Rapid, portable, one-pot CRISPR/Dx system for multiplex detection of human rhinovirus, adenovirus, and Mycoplasma pneumoniae.

BACKGROUND

Human rhinovirus (HRV), human adenovirus (HAdV), and Mycoplasma pneumoniae (Mp) are the primary pathogens responsible for childhood pneumonia cases. These pathogens often co-infect the respiratory system, and their infections exhibit highly similar symptoms, making a differential diagnosis challenging. The inability to identify the specific causative pathogen often results in the administration of empirical antibiotics. Empirical antibiotic treatment may not be effective and can cause adverse patient outcomes and drive antibiotic resistance.

METHOD: We developed a rapid and one-pot RPA-CRISPR/Cas12a system (CRISPR/Dx) for multiplex detection of HRV, HAdV, and Mp. It is also equipped with our customized miniature device to enable portable diagnostics and visible signal readout.

RESULTS

Our CRISPR/Dx system is able to detect 5 copies/μL of these respiratory pathogens, with a detection limit of 0.57 copies/μL for HRV, 0.18 copies/μL for HAdV, and 2.6 copies/μL for Mp. It completes the reaction and detection within 35 minutes, excluding sample preparation time or pretreatment steps. It has high detection specificity and no cross-reactivity between these respiratory pathogens. This CRISPR/Dx system could be integrated with a portable fluorescent detector to realize point-of-care testing of these pathogens.

CONCLUSION

Our CRISPR/Dx system allows multiplex detection, intuitive results readout, and obviates the necessity for large instruments, thereby making it well-suited for field-deployable and point-of-care diagnostics of infectious diseases.