Naked-eye detection with loop-mediated isothermal amplification for P. carotovorum subsp. carotovorum in agricultural products

Abstract

Pectobacterium carotovorum causing soft-rot disease requires on-site detection before the distribution of agricultural products. Loop-mediated isothermal amplification (LAMP), which is resistant to food inhibitors, is known for its high detection sensitivity for pathogens and when coupled with lateral flow immunoassay (LFA) enables visualizations. For detection of soft-rot disease, we developed a LAMP-LFA system targeting 16S ribosomal RNA, a partial sequence gene of P. carotovorum subsp. carotovorum. The LAMP-LFA was performed at 60 °C for 50 min followed by hybridization of digoxygenin-labeled LAMP amplicon and biotinylated probe. Detection sensitivity was 3.22 × 10¹ CFU/mL in pure culture, which specifically detected the target. In Chinese cabbage and potato, the target was detected up to low levels of 1.57 × 10² CFU/g and 1.29 × 10² CFU/g, respectively. This study showed potential applicability as a sensitive point-of-care system for soft-rot disease bacteria detection in agricultural products.

Supplementary information: The online version contains supplementary material available at 10.1007/s10068-023-01315-z.

Keywords: Agricultural product; Lateral flow immunoassay; Loop-mediated isothermal amplification; Pectobacterium carotovorum; Soft-rot disease; Visualization detection.

Fig. 1

The region of primers and probe sequence on the 16S rRNA gene, partial sequence gene of P. carotovorum subsp. Carotovorum

Fig. 2

Optimization of amplification temperature (A) and reaction time (B) of LAMP protocols. Lane M: 100-bp DNA ladder marker

Fig. 3

Specificity of LAMP-LFA by visualization of LFA strip. Lane 1: P. carotovorum subsp. carotovorum KACC 10,225; lane 2: P. carotovorum subsp. carotovorum Pcc3; lane 3: P. carotovorum subsp. brasiliense KCTC 17,662; lane 4: P. actinidiae KCTC 23,131; lane 5: D. chrysanthemi SL 3550; lane 6: D. chrysanthemi SL 4405; lane 7: D. chrysanthemi SL 4830; lane 8: B. cereus ATCC 11,778; lane 9: B. subtilis ATCC 6633; lane 10: E. coli ATCC 10,536; lane 11: E. faecium ATCC 19,434; lane 12: S. aureus KCTC 1624; lane 13: S. Typhimurium ATCC 14,028; lane 14: L. monocytogenes ATCC 19,114; and lane N: negative control

Fig. 4

Sensitivity of LAMP-LFA for detection of P. carotovorum subsp. carotovorum in pure culture. A Visualization of LFA strip. B Relative peak intensity on test line (B). Lane N: negative control. *p < 0.05

Fig. 5

The sensitivity of LAMP-LFA detecting P. carotovorum subsp. carotovorum in Chinese cabbage (A) and potato (B). Visualization result of LFA strip (top panel) and relative peak intensity of test line (bottom panel). Lane N: Negative control. *p < 0.05