Abstract

The aim of this study was to develop homogeneous and stable plasmid DNA reference materials for detecting the mechanisms of resistance to quinolones and fluoroquinolones in foodborne pathogens. The DNA fragments of 11 target genes associated with quinolone and fluoroquinolone resistance were artificially synthesized, inserted into plasmid vectors, and transferred into recipient cells. PCR and sequencing of DNA were performed to assess the genetic stability of the target DNA in recombinant Escherichia coli DH5α cells during subculturing for 15 generations. The limit of detection (LOD) of the target DNA was determined using PCR and real-time qualitative PCR (qPCR). The homogeneity and storage stability of plasmid DNA reference materials were evaluated in terms of plasmid DNA quantity, PCR-measured gene expression, and qPCR threshold cycle. All 11 target DNAs were successfully synthesized and inserted into vectors to obtain recombinant plasmids. No nucleotide mutations were identified in the target DNA being stably inherited and detectable in the corresponding plasmids during subculturing of recombinant strains. When the target DNA was assessed using PCR and qPCR, the LOD was ≤1.77 × 105 and 3.26 × 104 copies/μL, respectively. Further, when the reference materials were stored at 37 °C for 13 days, 4 °C for 90 days, and −20 °C for 300 days, each target DNA was detectable by PCR, and no mutations were found. Although the threshold cycle values of qPCR varied with storage time, they were above the LOD, and no significant differences were found in the quantity of each plasmid DNA at different timepoints. Further, the homogeneity and stability of the materials were highly consistent with the requirements of standard reference materials. To summarize, considering that our plasmid DNA reference materials conformed to standard requirements, they can be used to detect the mechanisms of quinolone and fluoroquinolone resistance in foodborne pathogens.

Highlights

  • According to the World Health Organization, nearly one-tenth of the global population get sick from foodborne diseases each year [1]

  • As a part of food safety inspection and for diagnosing clinical diseases caused by foodborne pathogens, homogeneous and stable plasmid DNA reference materials are required to ensure data accuracy when detecting the mechanisms of quinolone and fluoroquinolone resistance

  • We developed 11 qualitative plasmid DNA reference materials to study mechanisms associated with quinolone and fluoroquinolone resistance in foodborne pathogens for food safety inspection and molecular diagnosis of clinical diseases caused by foodborne pathogens

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Summary

Introduction

According to the World Health Organization, nearly one-tenth of the global population get sick from foodborne diseases each year [1]. This leads to 420,000 deaths annually, with the main reason being consumption of food contaminated with foodborne pathogens [2,3]. Quinolones (i.e., nalidixic acid) and fluoroquinolones (i.e., ciprofloxacin) are important synthetic antibiotics that are commonly used to treat diseases caused by foodborne pathogens. As a part of food safety inspection and for diagnosing clinical diseases caused by foodborne pathogens, homogeneous and stable plasmid DNA reference materials are required to ensure data accuracy when detecting the mechanisms of quinolone and fluoroquinolone resistance

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