Simultaneous detection ofListeriaspp. andListeria monocytogenesby reverse hybridization with 16S–23S rRNA spacer probes

1995 ◽  
Vol 9 (6) ◽  
pp. 423-432 ◽  
Author(s):  
Nancy P. Rijpens ◽  
Geert Jannes ◽  
Marina Van Asbroeck ◽  
Lieve M.F. Herman ◽  
Rudi Rossau
Keyword(s):  
Simultaneous Detection ◽  
23S Rrna ◽  
Reverse Hybridization

scholarly journals Rapid Detection, by PCR and Reverse Hybridization, of Mutations in the Helicobacter pylori 23S rRNA Gene, Associated with Macrolide Resistance

1999 ◽  
Vol 43 (7) ◽  
pp. 1779-1782 ◽  
Author(s):  
Leen-Jan van Doorn ◽  
Yvette J. Debets-Ossenkopp ◽  
Armelle Marais ◽  
Ricardo Sanna ◽  
Francis Mégraud ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Fragment Length Polymorphism ◽  
Simultaneous Detection ◽  
Point Mutations ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Rrna Gene ◽  
Reverse Hybridization ◽  
23S Rrna Gene ◽  
H Pylori

ABSTRACT A PCR-based reverse hybridization system (research prototype kit INNO-LiPA for H. pylori resistance) was developed and evaluated for simultaneous detection of 23S ribosomal DNA point mutations, associated with macrolide resistance in Helicobacter pylori. Fifty-seven H. pylori strains (51 natural, 6 laboratory-derived artificial, 52 resistant, and 5 susceptible strains) were tested by PCR-LiPA (detecting mutations A2115→G, G2141→A, A2142→G, A2142→C, A2143→G, A2143→C, and A2143→T), DNA sequencing, restriction fragment length polymorphism, and/or hybridization to oligonucleotide probes. Results were highly concordant, but PCR-LiPA appears to be more sensitive for the simultaneous detection of multiple mutants.

scholarly journals Prospective Evaluation of ResistancePlus MG, a New Multiplex Quantitative PCR Assay for Detection of Mycoplasma genitalium and Macrolide Resistance

2017 ◽  
Vol 55 (6) ◽  
pp. 1915-1919 ◽  
Author(s):  
S. N. Tabrizi ◽  
J. Su ◽  
C. S. Bradshaw ◽  
C. K. Fairley ◽  
S. Walker ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Clinical Performance ◽  
Simultaneous Detection ◽  
Mycoplasma Genitalium ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Rrna Gene ◽  
Resistance Mutations ◽  
Content Type ◽  
Conventional Culture

ABSTRACT Mycoplasma genitalium is a significant pathogen for which first-line treatment is becoming less effective due to increased resistance to macrolides. As conventional culture and antimicrobial susceptibility testing is not feasible for routine detection of this pathogen, molecular markers such as detection of mutations in the 23S rRNA gene have been described to predict resistance. Recently, a novel multiplex quantitative PCR (qPCR) assay, ResistancePlus MG, has been described for the simultaneous detection of Mycoplasma genitalium and macrolide resistance. In the current study, the clinical performance of the assay was evaluated on 1,089 consecutive urine and anogenital swab samples in symptomatic and asymptomatic male and female patients. Overall, 6.0% were positive for M. genitalium , with 63.1% having macrolide resistance-associated mutations. Compared to the laboratory-validated qPCR method targeting the 16S rRNA gene and Sanger sequencing to determine 23S rRNA mutations, the sensitivity and specificity of M. genitalium detection were 98.5% and 100% and for detection of macrolide resistance mutations were 100.0% and 96.2%, respectively. This assay offers a considerable advantage in clinical settings for M. genitalium testing by making the results of macrolide resistance and mutation analyses simultaneously available, which is increasingly important with escalating macrolide resistance.

scholarly journals Direct detection of Brucella spp. in raw milk by PCR and reverse hybridization with 16S-23S rRNA spacer probes.

1996 ◽  
Vol 62 (5) ◽  
pp. 1683-1688 ◽  
Author(s):  
N P Rijpens ◽  
G Jannes ◽  
M Van Asbroeck ◽  
R Rossau ◽  
L M Herman
Keyword(s):  
Direct Detection ◽  
Raw Milk ◽  
23S Rrna ◽  
Reverse Hybridization

scholarly journals Development of a multiplex qPCR assay for the simultaneous detection of Mycoplasma bovis, Mycoplasma species, and Acholeplasma laidlawii in milk

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11881
Author(s):  
Kanika Chauhan ◽  
Sharif S. Aly ◽  
Terry W. Lehenbauer ◽  
Karen H. Tonooka ◽  
Kathy Glenn ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Simultaneous Detection ◽  
Multiplex Assay ◽  
Qpcr Assay ◽  
23S Rrna ◽  
Mycoplasma Bovis ◽  
Amplification Efficiency ◽  
Rrna Gene ◽  
Multiplex Qpcr ◽  
Acholeplasma Laidlawii

Contagious bovine mastitis caused by Mycoplasma bovis and other Mycoplasma species including Mycoplasma californicum, Mycoplasma bovigenitalium, Mycoplasma alkalescens, Mycoplasma arginini, and Mycoplasma canadense is an economical obstacle affecting many dairy herds throughout California and elsewhere. Routine bacteriological culture-based assays for the pathogens are slow and subject to false-positive results due to the presence of the related, non-pathogenic species Acholeplasma laidlawii. To address the need for rapid and accurate detection methods, a new TaqMan multiplex, quantitative real-time PCR (qPCR) assay was developed that targets the 16S rRNA gene of Mycoplasma, rpoB gene of M. bovis, and the 16S to 23S rRNA intergenic transcribed spacer (ITS) region of A. laidlawii. qPCR amplification efficiency and range of detection were similar for individual assays in multiplex as when performed separately. The multiplex assay was able to distinguish between M. bovis and A. laidlawii as well as detect Mycoplasma spp. collectively, including Mycoplasma californicum, Mycoplasma bovigenitalium, Mycoplasma canadense, Mycoplasma arginini and Mycoplasma alkalescens. In milk, the lower limit of detection of M. bovis, M. californicum, and A. laidlawii with the multiplex assay was between 120 to 250 colony forming units (CFU) per mL. The assay was also able to simultaneously detect both M. bovis and A. laidlawii in milk when present in moderate (103 to 104 CFU/mL) to high (106 to 107 CFU/mL) quantities. Compared to laboratory culture-based methods, the multiplex qPCR diagnostic specificity (Sp) was 100% (95% CI [86.8–100]; n = 26) and diagnostic sensitivity (Se) was 92.3% (95% CI [74.9–99.1]; n = 26) for Mycoplasma species in milk samples collected from California dairy farms. Similarly, the Sp was 100% (95% CI [90.5–100]; n = 37) and Se was 93.3% (95% CI [68.1–99.8]; n = 15) for M. bovis. Our assay can detect and distinguish among M. bovis, other prevalent Mycoplasma spp., and non-pathogenic Acholeplasma laidlawii for effective identification and control of mycoplasma mastitis, ultimately supporting dairy cattle health and high-quality dairy products in California.

scholarly journals Evaluation of the ResistancePlus MG FleXible Assay for Detection of Wild-Type and 23S rRNA-Mutated Mycoplasma genitalium Strains

2020 ◽  
Vol 58 (3) ◽  
Author(s):  
Suhella Tulsiani Drud ◽  
Peter Njuguna ◽  
Samantha Ebeyan ◽  
Simon Erskine ◽  
Mette Holm ◽  
...  
Keyword(s):  
Simultaneous Detection ◽  
Mycoplasma Genitalium ◽  
Ease Of Use ◽  
23S Rrna ◽  
Analytical Sensitivity ◽  
Wild Type ◽  
Content Type ◽  
Clinical Sensitivity ◽  
Block Based ◽  
Sensitivity Specificity

ABSTRACT Antibiotic resistance in Mycoplasma genitalium is rising globally, and resistance-guided diagnostics can facilitate targeted and timely treatment. The ResistancePlus MG FleXible (RPMG Flex) assay for the detection of M. genitalium and macrolide resistance-mediating mutations (MRMM) was evaluated for analytical sensitivity, specificity, reproducibility, and inhibition in the presence of interfering substances by simulating M. genitalium-negative pooled urine and swab matrices with M. genitalium cultures. Furthermore, the clinical sensitivity of the assay was evaluated and compared with a reference real-time PCR assay. The analytical sensitivity of the RPMG Flex assay was 157 genomes/ml for wild-type (WT) and 387 genomes/ml for MRMM strains in both matrices. For clinical specimens, the RPMG assay had an overall sensitivity of 96.1% (95% urine: 10/10 WT, 9/10 MRMM; 96.5% swab: 25/26 WT, 26/29 MRMM) compared to 85.7% for the MgPa/MagNAPure24 assay (95% urine: 19/20; 87% swab: 48/57). Clinical specificity was 100% for urine and 98.5% for swab specimens, respectively. No inhibition due to the presence of any of the tested interfering substances was observed. The RPMG Flex assay was more sensitive than the reference MgPa assay, in particular, for swab specimens. The implementation of this assay may increase ease of use and considerably decrease hands-on time for sample preparation compared to a standard block-based assay. The RPMG Flex assay for the GeneXpert Dx system provides a much-needed platform for the simultaneous detection of MG and MRMM and may thereby facilitate resistance-guided therapy for M. genitalium infections.

scholarly journals Use of a DNA Microarray for Detection and Identification of Bacterial Pathogens Associated with Fishery Products

2011 ◽  
Vol 77 (23) ◽  
pp. 8219-8225 ◽  
Author(s):  
Boyang Cao ◽  
Rongrong Li ◽  
Songjin Xiong ◽  
Fangfang Yao ◽  
Xiangqian Liu ◽  
...  
Keyword(s):  
Dna Microarray ◽  
Bacterial Species ◽  
Simultaneous Detection ◽  
Detection Sensitivity ◽  
23S Rrna ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Content Type ◽  
Detection And Identification ◽  
Fishery Products

ABSTRACTWe established a microarray for the simultaneous detection and identification of diverse putative pathogens often associated with fishery products by targeting specific genes ofListeria monocytogenes,Salmonella,Shigella,Staphylococcus aureus,Streptococcus pyogenes,Vibrio cholerae,Vibrio parahaemolyticus,Vibrio vulnificus, andYersinia enterocoliticaand the 16S-23S rRNA gene internal transcribed spacer (ITS) region ofProteus mirabilisandProteus vulgaris. The microarray contained 26 specific probes and was tested against a total of 123 target bacterial strains that included 55 representative strains, 68 clinical isolates, and 45 strains of other bacterial species that belonged to 8 genera and 34 species, and it was shown to be specific and reproducible. A detection sensitivity of 10 ng DNA or 10 CFU/ml for pure cultures of each target organism demonstrated that the assay was highly sensitive and reproducible. Mock and real fishery product samples were tested by the microarray, and the accuracy was 100%. The DNA microarray method described in this communication is specific, sensitive, and reliable and has several advantages over traditional methods of bacterial culture and antiserum agglutination assays.

scholarly journals Rapid genetic testing for Gaucher disease by reverse hybridization

2003 ◽  
Vol 40 (4) ◽  
pp. 419-421 ◽  
Author(s):  
David J. Halsall ◽  
Gernot Kriegshäuser ◽  
Anne Moritz ◽  
Terence S. Elsey ◽  
Christian Oberkanins
Keyword(s):  
Polymerase Chain Reaction ◽  
Gaucher Disease ◽  
Simultaneous Detection ◽  
Restriction Digestion ◽  
Hybridization Method ◽  
Chain Reaction ◽  
Digestion Method ◽  
Reverse Hybridization ◽  
Polymerase Chain ◽  
Rapid Genetic Testing

Background: We evaluated a reverse hybridization method for the simultaneous detection of nine mutations that are associated with Gaucher disease. Results and conclusion: Results were in agreement with those obtained by a polymerase chain reaction-restriction digestion method. The hybridization method produced results more quickly and with less operator input.

scholarly journals Evaluation of the INNO-LiPA Rif. TB assay, a reverse hybridization assay for the simultaneous detection of Mycobacterium tuberculosis complex and its resistance to rifampin.

1997 ◽  
Vol 41 (10) ◽  
pp. 2093-2098 ◽  
Author(s):  
R Rossau ◽  
H Traore ◽  
H De Beenhouwer ◽  
W Mijs ◽  
G Jannes ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Susceptibility Testing ◽  
Simultaneous Detection ◽  
Nucleotide Sequencing ◽  
Tuberculosis Complex ◽  
Gene Encoding ◽  
In Vitro Susceptibility ◽  
Reverse Hybridization ◽  
In Vitro Susceptibility Testing

Mycobacterium tuberculosis resistance to rifampin results from nucleotide changes in the gene encoding the beta-subunit of the RNA polymerase (rpoB). We developed a reverse hybridization-based line probe assay (LiPA; the INNO-LiPA Rif. TB) carrying one oligonucleotide probe for the detection of M. tuberculosis complex strains and nine probes designed to detect nucleotide changes in the relevant part of rpoB. This assay was evaluated with 107 M. tuberculosis isolates with known rpoB sequences, 52 non-M. tuberculosis complex strains, and 61 and 203 clinical isolates found to be sensitive and resistant, respectively, by in vitro testing. The results indicated that (i) the M. tuberculosis complex probe was 100% specific, (ii) when compared to the results of nucleotide sequencing, no discrepancies with the results of INNO-LiPA Rif. TB were observed, (iii) all strains sensitive by in vitro susceptibility testing were correctly identified, and (iv) among the strains resistant by in vitro susceptibility testing, only 4 (2%) yielded conflicting results. The INNO-LiPA Rif. TB is therefore a reliable and widely applicable assay and a valuable tool for routine diagnostic use, given its simplicity and rapid performance.

scholarly journals Clinical Evaluation of Three Commercial PCR Assays for the Detection of Macrolide Resistance in Mycoplasma genitalium

2019 ◽  
Vol 58 (2) ◽  
Author(s):  
Chloé Le Roy ◽  
Cécile Bébéar ◽  
Sabine Pereyre
Keyword(s):  
Sensitivity And Specificity ◽  
Internal Control ◽  
Simultaneous Detection ◽  
Mycoplasma Genitalium ◽  
Macrolide Resistance ◽  
University Hospital ◽  
23S Rrna ◽  
Rrna Gene ◽  
Content Type ◽  
Commercial Kits

ABSTRACT As macrolide resistance in Mycoplasma genitalium is increasing worldwide, macrolide resistance-associated mutations should be assessed in M. genitalium-positive specimens. New commercial kits are available for detection of macrolide resistance concurrently with M. genitalium. We prospectively evaluated the handling and clinical performances of three commercial kits for detection of macrolide resistance in M. genitalium. Between August and December 2018, remnants of all urogenital specimens determined to be M. genitalium positive using an in-house real-time PCR assay were prospectively collected at the French National Reference Center for Bacterial Sexually Transmitted Infections, Bordeaux University Hospital, Bordeaux, France. The internal control of each kit was added to the primary specimen before DNA extraction, and the absence of amplification inhibition associated with the addition of the three internal controls was assessed. Specimens were evaluated with four assays: the ResistancePlus MG assay (SpeeDx), the S-DiaMGRes assay (Diagenode), the RealAccurate TVMGres assay (PathoFinder), and amplification and sequencing of the 23S rRNA gene (the reference assay). Overall, 195 M. genitalium-positive specimens were assessed. The positive agreement of M. genitalium detection for each kit ranged between 94.8% and 96.4%. Among 154 specimens with M. genitalium positivity as detected by the three commercial kits and 23S rRNA sequencing data, the clinical sensitivity and specificity ranges of the three commercial kits for detecting macrolide resistance-associated mutations were 95 to 100% and 94.6 to 97.3%, respectively. The sensitivity and specificity values were similar among the kits. The launch of three easy-to-use sensitive and specific commercial kits for simultaneous detection of M. genitalium and macrolide resistance will be useful for resistance-guided therapy.

scholarly journals Duplex Reverse-Hybridization Assay for The Simultaneous Detection ofKRAS/BRAFMutations in FFPE-extracted Genomic DNA from Colorectal Cancer Specimens

2013 ◽  
Vol 34 (3) ◽  
pp. 171-177 ◽  
Author(s):  
Veronika Buxhofer-Ausch ◽  
Christoph Ausch ◽  
Robert Zeillinger ◽  
Christian Oberkanins ◽  
Nadia Dandachi ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Genomic Dna ◽  
Simultaneous Detection ◽  
Test Strip ◽  
Hybridization Assay ◽  
Wild Type ◽  
Assay Sensitivity ◽  
Tissue Samples ◽  
Reverse Hybridization ◽  
Formalin Fixed Paraffin Embedded

We report the performance evaluation of a non-quantitative reverse-hybridization assay (KRAS-BRAFStripAssay) designed for the simultaneous detection of 10 mutations in codons 12 and 13 of theKRASgene andBRAFmutation V600E. Dilution experiments using DNA from tumor cell lines or from formalin-fixed paraffin-embedded (FFPE) colorectal cancer (CRC) tissue were performed to assess assay sensitivity. Using 50 ng of total DNA (mutant and wild-type), theKRAS-BRAFStripAssay demonstrated a detection limit of 1% mutant sequence in a background of wild-type DNA. With respect toBRAFV600E, theKRAS-BRAFStripAssay was evaluated using 60 FFPE CRC samples previously analyzed by high resolution melting (HRM). Test strip hybridization identified 2/60 (3%) samples to carry theBRAFV600E mutation, and results were in agreement with those obtained by HRM analysis. This work demonstrates theKRAS-BRAFStripAssay to be a robust and sensitive method for the detection of commonKRAS/BRAFmutations in genomic DNA isolated from FFPE tissue samples.

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