Engineering of the LAMP-CRISPR/Cas12b platform for Chlamydia psittaci detection

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 10 2 aM and 10 3 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.