis, with a median diagnostic time of 50 minutes (range, 35–124 minutes).

Conclusions

The RID-MyC assay, utilizing CRISPR-Cas12a technology, offers high diagnostic accuracy for FK. Its potential for point-of-care use could expedite and enhance the precision of fungal diagnostics, presenting a promising solution to current diagnostic challenges.