Tuberculosis Detection via ERA-CRISPR/Cas12a System: A Professional English Translation

Tuberculosis (TB), a chronic infectious disease caused by Mycobacterium tuberculosis, remains a major threat to global public health security. Worse still, the spread of drug-resistant tuberculosis is severely hindering the achievement of the "End TB Strategy". Conventional TB diagnostic methods, such as culture-based assays and drug susceptibility testing, are time-consuming (taking several weeks) and complex to perform, making them unable to meet the clinical demand for rapid diagnosis.

Enzymatic recombinase amplification (ERA) is an isothermal nucleic acid amplification technology, which can be likened to a genetic "photocopier". Compared with traditional nucleic acid amplification technologies, its greatest advantages are as follows: it operates at 37-42°C without complex temperature cycling, thus enabling the use of compact, portable instruments; additionally, it boasts extremely high amplification efficiency and can complete the amplification process within tens of minutes.

In this race against time, the powerful combination of ERA technology and the CRISPR/Cas12a system is constantly demonstrating enormous research potential for the detection of tuberculosis and its drug-resistant genes.

1. Development of a Rapid Detection System for Mycobacterium tuberculosis

The research team from the Affiliated Nanhua Hospital of University of South China has successfully developed an ERA-CRISPR/Cas12a dual detection system, providing a novel, rapid, highly sensitive and specific solution for the detection of Mycobacterium tuberculosis [1].

This system has yielded excellent validation results. The limit of detection (LOD) of the fluorescence detection system is as low as 9 copies/mL, and that of the lateral flow detection system is 90 copies/mL. No cross-reactivity was observed with 3 common respiratory pathogens (e.g., Staphylococcus aureus, Mycoplasma pneumoniae) and 6 non-tuberculous mycobacterial species, achieving 100% specificity. In 60 clinical sputum samples, the fluorescence system showed 100% positive and negative coincidence rates with commercial quantitative polymerase chain reaction (qPCR); the lateral flow system had a positive coincidence rate of 93.8% and a negative coincidence rate of 100%, which were highly consistent with the results of the gold standard assay. Results can be obtained in as little as 50 minutes, representing a significant speed improvement compared with traditional culture-based methods (taking several weeks) and conventional qPCR (taking several hours).

2. Development of a Rapid Detection System for Drug-Resistant Tuberculosis

Drug-resistant tuberculosis (DR-TB) is a serious public health issue that threatens the global prevention and control of tuberculosis.

The Cas12a_RR detection system developed by the research team from the Third People's Hospital of Shenzhen can recognize non-canonical protospacer adjacent motif (PAM) sequences, including TTCC, CTCC, TCCC and others. This significantly broadens the range of detectable targets and enables the effective identification of K43R and K88R mutations in the rpsL gene, as well as the G944C mutation in the katG gene. These mutations are closely associated with resistance to streptomycin and isoniazid. The system can detect drug-resistant gene mutations as low as 1% in mixed samples, demonstrating exceptional sensitivity [2,3].

3. Conclusion

Currently, the isothermal amplification-CRISPR/Cas12a system is still in continuous evolution. Research hotspots include: the development of a one-tube reaction system that enables amplification and cleavage to be performed in the same tube by optimizing the composition of reaction buffers or adding liquid phase barriers, thus avoiding aerosol contamination caused by tube opening; the construction of a multi-target simultaneous detection platform that leverages the orthogonal cleavage properties of Cas12a/Cas13a to detect Mycobacterium tuberculosis and multiple drug-resistant genes simultaneously; and the development of an integrated microfluidic chip system to realize fully automated detection from sample input to result output.

Compared with other reported isothermal amplification (LAMP, RPA, MCDA)-CRISPR/Cas12a methods, the ERA-CRISPR/Cas12a method exhibits great advantages in both detection performance and speed, further reflecting the excellent amplification efficiency and rapid amplification rate of the ERA system [1].

Let us join hands and use the light of science and technology to illuminate the final journey towards ending tuberculosis.

References

[1] Gan T, Yu J, Deng Z, et al. ERA-CRISPR/Cas12a system: a rapid, highly sensitive and specific assay for Mycobacterium tuberculosis[J]. Frontiers in Cellular and Infection Microbiology, 2024, 14: 1454076.

[2] Liu P, Wang X, Liang J, et al. A recombinase polymerase amplification-coupled Cas12a mutant-based module for efficient detection of streptomycin-resistant mutations in Mycobacterium tuberculosis[J]. Frontiers in Microbiology, 2022, 12: 796916.

[3] Liu P, Zhang J, Gong Y, et al. Application of engineered CRISPR/Cas12a variants with altered protospacer adjacent motif specificities for the detection of isoniazid resistance mutations in Mycobacterium tuberculosis[J]. Microbiology Spectrum, 2025, 13(10): e00165-25.