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

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.

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Molecular-Beacon Multiplex Real-Time PCR Assay for Detection of Vibrio cholerae

Applied and Environmental Microbiology ◽

10.1128/aem.02597-05 ◽

2006 ◽

Vol 72 (9) ◽

pp. 6424-6428 ◽

Cited By ~ 38

Author(s):

Aneta J. Gubala ◽

David F. Proll

Keyword(s):

Vibrio Cholerae ◽

Real Time ◽

Water Samples ◽

Real Time Pcr ◽

Molecular Beacon ◽

Environmental Water ◽

Molecular Beacons ◽

Pcr Assay ◽

Specificity And Sensitivity ◽

Pcr Assays

ABSTRACT A multiplex real-time PCR assay was developed using molecular beacons for the detection of Vibrio cholerae by targeting four important virulence and regulatory genes. The specificity and sensitivity of this assay, when tested with pure culture and spiked environmental water samples, were high, surpassing those of currently published PCR assays for the detection of this organism.

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Pathogen Identification by Multiplex LightMix Real-Time PCR Assay in Patients with Meningitis and Culture-Negative Cerebrospinal Fluid Specimens

Journal of Clinical Microbiology ◽

10.1128/jcm.01492-17 ◽

2017 ◽

Vol 56 (2) ◽

Cited By ~ 3

Author(s):

Karoline Wagner ◽

Burkard Springer ◽

Valeria P. Pires ◽

Peter M. Keller

Keyword(s):

Cerebrospinal Fluid ◽

Bacterial Meningitis ◽

Real Time ◽

Antimicrobial Therapy ◽

Real Time Pcr ◽

Bacterial Identification ◽

Rt Pcr ◽

Pcr Assay ◽

Content Type ◽

Culture Negative

ABSTRACT Acute bacterial meningitis is a medical emergency, and delays in initiating effective antimicrobial therapy result in increased morbidity and mortality. Culture-based methods, thus far considered the “gold standard” for identifying bacterial microorganisms, require 24 to 48 h to provide a diagnosis. In addition, antimicrobial therapy is often started prior to clinical sample collection, thereby decreasing the probability of confirming the bacterial pathogen by culture-based methods. To enable a fast and accurate detection of the most important bacterial pathogens causing meningitis, namely, Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Streptococcus agalactiae, and Listeria monocytogenes, we evaluated a commercially available multiplex LightMix real-time PCR (RT-PCR) in 220 cerebrospinal fluid (CSF) specimens. The majority of CSF samples were collected by lumbar puncture, but we also included some CSF samples from patients with symptoms of meningitis from the neurology department that were recovered from shunts. CSF samples were analyzed by multiplex RT-PCR enabling a first diagnosis within a few hours after sample arrival at our institute. In contrast, bacterial identification took between 24 and 48 h by culture. Overall, a high agreement of bacterial identification between culture and multiplex RT-PCR was observed (99%). Moreover, multiplex RT-PCR enabled the detection of pathogens, S. pneumoniae (n = 2), S. agalactiae (n = 1), and N. meningitidis (n = 1), in four culture-negative samples. As a complement to classical bacteriological CSF culture, the LightMix RT-PCR assay proved to be valuable by improving the rapidity and accuracy of the diagnosis of bacterial meningitis.

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Detection and Characterization of Diphtheria Toxin Gene-Bearing Corynebacterium Species through a New Real-Time PCR Assay

Journal of Clinical Microbiology ◽

10.1128/jcm.00639-20 ◽

2020 ◽

Vol 58 (10) ◽

Cited By ~ 1

Author(s):

Margaret M. Williams ◽

Jessica L. Waller ◽

Janessa S. Aneke ◽

Michael R. Weigand ◽

Maureen H. Diaz ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Toxin Production ◽

Toxin Gene ◽

Analytical Sensitivity ◽

Rt Pcr ◽

Pcr Assay ◽

Culture Methods ◽

Content Type ◽

Corynebacterium Ulcerans

ABSTRACT Respiratory diphtheria, characterized by a firmly adherent pseudomembrane, is caused by toxin-producing strains of Corynebacterium diphtheriae, with similar illness produced occasionally by toxigenic Corynebacterium ulcerans or, rarely, Corynebacterium pseudotuberculosis. While diphtheria laboratory confirmation requires culture methods to determine toxigenicity, real-time PCR (RT-PCR) provides a faster method to detect the toxin gene (tox). Nontoxigenic tox-bearing (NTTB) Corynebacterium isolates have been described, but impact of these isolates on the accuracy of molecular diagnostics is not well characterized. Here, we describe a new triplex RT-PCR assay to detect tox and distinguish C. diphtheriae from the closely related species C. ulcerans and C. pseudotuberculosis. Analytical sensitivity and specificity of the assay were assessed in comparison to culture using 690 previously characterized microbial isolates. The new triplex assay characterized Corynebacterium isolates accurately, with 100% analytical sensitivity for all targets. Analytical specificity with isolates was 94.1%, 100%, and 99.5% for tox, Diph_rpoB, and CUP_rpoB targets, respectively. Twenty-nine NTTB Corynebacterium isolates, representing 5.9% of 494 nontoxigenic isolates tested, were detected by RT-PCR. Whole-genome sequencing of NTTB isolates revealed varied mutations putatively underlying their lack of toxin production, as well as eight isolates with no mutation in tox or the promoter region. This new Corynebacterium RT-PCR method provides a rapid tool to screen isolates and identify probable diphtheria cases directly from specimens. However, the sporadic occurrence of NTTB isolates reinforces the viewpoint that diphtheria culture diagnostics continue to provide the most accurate case confirmation.

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Utility of Real-Time PCR for Detection of Exserohilum rostratum in Body and Tissue Fluids during the Multistate Outbreak of Fungal Meningitis and Other Infections

Journal of Clinical Microbiology ◽

10.1128/jcm.02443-14 ◽

2014 ◽

Vol 53 (2) ◽

pp. 618-625 ◽

Cited By ~ 13

Author(s):

Lalitha Gade ◽

Dale E. Grgurich ◽

Thomas M. Kerkering ◽

Mary E. Brandt ◽

Anastasia P. Litvintseva

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Response To Treatment ◽

Pcr Assay ◽

Content Type ◽

The Real ◽

Fungal Meningitis ◽

Exserohilum Rostratum ◽

Fungal Dna ◽

Pcr Assays

Exserohilum rostratumwas the major cause of the multistate outbreak of fungal meningitis linked to contaminated injections of methylprednisolone acetate produced by the New England Compounding Center. Previously, we developed a fungal DNA extraction procedure and broad-range andE. rostratum-specific PCR assays and confirmed the presence of fungal DNA in 28% of the case patients. Here, we report the development and validation of a TaqMan real-time PCR assay for the detection ofE. rostratumin body fluids, which we used to confirm infections in 57 additional case patients, bringing the total number of case patients with PCR results positive forE. rostratumto 171 (37% of the 461 case patients with available specimens). Compared to fungal culture and the previous PCR assays, this real-time PCR assay was more sensitive. Of the 139 identical specimens from case patients tested by all three methods, 19 (14%) were positive by culture, 41 (29%) were positive by the conventional PCR assay, and 65 (47%) were positive by the real-time PCR assay. We also compared the utility of the real-time PCR assay with that of the previously described beta-d-glucan (BDG) detection assay for monitoring response to treatment in case patients with serially collected CSF. Only the incident CSF specimens from most of the case patients were positive by real-time PCR, while most of the subsequently collected specimens were negative, confirming our previous observations that the BDG assay was more appropriate than the real-time PCR assay for monitoring the response to treatment. Our results also demonstrate that the real-time PCR assay is extremely susceptible to contamination and its results should be used only in conjunction with clinical and epidemiological data.

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Comparison of bluetongue virus detection and quantitation methods in south India

The Journal of Infection in Developing Countries ◽

10.3855/jidc.4681 ◽

2014 ◽

Vol 8 (10) ◽

pp. 1307-1312

Author(s):

Subhra Subhadra ◽

Subrat Kumar ◽

Veluvarthy VS Suryanarayana ◽

Daggupati Sreenivasulu

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Melting Curve ◽

Disease Diagnosis ◽

Sybr Green ◽

Melting Curve Analysis ◽

Rt Pcr ◽

Pcr Assay ◽

Animal Blood ◽

Specificity And Sensitivity

Introduction: Bluetongue (BT), a vector-borne viral disease, primarily affects sheep. Of the 26 serotypes of BTV identified so far, 22 are reported to be circulating in India. Due to an increase in vector population and delays in disease diagnosis, the BT control program heavily relies on rapid and confirmatory diagnosis. Polymerase chain reaction (PCR)-based real-time detection assays may be an ideal method to detect the BTV genome in animal blood at an early stage of infection. Methodology: In this study, a SYBR green-based real-time RT-PCR assay was evaluated, validated, and compared with conventional RT-PCR. The specificity and sensitivity of an assay using BTV-2 RNA extracted from tenfold serially diluted (starting from 1.0 TCID50/mL) cell culture virus was also evaluated. Results: While conventional RT-PCR could detect 3.16×102 TCID50 of virus/mL, the real-time PCR test had a detection limit of 3.16×10-4 TCID50/mL. Melting curve analysis indicated the absence of non-specific amplification (R2 = 0.987). Out of the 32 infected blood samples examined, 24 tested positive for BTV RNA. Seven that were found negative through conventional PCR tested positive through real-time PCR. Conclusions: These results showed that the SYBR green-based real-time PCR assay is rapid, sensitive, and equally specific in the diagnosis of BT in BTV-affected animals.

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

Journal of Clinical Microbiology ◽

10.1128/jcm.00855-17 ◽

2017 ◽

Vol 55 (11) ◽

pp. 3201-3209 ◽

Cited By ~ 11

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.

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A specific real-time PCR assay for the detection of Bordetella pertussis

Journal of Medical Microbiology ◽

10.1099/jmm.0.46947-0 ◽

2007 ◽

Vol 56 (7) ◽

pp. 918-920 ◽

Cited By ~ 19

Author(s):

Benoit Vincart ◽

Ricardo De Mendonça ◽

Sylvianne Rottiers ◽

Françoise Vermeulen ◽

Marc J. Struelens ◽

...

Keyword(s):

Real Time ◽

Bordetella Pertussis ◽

Real Time Pcr ◽

Analytical Sensitivity ◽

Rt Pcr ◽

Pcr Assay ◽

Pcr Assays ◽

Reference Strains ◽

Respiratory Specimens

A novel real-time PCR (RT-PCR) assay was developed for detection of Bordetella pertussis in respiratory specimens by targeting the pertactin gene. In vitro evaluation with reference strains and quality control samples showed analytical sensitivity equivalent to and specificity superior to those of PCR assays which target the IS481 element. The pertactin-based RT-PCR assay offers better discrimination between B. pertussis and other Bordetella species than previously described assays.

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Development and application of real-time PCR assay for detection of mutations associated with macrolide resistance in Mycoplasma genitalium directly in clinical specimens

10.26226/morressier.56d5ba28d462b80296c96082 ◽

2016 ◽

Cited By ~ 2

Author(s):

Inna Edelstein

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Mycoplasma Genitalium ◽

Macrolide Resistance ◽

Pcr Assay ◽

Clinical Specimens ◽

Detection Of Mutations

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Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽

10.3390/pathogens10020188 ◽

2021 ◽

Vol 10 (2) ◽

pp. 188

Author(s):

Tanja Hoffmann ◽

Andreas Hahn ◽

Jaco J. Verweij ◽

Gérard Leboulle ◽

Olfert Landt ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Real Time Pcr ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Pcr Assay ◽

Bead Beating ◽

Stool Samples ◽

Pcr Assays ◽

Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

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