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

Mol Biol Rep. 2021 Jan;48(1):1009-1016. doi: 10.1007/s11033-020-06116-9. Epub 2021 Jan 9.

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.

MeSH terms

  • Animals
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / isolation & purification
  • Bivalvia / microbiology
  • DNA, Bacterial / genetics
  • Food Analysis / instrumentation
  • Food Analysis / methods*
  • Food Contamination / analysis*
  • Hemolysin Proteins / genetics*
  • Hemolysin Proteins / isolation & purification
  • Humans
  • Molecular Diagnostic Techniques*
  • Nucleic Acid Amplification Techniques*
  • Penaeidae / microbiology
  • Point-of-Care Systems
  • Real-Time Polymerase Chain Reaction / methods
  • Seafood / analysis*
  • Sensitivity and Specificity
  • Vibrio parahaemolyticus / classification
  • Vibrio parahaemolyticus / genetics*
  • Vibrio parahaemolyticus / isolation & purification

Substances

  • Bacterial Proteins
  • DNA, Bacterial
  • Hemolysin Proteins
  • thermostable direct hemolysin-related hemolysin protein, Vibrio parahaemolyticus

Supplementary concepts

  • LAMP assay