Establishment of a rapid Bordetella pertussis detection method based on RPA-CRISPR-Cas12a technology

Whooping cough (pertussis) is an acute respiratory infectious disease caused by Bordetella pertussis (BP). It poses a risk to infants and young children. This investigation aimed to construct a simple, rapid, and accurate diagnostic protocol for BP detection that does not depend on complex equipment or large-scale instruments. This study combines Recombinase Polymerase Amplification (RPA) technology with the CRISPR/Cas12a system, utilizing immunochromatographic lateral flow strips (ILFS) test and fluorescence curves to observe data. This diagnostic strategy does not require complex equipment used in traditional diagnostic approaches (such as bacterial culture, pathogen detection, and molecular biology techniques), which has increased its accessibility and ease of use. The validation data indicate that the RPA-CRISPR/Cas12a-ILFS and RPA-CRISPR/Cas12a fluorescence detection analyses had a lower detection threshold of 10² copies/µL and did not cross-react with other prevalent infections. Furthermore, 40 clinical samples were evaluated and compared via qPCR, which revealed that the RPA-CRISPR/Cas12a method has 100% sensitivity and specificity. In addition, the RPA-CRISPR/Cas12a diagnostic platform showed significant potential for clinical application, specifically when resources are limited, enabling point-of-care testing. This platform’s simplicity, accuracy, and efficiency make it a powerful tool for pertussis diagnosis, which can improve patient care and public health outcomes.