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Engineering of the LAMP-CRISPR/Cas12b platform for Chlamydia psittaci detection
Rong Wang1, Xujian Mao2, Jian Xu2
1School of Public Health, Nanjing Medical University, Nanjing, Jiangsu 211166, PR China.
Insights
A new LAMP-CRISPR/Cas12b assay rapidly detects Chlamydia psittaci (C. psittaci), the cause of psittacosis. This sensitive and specific method offers quick diagnosis for this zoonotic infection.
Area of Science:
- Molecular Biology
- Infectious Diseases
- Biotechnology
Background:
- Chlamydia psittaci (C. psittaci) is a zoonotic bacterium causing psittacosis (parrot fever) in humans.
- Severe clinical manifestations include pneumonia, ARDS, and potential fatality, necessitating rapid diagnostics.
- Current detection methods may lack the speed, sensitivity, or specificity required for timely intervention.
Purpose of the Study:
- To engineer a novel LAMP-CRISPR/Cas12b platform for the rapid detection of C. psittaci.
- To establish both two-step and one-tube reaction systems for enhanced diagnostic capabilities.
Main Methods:
- Combined loop-mediated isothermal amplification (LAMP) with CRISPR-Cas12b assay.
- Designed specific LAMP primers and sgRNA targeting the conserved CPSIT_0429 gene of C. psittaci.
- Evaluated detection limits, specificity, and performance on clinical samples compared to qPCR.
Main Results:
- Developed two-step and one-tube LAMP-CRISPR/Cas12b assays capable of detection within 1 hour.
- Demonstrated high specificity with no cross-reactivity against non-C. psittaci templates.
- Achieved low detection limits of 10^2 aM (two-step) and 10^3 aM (one-tube), with results consistent with qPCR in 160 clinical samples.
Conclusions:
- The developed LAMP-CRISPR/Cas12b assay is a sensitive and specific tool for rapid C. psittaci detection.
- This platform provides crucial technical support for the timely diagnosis of psittacosis.
- Offers a promising advancement in molecular diagnostics for zoonotic bacterial infections.
Abstract:
Introduction. Chlamydia psittaci (C. psittaci) is a zoonotic infection, that causes psittacosis (parrot fever) in humans, leading to severe clinical manifestations, including severe pneumonia, adult respiratory distress syndrome, and, in rare cases, death.Gap Statement. Rapid, sensitive and specific detection of C. psittaci facilitates timely diagnosis and treatment of patients.Aim. This study aimed to engineer the LAMP-CRISPR/Cas12b platform for C. psittaci detection.Methodology. The loop-mediated isothermal amplification (LAMP) technique and clustered regularly interspaced short palindromic repeats-CRISPR associated protein 12b (CRISPR-Cas12b) assay were combined to establish two-step and one-tube LAMP-CRISPR/Cas12b reaction systems, respectively, for rapidly detecting C. psittaci.Results. The two-step and one-tube LAMP-CRISPR/Cas12b assay could complete detection within 1 h. No cross-reactivity was observed from non-C. psittaci templates with specific LAMP amplification primers and single-guide RNA (sgRNA) targeting the highly conserved short fragment CPSIT_0429 gene of C. psittaci. The detection limits of the two-step and one-tube LAMP-CRISPR/Cas12b reaction were 102 aM and 103 aM, respectively. The results were consistent with qPCR for nucleic acid detection in 160 clinical samples, including 80 suspected C. psittaci samples, kept in the laboratory.Conclusions. The LAMP-CRISPR/Cas12b assay developed in this study provides a sensitive and specific method for rapidly detecting C. psittaci and offers technical support for its rapid diagnosis.
