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Loop-mediated isothermal amplification method targets to the phoP gene for detection of Yersinia enterocolitica

Yongjun Li1, Min Jiang, Wei Liu

  • 1Department of Biochemistry and Molecular Biology, College of Life Sciences, Nankai University, Tianjin 300071, China.

Molecular and Cellular Probes
|October 17, 2009
PubMed

Insights

A new loop-mediated isothermal amplification (LAMP) assay rapidly detects Yersinia enterocolitica in food. This validated method offers 100% accuracy and significantly reduces analysis time compared to traditional culture methods.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Food Safety

Background:

  • Yersinia enterocolitica is a foodborne pathogen requiring accurate and rapid detection methods.
  • Traditional culture methods for Y. enterocolitica are time-consuming, delaying timely intervention.
  • Development of sensitive and specific molecular assays is crucial for effective food safety monitoring.

Purpose of the Study:

  • To develop and validate a loop-mediated isothermal amplification (LAMP) assay for the specific detection of Yersinia enterocolitica.
  • To evaluate the assay's performance in detecting Y. enterocolitica in various food samples.
  • To compare the LAMP assay's efficiency and speed against standard microbiological methods.

Main Methods:

  • Development of specific primers targeting the phoP gene of Yersinia enterocolitica for LAMP assay.
  • Sample preparation involving pre-enrichment in Luria-Bertani broth followed by DNA extraction and purification.
  • Validation using pure cultures of Y. enterocolitica and non-Y. enterocolitica strains, and analysis of 102 food samples (minced pork and powdered milk).
  • Comparison of LAMP assay results with the standard ISO 10273 method.

Main Results:

  • The LAMP assay demonstrated 100% specificity, correctly identifying all 37 Y. enterocolitica strains and 50 non-Y. enterocolitica strains.
  • A detection limit of 10(1) CFU of Y. enterocolitica was achieved with extracted DNA.
  • The assay accurately detected 2.2 CFU per 100 g of food samples after enrichment.
  • 100% accuracy was observed when compared to the standard ISO 10273 method.
  • The overall analysis time for the LAMP assay was approximately 24 hours, significantly faster than the 5-day traditional method.

Conclusions:

  • The developed LAMP assay is a highly specific and sensitive method for detecting Yersinia enterocolitica.
  • This rapid assay significantly reduces analysis time, offering a substantial improvement over conventional methods.
  • The combination of sample enrichment and LAMP assay provides a powerful tool for Yersinia enterocolitica detection in food safety diagnostics.
  • Further inter-laboratory validation could lead to the standardization of this LAMP assay for routine use in diagnostic laboratories.

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