Comparison of Biosensing Methods Based on Different Isothermal Amplification Strategies: A Case Study with Erwinia amylovora

Abstract

Isothermal amplifications allow for the highly sensitive detection of nucleic acids, bypassing the use of instrumental thermal cycling. This work aimed to carry out an experimental comparison of the four most promising techniques: recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) coupled with lateral flow test or coupled with additional amplification based on CRISPR/Cas12a resulting from the fluorescence of the Cas12a-cleaved probe. To compare the four amplification techniques, we chose the bacterial phytopathogen Erwinia amylovora (causative agent of fire blight), which has a quarantine significance in many countries and possesses a serious threat to agriculture. Three genes were chosen as the targets and primers were selected for each one (two for RPA and six for LAMP). They were functionalized by labels (biotin, fluorescein) at the 5′ ends for amplicons recognition by LFT. As a result, we developed LAMP-LFT, LAMP-CRISPR/Cas, RPA-LFT, and RPA-CRISPR/Cas for E. amylovora detection. The detection limit was 104 CFU/mL for LAMP-LFT, 103 CFU/mL for LAMP-CRISPR/Cas, and 102 CFU/mL for RPA-LFT and RPA-CRISPR/Cas. The results of four developed test systems were verified by qPCR on a panel of real samples. The developed assays based on RPA, LAMP, CRISPR/Cas12a, and LFT are rapid (30–55 min), user-friendly, and highly sensitive for E. amylovora detection. All proposed detection methods can be applied to fire blight diagnosis and effective management of this disease.

Comparison of Single-Stranded DNA Probes Conjugated with Magnetic Particles for Trans-Cleavage in Cas12a-Based Biosensors

“…Two cis-targets for recognizing gRNA-Cas12a were used: ribosomal intergenic spacer (IGS) from D. solani and recombinase A (RecA) from E. amylovora (sequences are presented in Section S2, Supplementary Materials). The dsDNA fragments (596 bp of IGS, 432 bp of RecA) were amplified for further application using PCR according to protocols described in [27,28]. Details of the syntheses are described in Section S2, Supplementary Materials.…”

“…The design, syntheses, and purification of gRNA for recognition of IGS of D. solani and gRNA for recognition of RecA of E. amylovora are precisely described in [26,28]. See also Section S3, Supplementary Materials, for more details regarding gRNA syntheses.…”

DNA Probes for Cas12a-Based Assay with Fluorescence Anisotropy Enhanced Due to Anchors and Salts

“…PCR to produce the cis-target DNA fragment of the target AMY1267 gene of Erwinia amylovora (strain CFBP 1430) was performed according to [32]. Briefly, PCR was carried out using 500 nM of forward and reverse primers, 200 µM dNTP, Tersus polymerase, 5 × 10 8 CFU/mL of E. amylovora CFBP 1430 (from the collection of the All-Russian Plant Quarantine Center, Bykovo, Russia), and the BioRad T100 Thermal Cycler (Hercules, CA, USA) (38 cycles including 30 s of denaturation at 95 • C, 30 s of annealing at 55 • C, and 60 s of elongation at 72 • C).…”

“…RPA was performed using the RPA basic kit (TwistDx, Cambridge, UK) according to [32]. Briefly, labeled primers were mixed with the rehydration buffer to a final concentration of 300 nM.…”

“…As the cis-target DNA, we used a gene fragment of E amylovora (phytopathogen that causes fire blight [37]), which enables a specific diagnosis in a CRISPR/Cas12a-based assay with RPA [32].…”