Evaluation of loop-mediated isothermal amplification assay along with conventional and real-time PCR assay for sensitive detection of pathogenic Vibrio parahaemolyticus from seafood sample without enrichment

Abstract

The primary reason for foodborne illness is improper seafood safety testing, and hence, an appropriate tool for testing is the key to control the outbreaks. The current study aimed to develop a loop-mediated isothermal amplification (LAMP) assay to detect pathogenic Vibrio parahaemolyticus, important foodborne pathogen, targeting tdh, and trh genes. The specificity of the LAMP assay was good without any false-positive and false-negative results. The assay was highly sensitive and could detect the pathogenic V. parahaemolyticus as low as 1 CFU/reaction in spiked seafood samples and 1 pg of extracted DNA. Out of 62 seafood samples from India's southwest coastal region tested with LAMP assay, eight (12.9%) were positive for trh, and seven (11.29%) samples were positive tdh gene. LAMP-based on tdh and trh was found to be significantly more sensitive (p < 0.05) than conventional PCR and nearly equal sensitive as real-time PCR (RT-PCR) for the detection of pathogenic V. parahaemolyticus. Our study shows that LAMP assay can be a better approach as a point-of-care (POC) diagnostic tool and could detect pathogenic V. parahaemolyticus on seafood samples directly without enrichment and isolation. The high sensitivity and simplicity make LAMP assay a better alternative method than the conventional method and RT-PCR for the detection of pathogens. LAMP assay can be considered as a good alternative to PCR for the routine detection of pathogenic V. parahaemolyticus in seafood.

Keywords: LAMP assay; Seafood; Sensitive detection; V. parahaemolyticus; Without enrichment.

Fig. 1

Determination of sensitivity test by artificial contamination of clam targeting trh⁺ and tdh⁺ V. parahaemolyticus culture. (A1, B1) Conventional PCR amplification targeting trh using Tada et al. [19] and F3–B3. Lane M: 100 bp marker, Lane 1: Negative control, Lane 2: Positive control: 2.35 × 10⁷ CFU/ml. Lanes 3–9: Reaction carried out with spiked suspensions containing 10⁶, to 0 CFU/ml cells. (A2, B2) Conventional PCR amplification targeting tdh using Tada et al. [19] and F3–B3. Lane M: 100 bp marker, Lane 1: Negative control, Lane 2: Positive control: 2.88 × 10⁷ CFU/ml. Lanes 3–9: Reaction carried out with spiked suspensions containing 10⁶, to 0 CFU/ml cells. (C1, C2) LAMP amplification targeting trh and tdh. Lane M: 100 bp marker, Lane 1: Positive control (trh: 2.35 × 10⁷ CFU/ml and tdh^: 2.88 × 10⁷ CFU/ml), Lane 2: Negative control, Lanes 3–9: Reaction carried out with spiked suspensions containing 10⁶, to 0 CFU/ml cells. (D1, D2) Results of RT-PCR showing sensitivity for trh and tdh (F3 and B3) in the spiked sample

Fig. 2

Comparison between LAMP, conventional PCR assay, and RT-PCR of trh gene (a) and tdh gene (b). *Significant difference between LAMP and conventional PCR assay (p < 0.05). ^#No significant difference between LAMP and real-time PCR assay