A locked nucleic acid (LNA)-based real-time PCR assay for the rapid detection of multiple Bacterial antibiotic resistance genes directly from positive blood culture

Lingxiang Zhu, Dingxia Shen, Qiming Zhou, Zexia Li, Xiangdong Fang, Quan Zhen Li

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Bacterial strains resistant to various antibiotic drugs are frequently encountered in clinical infections, and the rapid identification of drug-resistant strains is highly essential for clinical treatment. We developed a locked nucleic acid (LNA)-based quantitative real-time PCR (LNA-qPCR) method for the rapid detection of 13 antibiotic resistance genes and successfully used it to distinguish drug-resistant bacterial strains from positive blood culture samples. A sequence-specific primer-probe set was designed, and the specificity of the assays was assessed using 27 ATCC bacterial strains and 77 negative blood culture samples. No cross-reaction was identified among bacterial strains and in negative samples, indicating 100% specificity. The sensitivity of the assays was determined by spiking each bacterial strain into negative blood samples, and the detection limit was 1-10 colony forming units (CFU) per reaction. The LNA-qPCR assays were first applied to 72 clinical bacterial isolates for the identification of known drug resistance genes, and the results were verified by the direct sequencing of PCR products. Finally, the LNA-qPCR assays were used for the detection in 47 positive blood culture samples, 19 of which (40.4%) were positive for antibiotic resistance genes, showing 91.5% consistency with phenotypic susceptibility results. In conclusion, LNA-qPCR is a reliable method for the rapid detection of bacterial antibiotic resistance genes and can be used as a supplement to phenotypic susceptibility testing for the early detection of antimicrobial resistance to allow the selection of appropriate antimicrobial treatment and to prevent the spread of resistant isolates.

Original languageEnglish (US)
Article numbere0120464
JournalPLoS One
Volume10
Issue number3
DOIs
StatePublished - Mar 16 2015

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Bacterial Drug Resistance
Cell culture
antibiotic resistance
nucleic acids
Real-Time Polymerase Chain Reaction
Assays
quantitative polymerase chain reaction
Blood
Genes
Anti-Bacterial Agents
blood
assays
Microbial Drug Resistance
genes
Pharmaceutical Preparations
rapid methods
drugs
Cross Reactions
sampling
Drug Resistance

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

A locked nucleic acid (LNA)-based real-time PCR assay for the rapid detection of multiple Bacterial antibiotic resistance genes directly from positive blood culture. / Zhu, Lingxiang; Shen, Dingxia; Zhou, Qiming; Li, Zexia; Fang, Xiangdong; Li, Quan Zhen.

In: PLoS One, Vol. 10, No. 3, e0120464, 16.03.2015.

Research output: Contribution to journalArticle

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