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Updated: Jun 29, 2025
Author Spotlight: Development of Simplified CRISPR-Based Tests for Rapid Detection of Infectious Diseases
Published on: August 16, 2024
An isothermal CRISPR- based lateral flow assay for detection of Neisseria meningitidis
Dao Thi Huyen1, Julien Reboud2, Dao Thanh Quyen1,3
1Vietnamese - German Center for Medical Research (VG-CARE), 108 Military Central Hospital, Nr 1, Tran Hung Dao Street, Hai Ba Trung Dist., Hanoi, 10000, Vietnam.
Background:
Neisseria meningitidis can cause life-threatening meningococcal meningitis and meningococcemia. Old standard microbiological results from CSF/blood cultures are time consuming. This study aimed to combine the sensitivity of loop-mediated isothermal nucleic acid amplification (LAMP) with the specificity of CRISPR/Cas12a cleavage to demonstrate a reliable diagnostic assay for rapid detection of N. meningitidis.
Methods:
A total of n = 139 samples were collected from patients with suspected meningococcal disease and were used for evaluation. The extracted DNA was subjected to qualitative real-time PCR, targeting capsular transporter gene (ctrA) of N. meningitidis. LAMP-specific primer pairs, also targeting the ctrA, were designed and the LAMP products were subjected to CRISPR/Cas12 cleavage reaction. the readout was on a lateral flow strip. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of LAMP-CRISPR/Cas was compared with real-time PCR assays. The limit of detection (LOD) was established with serial dilutions of the target N. meningitidis DNA and calculated by Probit regression analysis.
Results:
Six LAMP assay-specific primers were developed targeting the ctrA gene of N. meningitidis, which is conserved in all meningococcal serogroups. The LAMP primers did not amplify DNA from other bacterial DNA tested, showing 100% specificity. The use of 0.4 M betaine increased the sensitivity and stability of the reaction. LAMP-CRISPR/Cas detected meningococcal serogroups (B, C, W). The assay showed no cross-reactivity and was specific for N. meningitidis. The LOD was 74 (95% CI: 47-311) N. meningitidis copies. The LAMP-CRISPR/Cas performed well compared to the gold standard. In the 139 samples from suspected patients, the sensitivity and specificity of the test were 91% and 99% respectively.
Conclusion:
This developed and optimized method can complement for the available gold standard for the timely diagnosis of meningococcal meningitis and meningococcemia.
Insights
A novel diagnostic assay combining loop-mediated isothermal nucleic acid amplification (LAMP) and CRISPR/Cas12a cleavage rapidly and accurately detects Neisseria meningitidis. This method offers a sensitive and specific alternative for diagnosing meningococcal disease, improving upon traditional culture methods.
Area of Science:
- Molecular Diagnostics
- Microbiology
- Biotechnology
Background:
- Neisseria meningitidis causes severe meningococcal meningitis and meningococcemia.
- Conventional microbiological culture methods for diagnosis are time-consuming.
- Rapid and reliable diagnostic tools are crucial for timely intervention.
Purpose of the Study:
- To develop and validate a rapid diagnostic assay for Neisseria meningitidis.
- To combine loop-mediated isothermal nucleic acid amplification (LAMP) with CRISPR/Cas12a cleavage.
- To achieve sensitive and specific detection of N. meningitidis.
Main Methods:
- Developed six LAMP primer pairs targeting the conserved ctrA gene of N. meningitidis.
- Optimized LAMP reaction conditions with 0.4 M betaine.
- Utilized CRISPR/Cas12a cleavage for signal amplification and detection on a lateral flow strip.
- Evaluated assay performance on 139 clinical samples from suspected meningococcal disease patients.
Main Results:
- The LAMP assay demonstrated 100% specificity against other bacterial DNA.
- The optimized LAMP-CRISPR/Cas assay detected N. meningitidis serogroups B, C, and W.
- The limit of detection (LOD) was 74 N. meningitidis copies.
- Compared to real-time PCR, the assay showed 91% sensitivity and 99% specificity on clinical samples.
Conclusions:
- The developed LAMP-CRISPR/Cas assay is a reliable and rapid method for diagnosing N. meningitidis.
- This assay can serve as a valuable complement to existing gold-standard diagnostic methods.
- The optimized assay facilitates timely diagnosis of meningococcal meningitis and meningococcemia.
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