The ring rot of potato caused by the bacterial pathogen Clavibacter sepedonicus is a quarantine disease posing a threat to the potato industry worldwide. The sensitive and selective detection of C. sepedonicus is of a high importance for its effective control. Here, the detection system is reported to determine viable bacteria of C. sepedonicus in potato tubers, based on the coupling of CRISPR/Cas13a nuclease with NASBA (Nucleic Acid Sequence Based Amplification)—the method of isothermal amplification of RNA. Detection can be conducted using both instrumental and non-instrumental (visual inspection of test tubes under blue light) modes. When NASBA and Cas13a analyses were carried out in separate test tubes, the limit of detection (LOD) for the system was 1000 copies of purified target 16S rRNA per NASBA reaction or about 24 colony-forming units (CFUs) of C. sepedonicus per 1 g of tuber tissue. The testing can also be conducted in the “one-pot” format (a single test tube), though with lower sensitivity: LOD was 10,000 copies of target RNA or about 100 CFU per 1 g of tuber tissue for both instrumental and visual detection modes. The overall time of NASBA/Cas13a analysis did not exceed 2 h. The developed NASBA/Cas13a detection system has the potential to be employed as a routine test of C. sepedonicus, especially for on-site testing.

A straightforward approach is suggested to selectively recognize specific products of loop-mediated isothermal amplification (LAMP) with the Cas12a nuclease without a need for a protospacer adjacent motif (PAM) in the sequence of LAMP amplicons (LAMPlicons). This strategy is based on the presence of single-stranded DNA loops in LAMPlicons and the ability of Cas12a to be trans-activated via the binding of guide RNA (gRNA) to single-stranded DNA in the absence of PAM. The approach feasibility is demonstrated on Clavibacter species—multiple bacterial plant pathogens that cause harmful diseases in agriculturally important plants. For Clavibacter species, the detection sensitivity of the developed PAM-independent LAMP/Cas12a system was determined by that of LAMP. The overall detection selectivity was enhanced by the Cas12a analysis of LAMPlicons. It was shown that the LAMP/Cas12a detection system can be fine-tuned by carefully designing gRNA to selectively distinguish C. sepedonicus from other Clavibacter species based on single nucleotide substitutions in the targeted LAMPlicon loop. The suggested loop-based Cas12a analysis of LAMPlicons was compatible with the format of a single test tube assay with the option of naked-eye detection. The findings broaden the palette of approaches to designing PAM-independent LAMP/Cas12a detection systems with potential for on-site testing.

A straightforward approach is suggested to selectively recognize specific products of loop-mediated isothermal amplification (LAMP) by Cas12a nuclease without a need for a protospacer adjacent motif (PAM) in the sequence of LAMP amplicons (LAMPlicons). The strategy is based on the presence of single-stranded DNA loops in LAMPlicons and the ability of Cas12a to be trans-activated via binding of guide RNA (gRNA) to single-stranded DNA in the absence of PAM. The approach feasibility is demonstrated on Clavibacter species – a bunch of bacterial plant pathogens causing harmful diseases of agriculturally important plants. In regard to Clavibacter species, the detection sensitivity of the developed PAM-independent LAMP/Cas12a system was determined by that of LAMP, while the overall detection selectivity was enhanced by the Cas12a analysis of LAMPlicons. It was shown that the LAMP/Cas12a detection system can be fine-tuned by carefully designing gRNA so to selectively distinguish C. sepedonicus among other Clavibacter species, based on single nucleotide substitutions in the targeted LAMPlicon loop. The suggested loop-based Cas12a analysis of LAMPlicons was compatible with a format of single test tube assay with the option of naked-eye detection. The findings broaden the palette of approaches to designing PAM-independent LAMP/Cas12a detection systems with a potential for on-site testing.

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.

A biosensing system relying on asymmetric PCR (aPCR) and PAM-independent recognition of single-stranded DNA amplicons by a Cas12a/gRNA complex is adapted for on-site nucleic acid testing (NAT). aPCR was performed on a low-cost, portable, smartphone-controlled thermocycler operating in endpoint mode. Subsequently, a selective visual readout of the aPCR outcome was achieved using a Cas12a-based assay. A mechanism to prevent false positives was incorporated into the aPCR/Cas12a biosensing system by introducing uracil into the amplicons produced during amplification. Pectobacterium species—harmful bacterial plant pathogens—and genus-specific primers Y1 and Y2 against Pectobacterium spp. were utilized as a convenient model. The absence of a PAM requirement allowed for fine-tuning the selectivity of the Cas12a assay against a strain of a particular Pectobacterium species, P. polaris, by properly positioning the gRNA spacer on the ssDNA. P. polaris was detected down to 10 copies of the bacterial genome per reaction, with an overall testing time of about 2 h and either instrumental or visual readouts. Similar selectivity and sensitivity toward a P. polaris strain were observed with the aPCR/Cas12a biosensing system for potato samples artificially contaminated with Pectobacterium species. The overall findings indicate aPCR/Cas12a-based biosensing as a technique suitable for on-site NAT.