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One-Pot Photocontrolled CRISPR-Cas12b Coupled with Loop-Mediated Isothermal Amplification Assay for Point-of-Care
Wenhui Zhu1,2, Tao Qi1,2, Ze Wu1,2
1Department of Laboratory Medicine, Guangdong Provincial Key Laboratory of Precision Medical Diagnostics, Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensors, Guangdong Provincial Key Laboratory of Single-Cell and Extracellular Vesicles, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
Abstract:
Mycoplasma pneumoniae (MP) is a highly prevalent respiratory pathogen, making the development of point-of-care testing (POCT) methods for its detection essential. The integration of loop-mediated isothermal amplification (LAMP) with CRISPR-Cas12b systems demonstrates remarkable specificity and offers promising potential for MP POCT application. However, the current one-pot LAMP/CRISPR-Cas12b system (HOLMESv2) faces the challenge of low sensitivity due to the premature cleavage of the template by CRISPR, which limits its practical utility. To address this, this study introduces a photocontrolled HOLMESv2 (pHOLMESv2) assay using gRNA with an NPOM-modified spacer region. This modification prevents full base pairing between the gRNA and MP DNA, thereby keeping the CRISPR-Cas12b system in an inactive condition during the LAMP reaction and avoiding unintended cleavage of the DNA template. After completion of the LAMP reaction, light irradiation eliminates the NPOM group from the gRNA, restoring its activity to cleave the LAMP products, resulting in a fluorescence signal. The pHOLMESv2 assay successfully addresses the issue of premature DNA template cleavage, improving the limit of detection (LoD) by 133-fold (7.5 copies/μL). Additionally, this method enables direct detection of samples treated with nucleic acid release agents, eliminating the need for complex extraction, and features lyophilized reagents for enhanced stability, storage, and transport. The efficacy of pHOLMESv2 was assessed with 160 clinical MP samples, achieving a sensitivity of 99.0% and a specificity of 100.0%. The pHOLMESv2 assay, when combined with the developed smartphone-based amplification reader, provides a highly sensitive, specific, portable, and cost-effective MP detection, indicating its potential for significant diagnostic use.
Insights
A new photocontrolled assay (pHOLMESv2) enhances Mycoplasma pneumoniae detection sensitivity by 133-fold. This point-of-care test offers rapid, accurate results for respiratory pathogen diagnosis without complex sample prep.
Area of Science:
- Molecular Diagnostics
- Isothermal Amplification Technologies
- CRISPR-based Detection Systems
Background:
- Mycoplasma pneumoniae (MP) is a common respiratory pathogen necessitating sensitive point-of-care testing (POCT).
- Existing one-pot LAMP/CRISPR-Cas12b (HOLMESv2) systems for MP detection suffer from low sensitivity due to premature template cleavage.
- This limitation hinders the practical application of these promising diagnostic tools.
Purpose of the Study:
- To develop a photocontrolled loop-mediated isothermal amplification (LAMP) and CRISPR-Cas12b assay (pHOLMESv2) to overcome sensitivity limitations in Mycoplasma pneumoniae detection.
- To improve the limit of detection (LoD) and enable direct sample analysis for simplified POCT.
- To validate the performance of the pHOLMESv2 assay using clinical samples.
Main Methods:
- A photocontrolled HOLMESv2 (pHOLMESv2) assay was developed using a photocaged guide RNA (gRNA) with an NPOM-modified spacer region.
- The photocaging prevented premature CRISPR-Cas12b activity during the LAMP reaction, preserving the DNA template.
- Light irradiation post-LAMP activated the gRNA to cleave amplified products, generating a detectable signal.
Main Results:
- The pHOLMESv2 assay demonstrated a 133-fold improvement in LoD (7.5 copies/μL) compared to the original HOLMESv2 system.
- The assay allowed direct detection from samples treated with nucleic acid release agents, bypassing complex extraction steps.
- Clinical evaluation with 160 samples yielded 99.0% sensitivity and 100.0% specificity for Mycoplasma pneumoniae detection.
Conclusions:
- The photocontrolled pHOLMESv2 assay effectively prevents premature template cleavage, significantly enhancing sensitivity for Mycoplasma pneumoniae detection.
- This method offers a simplified, direct detection approach with stable, lyophilized reagents, suitable for POCT.
- Combined with a smartphone reader, pHOLMESv2 presents a highly sensitive, specific, portable, and cost-effective diagnostic solution for Mycoplasma pneumoniae.
