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ERA-CRISPR/Cas12a system: a rapid, highly sensitive and specific assay for Mycobacterium tuberculosis

Published by: Team from Hengyang Medical School, University of South China

Published in: Frontiers in Cellular and Infection Microbiology

1. Target Gene Selection The unique and highly conserved IS1081 gene of Mycobacterium tuberculosis was chosen as the detection target. This gene contains 5–7 repeat sequences and covers all Mycobacterium tuberculosis strains (including those lacking the IS6110 gene), thus avoiding false negatives.
2. Isothermal Amplification Using ERA technology, trace amounts of target DNA were rapidly amplified within 15–20 minutes at a constant temperature of 37–42 °C, without the need for expensive instruments such as PCR thermal cyclers.
3. CRISPR-Mediated Specific Recognition Optimized gRNA was designed to guide the Cas12a protein in recognizing the amplification products, activating its non-specific single-stranded DNA cleavage activity and achieving signal amplification.
4. Dual-Mode Detection Fluorescence signals were monitored in real time via fluorescent F-Q probes, or results were read visually using lateral flow strips with F-B probes, adapting to diverse application scenarios.
   
   

This study utilized two core products from Suzhou GenDx Biotech Co., Ltd. (GenDx Biotech), which provided critical support for the system’s performance:

1. ERA Basic Kit (Cat. No.: KS101)
   Used for isothermal amplification of target DNA, supplying core components including enzymes and buffer required for the reaction.
2. CRISPR Lateral Flow Strip (Cat. No.: TS104)
   Used for visual detection, enabling rapid qualitative reading of results.

The application of these two products not only streamlined the experimental workflow but also ensured high amplification efficiency and detection stability, serving as key support for the rapid implementation of this dual-mode detection system.

Sensitivity

The limit of detection (LOD) of the fluorescence detection system was as low as 9 copies/mL, while the lateral flow detection system reached 90 copies/mL.The fluorescence system also enabled semi‑quantitative analysis of low concentrations of M. tuberculosis DNA, with a linear equation ofY = 655994X − 229562 and a correlation coefficient R² = 0.9729.

Specificity

No cross-reactivity was observed with 3 common respiratory pathogens (e.g., Staphylococcus aureus, Mycoplasma pneumoniae) and 6 non-tuberculous mycobacterial species. The specificity reached 100%, effectively avoiding misdiagnosis.

Clinical Consistency

• The fluorescence detection system showed 100% positive agreement and 100% negative agreement with commercial qPCR.

• The lateral flow detection system showed 93.8% positive agreement and 100% negative agreement, demonstrating high consistency with gold-standard diagnostic methods.

Detection Speed

Results can be obtained in as fast as 50 minutes (20 min ERA amplification + 30 min CRISPR detection), representing a significant time reduction compared with traditional culture (several weeks) and conventional qPCR (several hours).

The ERA Basic Kit and CRISPR lateral flow strips from GenDx Biotech exhibited distinct advantages in this study, serving as a critical guarantee for the technological breakthrough.

High Amplification Efficiency

The ERA Basic Kit enables rapid amplification of trace target sequences under isothermal conditions, completing nucleic acid amplification within 20 minutes. The enzyme‑driven reaction features high specificity, minimizing false positives caused by non‑specific amplification.

Strong Compatibility

It is perfectly compatible with the CRISPR/Cas12a system. Amplification products can be directly used in the subsequent cleavage reaction without complex purification steps, greatly simplifying the experimental workflow.

Operational Convenience

No large‑scale sophisticated instruments are required. Amplification can be achieved using only a simple water bath or heating block. Combined with lateral flow strips, results can be rapidly read without specialized personnel, making it ideal for on‑site rapid testing.

Reliable Stability

All components have undergone rigorous optimization, delivering excellent experimental reproducibility. It provides stable performance support for clinical sample detection and ensures the accuracy of diagnostic results.

The ERA-CRISPR/Cas12a dual‑mode detection system developed in this study not only overcomes the long‑standing limitations of conventional tuberculosis detection—slow speed, insufficient sensitivity, and inadequate specificity—but also benefits from the support of GenDx Biotech products, featuring controllable cost, portability, and ease of use. It is particularly suitable for tuberculosis screening and diagnosis in resource‑limited settings.