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

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.

NDM Metallo-β-Lactamases and Their Bacterial Producers in Health Care Settings

“…Both assays have 100% sensitivity and specificity for detecting bla NDM (218)(219)(220). A locked nucleic acid (LNA)-based quantitative real-time PCR assay has been developed to simultaneously detect multiple antimicrobial resistance genes, including bla NDM , directly from positive blood cultures but has been tested only on several NDM-positive strains (221). A long-fragment real-time quantitative PCR-combined in vitro protein expression (PCR-P) method has been developed for detection of bla NDM-1 .…”

Standardization of an LNA-based TaqMan assay qPCR analysis for Aspiculuris tetraptera DNA in mouse faeces

“…Lastly, among the various treatments performed, the ethanol precipitation treatment substituting the components in the dilutions was found to increase detection sensitivity, although the addition of TritonX-100 or Tween-20 inhibited secondary formation of genomic DNA in the diluted samples, as the heat treatment exhibited no effect on the PCR products. Various components, such as polysaccharides and Ca 2+ , in the faeces have been reported to inhibit the PCR process [ 20 , 21 ].…”

“…The LNA has also been employed to detect microorganisms, such as the hepatitis B virus [ 14 ], S almonella [ 15 ], unicellular parasites Giardia and Cryptosporidium [ 16 ] , and nematode Meloidogyne enterolobii [ 17 ]. As a quantitative PCR (qPCR) method, the TaqMan probe can be utilized to detect the dye of DNA, instead of using SYBR Green [ 18 – 20 ]. The probe used in the TaqMan assay inserts a reporter fluorescent dye into the 5′ -end of the probe and a quencher dye is linked to the 3′ -end, and the 3′ -terminal end is chemically phosphorylated to inhibit extension from the probe during PCR.…”

Rapid Identification of Bacterial Antibiotic Resistance by qPCR in Infants with Gram-Negative Septicaemia: A Proof-of-Concept Study

“…In another recent study, researchers spiked blood specimens with bacteria which had known antibiotic resistance profiles and successfully identified the resistance genes from whole blood [25]. Although they demonstrated that a PCR-based technique could identify resistance genes and potentially predict antibiotic resistance directly from blood samples, the quantity of bacteria added to these blood samples was artificially predefined and does not represent clinical blood samples of patients with Gram-negative septicaemia, as the concentration of bacteria in individual samples may vary substantially.…”