Combining Asymmetric PCR with PAM-Independent Cas12a Analysis of Single-Stranded DNA Amplicons for Programmable Biosensing: Application to Pectobacterium polaris Detection

Résumé fourni par la source

A programmable Cas12a-based biosensing system is developed for sensitive and se-lective detection of Pectobacterium polaris, a newly emerging pathogen affecting a wide range of agriculturally important plants. The system relies on asymmetric PCR (aPCR) and PAM-independent recognition of single-stranded DNA by a Cas12a/gRNA complex and allowed to determine P. polaris with limit of detection of 10 copies of bacterial genome per reaction. The overall assay time is about 2 h. The detection is achieved by using PCR primers specific for Pectobacterium species and gRNA selectively recognizing sequence unique for P. polaris. With no PAM requirement, the system selectivity was fine-turned by properly positioning gRNA spacer on single-stranded amplicons. The designed aPCR/Cas12a biosensing system is compatible with the introduction of uracil into ampli-cons to prevent carryover contamination and with visual detection. In a broad context, the findings suggest that PCR tests for pathogen detection can be easily adapted to a format of aPCR/Cas12a programmable biosensing by converting PCR into aPCR and by coupling aPCR to PAM-independent Cas12a analysis of single-stranded amplicons. This may pave the way to modify current laboratory-based PCR tests into on-site tests conducted on con-ventional inexpensive thermocyclers with subsequent visual detection for on-site screen-ing or initial decision-making.

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