CRISPR-Cas14a with competitive isothermal amplification for rapid visual pathogen diagnosis

The aquatic foodborne pathogen-induced infectious disease is becoming one of the killers to people's health worldwide, attributed to its long latent period and high infectivity. It is urgently desiderated to develop simple, rapid, sensitive and universal detection techniques for early diagnosis and timely treatment of pathogens. Here, we present a novel visible CRISPR biosensing pathogen detection system named integrating competitive annealing mediated isothermal amplification (CAMP) -mediated CRISPR-Cas14a isothermal detection platform (CCIP) through CAMP with CRISPR-Cas14a. Initially, in the presence of target gene, CAMP was conducted at constant temperature to produce folded CAMP amplicon with abundant single-stranded DNA (ssDNA) target sites. When Cas14a-small guide RNA (sgRNA) binary complex specially recognized target sequences, it would trigger the cleavage collateral activity on nonspecific ssDNA fluorescence probes to produce optical signal with a quite high turnover. Furthermore, the results could be easily observed by naked eyes in an hour with high specificity and sensitivity of 102 aM. Furthermore, the method was verified with a pathogen detection result of 100% accuracy for 15 whole blood samples. Thus, CCIP is a promising point-of-care testing (POCT) technique for practical pathogen diagnosis.