Detection of vancomycin resistant genesvanAandvanBby Cycling Probe Technology

Modrušan, Zora; Marlowe, C.; Wheeler, Donald E.; Pirseyedi, Mona; Bryan, R. Nick

doi:10.1006/mcpr.1999.0242

Cited by 16 publications

(4 citation statements)

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“…The strains were identified with API 20S Streptococcus identification kits (bioMerieux, Marcy l'Etoile, France), with additional tests needed for a final identification of some strains. Genotypes were identified by multiplex PCR as previously described (5).…”

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confidence: 99%

Evaluation of the Velogene Genomic Assay for Detection of vanA and vanB Genes in Vancomycin-Resistant Enterococcus Species

2004

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The Velogene (VEL) genomic assay is a qualitative DNA probe for vanA and vanB in enterococci. 150 clinical isolates were tested with the VEL assay and characterized by pigment production, catalase levels, motility, growth in 6 g of vancomycin/ml, vancomycin and teicoplanin susceptibility, API 20S assay, and genotyping by multiplex PCR. The VEL assay identified all enterococcal strains with vanA and vanB genes.During the last three decades, the prevalence of enterococci as nosocomial pathogens has been increasing. Vancomycinresistant enterococci (VRE) were first recognized in 1986 (4) and are now a worldwide clinical problem, especially in acute care hospitals and intensive care units (3).There are three predominant genotypes of VRE (2). The VanA gene confers high-level vancomycin resistance (vancomycin MIC Ն 64 g/ml), with accompanying teicoplanin resistance (Ͼ16 g/ml). The vanB gene confers low-to high-level vancomycin resistance (vancomycin MICs between 16 and Ն1,000 g/ml) and no teicoplanin resistance. Both vanA and vanB are acquired and transferable and are most commonly seen in Enterococcus faecalis and E. faecium. VanC1 (associated with E. casseliflavus), vanC2 (associated with E. gallinarum), and vanC3 (associated with E. flavescens) confer intrinsically low-level vancomycin resistance (3, 6).Conventional testing with the vancomycin screen agar containing 6 g of vancomycin/ml and conventional vancomycin susceptibility tests do not reliably differentiate between vanC and low-level vanB resistance. Identification of the isolated species of VRE requires a minimum of 18 to 24 h. Hospitals that would implement infection control measures to control VRE with vanA and vanB genes, but not those with vanC genes, need the differentiation. Homebrew PCR assays may detect resistance genes, but they are available only to those laboratories with the expertise to develop their own tests. An efficient, simple, and reliable test to differentiate genotypes would help rapid treatment and initiation of appropriate infection control procedures (3).The Velogene (VEL) assay (ID Biomed, Bothell, Wash.) is based on a cycling probe technology that can be used to detect small amounts of DNA (1). Bacterial DNA is released from cells by suspending 1 loopful of growth from an 18-to 24-h culture in a lysing reagent that contains 20 mM TES [N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid] (pH 6.8), 0.05% Triton X-100, 150 U of achromopeptidase/ml, and 50 U of mutanolysin/ml. The DNA is denatured at 95°C for 5 min. When the target DNA (vanA and vanB genes) is hybridized with a fluorescein-labeled, biotinylated DNA-RNA-DNA chimeric probe, the RNase H cleaves the RNA portion of the probe. The mixture is transferred into streptavidin-coated microwells, and the uncleaved probe (negative reaction) is detected with the addition of fluorescein antibody conjugated with horseradish peroxidase, which converts the substrate to a blue end product. A positive-reaction product is colorless. The results can be detected visually or by spectrophotom...

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confidence: 99%

Evaluation of the Velogene Genomic Assay for Detection of vanA and vanB Genes in Vancomycin-Resistant Enterococcus Species

2004

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“…After hybridization the RNA part is susceptible to RNase H degradation and results in a cleaved probe that can be detected easily. The results obtained with this assay showed that 11 isolates of a total of 440 clinical isolates were were discrepant, but PCR confirmed the results obtained with the probe (200). The use of molecular techniques to detect transposon Tn1546 that largely encodes VanA resistance for epidemiological purposes has been reviewed recently (79).…”

Section: Detection Of Resistancementioning

confidence: 54%

Molecular Detection of Antimicrobial Resistance

Merawi¹,

Girma²

2017

J. Anim. Sci. Adv.

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“…Usually, the chimeric probe is labeled with radioisotope [ γ -32 P], and the cleaved probe percentage is evaluated by electrophoresis on a polyacrylamide gel (1,7,9,12,13). This method is time consuming, limits the number of samples that can be simultaneously analyzed and is not easily automated.…”

Section: Introductionmentioning

confidence: 99%

Colorimetric Detection of the Tuberculosis Complex Using Cycling Probe Technology and Hybridization in Microplates

et al. 2000

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Cycling probe technology (CPT) is a simple signal amplification method for the detection of specific target DNA sequences. CPT uses a chimeric DNA-RNA-DNA probe that is cut by RNase H when bound to its complementary target sequence. In this study, a hybridization assay was developed to detect biotinylated CPT products that result from the amplification of a Mycobacterium tuberculosis complex sequence. The chimeric probe was specifically designed to avoid the formation of secondary structures. The chosen capture probe was perfectly complementary to and was the same size as OL2, one of the two CPT products. The assay was based on the observation that a long sequence, such as the initial probe, was destabilized when bound to a small capture probe as a result of steric hindrance. The capture probe preferentially bound OL2 rather than the long initial probe. We added a prehybridization step with a helper DNA to enhance this discrimination between the two sequences. Colorimetric detection was performed using a peroxidase-streptavidin conjugate. After optimization, the non-isotopic hybridization assay allowed the detection of around 10 amol of target DNA. Besides being faster and easier to perform, this detection method was compared to electrophoresis separation and gave similar results.

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Detection of vancomycin resistant genesvanAandvanBby Cycling Probe Technology

Cited by 16 publications

References 0 publications

Evaluation of the Velogene Genomic Assay for Detection of vanA and vanB Genes in Vancomycin-Resistant Enterococcus Species

Evaluation of the Velogene Genomic Assay for Detection of vanA and vanB Genes in Vancomycin-Resistant Enterococcus Species

Molecular Detection of Antimicrobial Resistance

Colorimetric Detection of the Tuberculosis Complex Using Cycling Probe Technology and Hybridization in Microplates

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