scholarly journals Performance and Verification of a Real-Time PCR Assay Targeting thegyrAGene for Prediction of Ciprofloxacin Resistance in Neisseria gonorrhoeae: TABLE 1

2016 ◽  
Vol 54 (3) ◽  
pp. 805-808 ◽  
Author(s):  
P. Hemarajata ◽  
S. Yang ◽  
O. O. Soge ◽  
R. M. Humphries ◽  
J. D. Klausner
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Real Time ◽  
Real Time Pcr ◽  
The United States ◽  
Test Results ◽  
Pcr Assay ◽  
Content Type ◽  
Agar Dilution ◽  
Ciprofloxacin Resistance ◽  
To Receive

In the United States, 19.2% ofNeisseria gonorrhoeaeisolates are resistant to ciprofloxacin. We evaluated a real-time PCR assay to predict ciprofloxacin susceptibility using residual DNA from the Roche Cobas 4800 CT/NG assay. The results of the assay were 100% concordant with agar dilution susceptibility test results for 100 clinical isolates. Among 76 clinical urine and swab specimens positive forN. gonorrhoeaeby the Cobas assay, 71% could be genotyped. The test took 1.5 h to perform, allowing the physician to receive results in time to make informed clinical decisions.

scholarly journals Evaluation of the New BD Max GC Real-Time PCR Assay, Analytically and Clinically as a Supplementary Test for the BD ProbeTec GC Qx Amplified DNA Assay, for Molecular Detection of Neisseria gonorrhoeae

2015 ◽  
Vol 53 (12) ◽  
pp. 3935-3937 ◽  
Author(s):  
Daniel Golparian ◽  
Stina Boräng ◽  
Martin Sundqvist ◽  
Magnus Unemo
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Real Time ◽  
Sensitivity And Specificity ◽  
Real Time Pcr ◽  
Molecular Detection ◽  
Analytical Sensitivity ◽  
Pcr Assay ◽  
Content Type ◽  
Dna Assay ◽  
Supplementary Test

The new BD Max GC real-time PCR assay showed high clinical and analytical sensitivity and specificity. It can be an effective and accurate supplementary test for the BD ProbeTec GC Qx amplified DNA assay, which had suboptimal specificity, and might also be used for initial detection ofNeisseria gonorrhoeae.

scholarly journals Molecular Assay for Detection of Genetic Markers Associated with Decreased Susceptibility to Cephalosporins in Neisseria gonorrhoeae

2015 ◽  
Vol 53 (7) ◽  
pp. 2042-2048 ◽  
Author(s):  
S. W. Peterson ◽  
I. Martin ◽  
W. Demczuk ◽  
A. Bharat ◽  
L. Hoang ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Real Time ◽  
Dna Sequence ◽  
Real Time Pcr ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Specific Marker ◽  
Molecular Assay ◽  
Pcr Assay ◽  
Content Type

The incidence of antimicrobial-resistantNeisseria gonorrhoeaecontinues to rise in Canada; however, antimicrobial resistance data are lacking for approximately 70% of gonorrhea infections that are diagnosed directly from clinical specimens by nucleic acid amplification tests (NAATs). We developed a molecular assay for surveillance use to detect mutations in genes associated with decreased susceptibility to cephalosporins that can be applied to both culture isolates and clinical samples. Real-time PCR assays were developed to detect single nucleotide polymorphisms (SNPs) inponA,mtrR,penA,porB, and oneN. gonorrhoeae-specific marker (porA). We tested the real-time PCR assay with 252 gonococcal isolates, 50 nongonococcal isolates, 24N. gonorrhoeae-negative NAAT specimens, and 34N. gonorrhoeae-positive NAAT specimens. Twenty-four of theN. gonorrhoeae-positive NAAT specimens had matched culture isolates. Assay results were confirmed by comparison with whole-genome sequencing data. For 252N. gonorrhoeaestrains, the agreement between the DNA sequence and real-time PCR was 100% forporA,ponA, andpenA, 99.6% formtrR, and 95.2% forporB. The presence of ≥2 SNPs correlated with decreased susceptibility to ceftriaxone (sensitivities of >98%) and cefixime (sensitivities of >96%). Of 24 NAAT specimens with matched cultures, the agreement between the DNA sequence and real-time PCR was 100% forporB, 95.8% forponAandmtrR, and 91.7% forpenA. We demonstrated the utility of a real-time PCR assay for sensitive detection of known markers for the decreased susceptibility to cephalosporins inN. gonorrhoeae. Preliminary results with clinical NAAT specimens were also promising, as they correlated well with bacterial culture results.

scholarly journals Development of a Novel Real-Time PCR Assay with High-Resolution Melt Analysis To Detect and Differentiate OXA-48-Like β-Lactamases in Carbapenem-Resistant Enterobacteriaceae

2015 ◽  
Vol 59 (9) ◽  
pp. 5574-5580 ◽  
Author(s):  
Peera Hemarajata ◽  
Shangxin Yang ◽  
Janet A. Hindler ◽  
Romney M. Humphries
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Real Time Pcr ◽  
Hydrolytic Activity ◽  
The United States ◽  
Pcr Assay ◽  
High Resolution Melt ◽  
Content Type ◽  
High Resolution Melt Analysis ◽  
Carbapenem Resistant

ABSTRACTThe rapid global spread of carbapenem-resistantEnterobacteriaceae(CRE) poses an urgent threat to public health. More than 250 class D β-lactamases (OXAs) have been described in recent years, with variations in hydrolytic activity for β-lactams. The plasmid-borne OXA-48 β-lactamase and its variants are identified only sporadically in the United States but are common in Europe. Recognition of these OXA-48-like carbapenemases is vital in order to control their dissemination. We developed a real-time PCR assay based on high-resolution melt analysis, usingblaOXA-48-like-specific primers coupled with an unlabeled 3′-phosphorylated oligonucleotide probe (LunaProbe) homologous to OXA-48-like carbapenemase genes. The assay was validated using genomic DNA from 48 clinical isolates carrying a variety of carbapenemase genes, includingblaKPC,blaSME,blaIMP,blaNDM-1,blaVIM,blaOXA-48,blaOXA-162,blaOXA-181,blaOXA-204,blaOXA-244,blaOXA-245, andblaOXA-232. Our assay identified the presence ofblaOXA-48-likeβ-lactamase genes and clearly distinguished betweenblaOXA-48and its variants in control strains, including betweenblaOXA-181andblaOXA-232, which differ by only a single base pair in the assay target region. This approach has potential for use in epidemiological investigations and continuous surveillance to help control the spread of CRE strains producing OXA-48-like enzymes.

scholarly journals Multiplex Real-Time PCR Assay for Simultaneous Identification of Neisseria gonorrhoeae and Its Ciprofloxacin Susceptibility Status

2017 ◽  
Vol 55 (11) ◽  
pp. 3201-3209 ◽  
Author(s):  
Sumudu R. Perera ◽  
Nurul H. Khan ◽  
Irene Martin ◽  
Ali Taheri ◽  
Rajinder P. Parti ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Real Time ◽  
Sensitivity And Specificity ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Content Type ◽  
Simultaneous Identification ◽  
Susceptibility Status

ABSTRACTA real-time PCR (RT-PCR) assay was designed for the simultaneous identification ofNeisseria gonorrhoeaeand its ciprofloxacin susceptibility status. A SYBR green-based multiplex RT-PCR format was used; it comprised two different forward primers and a common reverse primer to detect single nucleotide polymorphisms (SNPs) ingyrAofN. gonorrhoeae. The primer pairs were evaluated for their sensitivity and specificity using genomic DNA from 254N. gonorrhoeaeisolates (82 were ciprofloxacin susceptible and 172 were ciprofloxacin resistant) and 23 non-N. gonorrhoeaespecies isolates. The performance of the primers was validated using genomic DNA from 100 differentN. gonorrhoeaeisolates (46 were ciprofloxacin susceptible and 54 were ciprofloxacin resistant) and 52 non-N. gonorrhoeaeisolates. The latter panel was revalidated by testing 99 (46 isolates were ciprofloxacin susceptible and 53 isolates were ciprofloxacin resistant) of theN. gonorrhoeaeisolates and 23 non-N. gonorrhoeaeisolates. These primers detectedN. gonorrhoeaeand its ciprofloxacin susceptibility status with over 99% sensitivity and specificity for all panels tested. This assay has the potential to be an inexpensive and rapid test for the simultaneous identification ofN. gonorrhoeaeand its ciprofloxacin susceptibility status.

scholarly journals Abilities of the mCP Agar Method and CRENAME Alpha Toxin-Specific Real-Time PCR Assay To Detect Clostridium perfringens Spores in Drinking Water

2013 ◽  
Vol 79 (24) ◽  
pp. 7654-7661 ◽  
Author(s):  
Andrée F. Maheux ◽  
Ève Bérubé ◽  
Dominique K. Boudreau ◽  
Romain Villéger ◽  
Philippe Cantin ◽  
...  
Keyword(s):  
Drinking Water ◽  
Water Sample ◽  
Real Time ◽  
Clostridium Perfringens ◽  
Real Time Pcr ◽  
Potable Water ◽  
Alpha Toxin ◽  
Pcr Assay ◽  
Content Type ◽  
The Public

ABSTRACTWe first determined the analytical specificity and ubiquity (i.e., the ability to detect all or most strains) of aClostridium perfringens-specific real-time PCR (rtPCR) assay based on thecpagene (cpartPCR) by using a bacterial strain panel composed ofC. perfringensand non-C. perfringens Clostridiumstrains. All non-C. perfringens Clostridiumstrains tested negative, whereas allC. perfringensstrains tested positive with thecpartPCR, for an analytical specificity and ubiquity of 100%. ThecpartPCR assay was then used to confirm the identity of 116 putativeC. perfringensisolates recovered after filtration of water samples and culture on mCP agar. Colonies presenting discordant results between the phenotype on mCP agar andcpartPCR were identified by sequencing the 16S rRNA andcpagenes. Four mCP−/rtPCR+colonies were identified asC. perfringens, whereas 3 mCP+/rtPCR−colonies were identified as non-C. perfringens. ThecpartPCR was negative with all 51 non-C. perfringensstrains and positive with 64 of 65C. perfringensstrains. Finally, we compared mCP agar and a CRENAME (concentration andrecovery of microbial particles,extraction ofnucleicacids, andmolecularenrichment) procedure pluscpartPCR (CRENAME +cpartPCR) for their abilities to detectC. perfringensspores in drinking water. CRENAME +cpartPCR detected as few as oneC. perfringensCFU per 100 ml of drinking water sample in less than 5 h, whereas mCP agar took at least 25 h to deliver results. CRENAME +cpartPCR also allows the simultaneous and sensitive detection ofEscherichia coliandC. perfringensfrom the same potable water sample. In itself, it could be used to assess the public health risk posed by drinking water potentially contaminated with pathogens more resistant to disinfection.

scholarly journals A New Highly Sensitive and Specific Real-Time PCR Assay Targeting the Malate Dehydrogenase Gene ofKingella kingaeand Application to 201 Pediatric Clinical Specimens

2018 ◽  
Vol 56 (8) ◽  
Author(s):  
Nawal El Houmami ◽  
Guillaume André Durand ◽  
Janek Bzdrenga ◽  
Anne Darmon ◽  
Philippe Minodier ◽  
...  
Keyword(s):  
Real Time ◽  
Malate Dehydrogenase ◽  
Real Time Pcr ◽  
Detection Sensitivity ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Content Type ◽  
Clinical Specimens ◽  
Specificity And Sensitivity ◽  
Pcr Assays

ABSTRACTKingella kingaeis a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium using culture and broad-range 16S rRNA gene PCR assays from clinical specimens have proven unsatisfactory; therefore, by the late 2000s, these were gradually phased out to explore the benefits of specific real-time PCR tests targeting thegroELgene and the RTX locus ofK. kingae. However, recent studies showed that real-time PCR (RT-PCR) assays targeting theKingellasp. RTX locus that are currently available for the diagnosis ofK. kingaeinfection lack specificity because they could not distinguish betweenK. kingaeand the recently describedKingella negevensisspecies. Furthermore,in silicoanalysis of thegroELgene from a large collection of 45K. kingaestrains showed that primers and probes fromK. kingaegroEL-based RT-PCR assays display a few mismatches withK. kingae groELvariations that may result in decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative togroEL- and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, aK. kingae-specific RT-PCR assay targeting the malate dehydrogenase (mdh) gene was developed for predicting no mismatch between primers and probe and 18 variants of theK. kingae mdhgene from 20 distinct sequence types ofK. kingae. This novelK. kingae-specific RT-PCR assay demonstrated high specificity and sensitivity and was successfully used to diagnoseK. kingaeinfections and carriage in 104 clinical specimens from children between 7 months and 7 years old.

scholarly journals A Real-Time PCR Assay for Simultaneous Detection and Differentiation of Four Common Entamoeba Species That Infect Humans

2020 ◽  
Vol 59 (1) ◽  
pp. e01986-20
Author(s):  
Ibne Karim M. Ali ◽  
Shantanu Roy
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Small Subunit ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Content Type ◽  
Rdna Sequences ◽  
Appropriate Treatment

ABSTRACTThere are over 40 species within the genus Entamoeba, eight of which infect humans. Of these, four species (Entamoeba histolytica, E. dispar, E. moshkovskii, and E. bangladeshi) are morphologically indistinguishable from each other, and yet differentiation is important for appropriate treatment decisions. Here, we developed a hydrolysis probe-based tetraplex real-time PCR assay that can simultaneously detect and differentiate these four species in clinical samples. In this assay, multicopy small-subunit (SSU) ribosomal DNA (rDNA) sequences were used as targets. We determined that the tetraplex real-time PCR can detect amebic DNA corresponding to as little as a 0.1 trophozoite equivalent of any of these species. We also determined that this assay can detect E. histolytica DNA in the presence of 10-fold more DNA from another Entamoeba species in mixed-infection scenarios. With a panel of more than 100 well-characterized clinical samples diagnosed and confirmed using a previously published duplex real-time PCR (capable of detecting E. histolytica and E. dispar), our tetraplex real-time PCR assay demonstrated levels of sensitivity and specificity comparable with those demonstrated by the duplex real-time PCR assay. The advantage of our assay over the duplex assay is that it can specifically detect two additional Entamoeba species and can be used in conventional PCR format. This newly developed assay will allow further characterization of the epidemiology and pathogenicity of the four morphologically identical Entamoeba species, especially in low-resource settings.

scholarly journals Use of Multiplex Real-Time PCR To Diagnose Scrub Typhus

2017 ◽  
Vol 55 (5) ◽  
pp. 1377-1387 ◽  
Author(s):  
Wiwit Tantibhedhyangkul ◽  
Ekkarat Wongsawat ◽  
Saowaluk Silpasakorn ◽  
Duangdao Waywa ◽  
Nuttawut Saenyasiri ◽  
...  
Keyword(s):  
Early Diagnosis ◽  
Real Time ◽  
Real Time Pcr ◽  
Scrub Typhus ◽  
Febrile Illness ◽  
Asia Pacific ◽  
Diagnostic Tools ◽  
Pcr Assay ◽  
Content Type ◽  
Serologic Tests

ABSTRACTScrub typhus, caused byOrientia tsutsugamushi, is a common cause of acute undifferentiated febrile illness in the Asia-Pacific region. However, its nonspecific clinical manifestation often prevents early diagnosis. We propose the use of PCR and serologic tests as diagnostic tools. Here, we developed a multiplex real-time PCR assay using hydrolysis (TaqMan) probes targetingO. tsutsugamushi47-kDa,groEL, and human interferon beta (IFN-β gene) genes to improve early diagnosis of scrub typhus. The amplification efficiency was higher than 94%, and the lower detection limit was 10 copies per reaction. We used a human gene as an internal DNA quality and quantity control. To determine the sensitivity of this PCR assay, we selected patients with confirmed scrub typhus who exhibited a clear 4-fold increase in the level of IgG and/or IgM. The PCR assay result was positive in 45 of 52 patients, indicating a sensitivity of 86.5% (95% confidence interval [CI]: 74.2 to 94.4). The PCR assessment was negative for all 136 non-scrub typhus patients, indicating a specificity of 100% (95% CI: 97.3 to 100). In addition, this test helped diagnose patients with inconclusive immunofluorescence assay (IFA) results and using single blood samples. In conclusion, the real-time PCR assay proposed here is sensitive and specific in diagnosing scrub typhus. Combining PCR and serologic tests will improve the diagnosis of scrub typhus among patients presenting with acute febrile illness.

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