scholarly journals Molecular diagnosis of Kingella kingae osteoarticular infections by specific real-time PCR assay

2009 ◽  
Vol 58 (1) ◽  
pp. 65-68 ◽  
Author(s):  
Abdessalam Cherkaoui ◽  
Dimitri Ceroni ◽  
Stéphane Emonet ◽  
Yan Lefevre ◽  
Jacques Schrenzel
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Cell Types ◽  
Cross Reactivity ◽  
Toxin Gene ◽  
Rrna Gene ◽  
Putative Virulence Factor ◽  
Pcr Assay ◽  
Kingella Kingae ◽  
Rtx Toxin

Kingella kingae is an emerging pathogen that is recognized as a causative agent of septic arthritis and osteomyelitis, primarily in infants and children. The bacterium is best detected by rapid inoculation in blood culture systems or by real-time PCR assays. Pathogenesis of the agent was linked recently to the production of a potent cytotoxin, known as RTX, which is toxic to a variety of human cell types. The locus encoding the RTX toxin is thought to be a putative virulence factor, and is, apparently, essential for inducing cytotoxic effects on respiratory epithelial, synovial and macrophage-like cells. Herein, we describe a novel real-time PCR assay that targets the RTX toxin gene and illustrate its use in two clinical cases. The assay exhibited a sensitivity of 30 c.f.u., which is 10-fold more sensitive than a previously published semi-nested broad-range 16S rRNA gene PCR, and showed no cross-reactivity with several related species and common osteoarticular pathogens.

Real-Time PCR Assay for Detection of Kingella kingae in Children

2018 ◽  
Vol 13 (03) ◽  
pp. 216-223
Author(s):  
Theresa Madigan ◽  
Scott Cunningham ◽  
Poornima Ramanan ◽  
Micah Bhatti ◽  
Robin Patel
Keyword(s):  
Synovial Fluid ◽  
Septic Arthritis ◽  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Toxin Gene ◽  
Pcr Assay ◽  
Kingella Kingae ◽  
The Real ◽  
Osteoarticular Infections

Background Kingella kingae is a known cause of osteoarticular infections in children younger than 4 years of age, but it is not always recoverable in culture. Molecular methods are increasingly used for diagnosis. Methods To facilitate diagnosis of K. kingae septic arthritis, we developed a real-time polymerase chain reaction (PCR) assay for the detection of K. kingae that targets the repeat-in-toxin gene (rtxB). Results We present three pediatric patients with K. kingae septic arthritis at our institution who were diagnosed using the real-time PCR assay. All underwent arthrotomy with irrigation and debridement and were symptom-free after 3 weeks of therapy with β-lactam antibiotics. Cultures of synovial fluid or tissue grew K. kingae in two of three; K. kingae real-time PCR was positive in all three patients. In addition, 11 cases of K. kingae osteoarticular infection were diagnosed through Mayo Medical Laboratories using this assay. The limit of detection of the real-time PCR assay was 73.7 colony-forming unit (CFU)/µL for tissue and 1.3 CFU/µL for synovial fluid. Conclusions PCR-based detection methods are faster and more sensitive than conventional culture-based methods for the diagnosis of K. kingae osteoarticular infections in children.

scholarly journals Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

2003 ◽  
Vol 69 (12) ◽  
pp. 7430-7434 ◽  
Author(s):  
Trevor G. Phister ◽  
David A. Mills
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Primers ◽  
Rrna Gene ◽  
Dekkera Bruxellensis ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Spoilage Yeast ◽  
Pcr Method ◽  
26S Rrna

ABSTRACT Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.

scholarly journals Laboratory diagnosis of 37 cases of Bartonella endocarditis based on enzyme immunoassay and real-time PCR

2021 ◽  
Author(s):  
Lev Shapira ◽  
Michal Rasis ◽  
Inbal Binsky Ehrenreich ◽  
Yasmin Maor ◽  
Eugene A. Katchman ◽  
...  
Keyword(s):  
Enzyme Immunoassay ◽  
Real Time ◽  
Real Time Pcr ◽  
Heart Valves ◽  
Q Fever ◽  
Laboratory Diagnosis ◽  
Cross Reactivity ◽  
Immunofluorescence Assay ◽  
Complete Agreement ◽  
Pcr Assay

Bartonella spp., mostly B. quintana and B. henselae, are a common cause of culture-negative endocarditis. Serology, using immunofluorescence assay (IFA) and PCR performed on cardiac tissues are the mainstays of diagnosis. We developed an enzyme immunoassay (EIA) and a novel multiplex real-time PCR assay, utilizing Bartonella genus-specific, B. henselae-specific and B. quintana-specific SimpleProbe probes, for diagnosis of Bartonella endocarditis. We aimed to evaluate the performance of these assays. Thirty-seven patients with definite endocarditis, 18 with B. henselae, 18 with B. quintana and one with B. koehlerae were studied. Diagnosis was confirmed by conventional PCR and DNA sequencing of surgical cardiac specimens. Similarly to IFA, anti-Bartonella IgG titers ≥1:800 were found in 94% of patients by EIA; cross-reactivity between B. henselae and B. quintana precluded species-specific serodiagnosis, and frequent (41%) but low-titer cross-reactivity between Coxiella burnetii antibodies and B. henselae antigen was found in patients with Q fever endocarditis. Low-titer (1:100) cross-reactivity was uncommonly found also in patients with brucellosis and culture-positive endocarditis, particularly Enterococcus faecalis endocarditis. Real-time PCR performed on explanted heart valves/vegetations was in complete agreement with results of sequence-based diagnosis with characteristic melting curves. The genus-specific probe identified five additional endocarditis-associated Bartonella spp. at the genus level. In conclusion, EIA coupled with a novel real-time PCR assay can play an important role in Bartonella endocarditis diagnosis and expand the diagnostic arsenal at the disposal of the clinical microbiologist. Since serology remains a major diagnostic tool, recognizing its pitfalls is essential to avoid incorrect diagnosis.

Improving the specific diagnosis of trematode, cestode and nematode infections by a multiplex single-tube real-time PCR assay

2019 ◽  
Vol 72 (7) ◽  
pp. 487-492 ◽  
Author(s):  
Samson S Y Wong ◽  
Rosana W S Poon ◽  
Kelvin K W To ◽  
Jasper F W Chan ◽  
Gang Lu ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Molecular Diagnostic ◽  
Rrna Gene ◽  
28S Rrna ◽  
Pcr Assay ◽  
Single Tube ◽  
Histological Sections ◽  
Pcr Products ◽  
Stool Samples

AimsHelminth infections are becoming uncommon in high-income countries and laboratory staff may lose expertise in their morphological identification, especially in histological sections where speciation of helminths is challenging. Commercially available molecular diagnostic panels for faecal specimens only offer tests for protozoa but not helminths. We aim to improve the identification accuracy of helminths using a multiplex PCR assay.MethodsWe designed three pairs of PCR primers and probes targeting multicopy genes for a multiplex single-tube real-time PCR assay which covers 16 trematode (28S rRNA gene), 24 cestode (cox1 gene) and 33 nematode (cox1 gene) species. Helminths (n=27) from faecal samples (n=10), fresh parasites (n=11), formalin-fixed specimens (n=4), cerebrospinal fluid (n=1) and bile (n=1) were examined morphologically and tested by PCR. Fifty stool samples negative for parasites by microscopy were also tested.ResultsThe PCR assay correctly identified the genera of all tested helminths. Agarose gel electrophoresis and sequencing of the purified PCR amplicons confirmed that the PCR products were of correct sizes with 100% correlation with the respective species. Sequencing of the cox1 gene failed to identify Capillaria spp. in one sample owing to the lack of corresponding sequences in GenBank. PCR and sequencing of the nematode 18S rRNA gene using consensus primers showed 100% homology with Capillaria spp. sequence. No positive PCR products were found in the negative stool samples.ConclusionsThe highly specific test correctly identified all helminths in our cohort. It is a useful adjunct to helminth identification in difficult situations such as histological sections.

scholarly journals Development of a DNA-Based Detection Method for Cocos Nucifera Using TaqMan™ Real-Time PCR

Foods ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 332
Author(s):  
Jasmin Wrage ◽  
Oxana Kleyner ◽  
Sascha Rohn ◽  
Jürgen Kuballa
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Detection Method ◽  
Immunoglobulin E ◽  
Limit Of Detection ◽  
Cocos Nucifera ◽  
Cross Reactivity ◽  
Pcr Assay ◽  
Probe System ◽  
Ige Mediated

So far, only a few cases of immunoglobulin E (IgE)-mediated coconut allergies have been described in the literature. Due to a growing consumption of coconut-containing foods in occidental countries, the number of coconut allergies may also increase. As there is no causative immunotherapy in clinical routine, appropriate food labelling is particularly important, also with regard to cross-contamination, to prevent serious health consequences. The purpose of this study was to develop a DNA-based detection method for coconut (Cocos nucifera). Initially, three sets of coconut-specific primers were designed and tested. A TaqMan™ probe was then developed to identify and quantify coconut by real-time PCR assay. With 27 other plant and animal species, the specificity of the primer/probe system was tested and cross reactivity was excluded. In a dilution series, a limit of detection of 1 pg/µL was determined. Thus, the developed real-time PCR assay is a suitable method to detect coconut in food.

scholarly journals Real-Time PCR Detection of Parapoxvirus DNA,

2006 ◽  
Vol 52 (2) ◽  
pp. 316-319 ◽  
Author(s):  
Andreas Nitsche ◽  
Mathias Büttner ◽  
Sonja Wilhelm ◽  
Georg Pauli ◽  
Hermann Meyer
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Cross Reactivity ◽  
Probit Analysis ◽  
Animal Origin ◽  
Pcr Assay ◽  
Medical Practitioners ◽  
B2l Gene ◽  
Zoonotic Infections

Abstract Background: Detection of parapoxviruses is important in various animals as well as in humans as zoonotic infections. Reliable detection of parapoxviruses is fundamental for the exclusion of other rash-causing illnesses, for both veterinarians and medical practitioners. To date, however, no real-time PCR assay for the detection of parapoxviruses has been reported. Methods: A minor groove binder–based quantitative real-time PCR assay targeting the B2L gene of parapoxviruses was developed on the ABI Prism and the LightCycler platforms. Results: The real-time PCR assay successfully amplified DNA fragments from a total of 41 parapoxvirus strains and isolates representing the species orf virus, bovine papular stomatitis virus, pseudocowpoxvirus, and sealpoxvirus. Probit analysis gave a limit of detection of 4.7 copies per assay (95% confidence interval, 3.7–6.8 copies per reaction). Scabs contain a sufficient amount of parapoxvirus DNA and can therefore be used for PCR without any DNA preparation step. No cross-reactivity to human, bovine, or sheep genomic DNA or other DNA viruses, including orthopoxviruses, molluscum contagiosum viruses, and yaba-like disease viruses, was observed. Conclusion: The presented assay is suitable for the detection of parapoxvirus infections in clinical material of human and animal origin.

Development of a Quantitative Real-Time PCR Method To Enumerate Total Bacterial Counts in Ready-to-Eat Fruits and Vegetables

2006 ◽  
Vol 69 (10) ◽  
pp. 2504-2508 ◽  
Author(s):  
HAJIME TAKAHASHI ◽  
HIROTAKA KONUMA ◽  
YUKIKO HARA-KUDO
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Fruits And Vegetables ◽  
Bacterial Species ◽  
Culture Method ◽  
Plate Count ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Cycle Number ◽  
Wide Range

A newly developed real-time PCR assay rapidly quantifies the total bacterial numbers in contaminated ready-to-eat vegetables and fruits compared with the standard plate count method. Primers targeting the rpoB gene, which encodes for the β subunit of the bacterial RNA polymerase and which is common to most bacterial species, was used instead of the 16S rRNA gene, which has multiple copies and varies among bacterial species. A primer pair specific for rpoB was confirmed to amplify rpoB in a wide range of bacterial species after we assessed 49 strains isolated from five kinds of fruits and vegetables. We purchased fruits and vegetables from retail shops and enumerated the bacteria associated with them by use of real-time PCR and compared this to the number found by the culture method. We found a high correlation between the threshold PCR cycle number when compared with the plate count culture number. The real-time PCR assay developed in this study can enumerate the dominant bacterial species in ready-to-eat fruits and vegetables.

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

2020 ◽  
Vol 58 (10) ◽  
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.

PCR and culture for diagnosis of Acanthamoeba keratitis

2020 ◽  
pp. bjophthalmol-2020-316730
Author(s):  
Helene Yera ◽  
Vichita Ok ◽  
Fiona Lee Koy Kuet ◽  
Naima Dahane ◽  
Frédéric Ariey ◽  
...  
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Real Time Pcr ◽  
Latent Class ◽  
Rrna Gene ◽  
Target Sequence ◽  
Small Subunit Rrna ◽  
Pcr Assay ◽  
Corneal Scraping ◽  
Pcr Assays

Background/AimsAcanthamoeba keratitis (AK) is a rare but sight-threatening infection. Molecular diagnosis of corneal scraping has improved the diagnosis of AK. Different molecular targets and conditions have been used in diagnosis thus far. In this study, we prospectively compared the performance of five PCR assays on corneal samples for the diagnosis of AK.Methods1217 corneal scraping samples were obtained from patients, for whom an AK was suspected. Sample processing involved both molecular diagnostics and culture. Acanthamoeba PCR assays detected different regions of the Acanthamoeba nuclear small-subunit rRNA gene: three final point PCR assays using Nelson, ACARNA and JDP1–JDP2 pairs of primers, and two real-time PCR assays using Acant primer-probe. Human DNA and internal control were co-amplified in the real-time PCR assay to ensure scraping quality and the absence of inhibitors. In the absence of a gold standard, the performance of each test was evaluated using latent class analysis. Genotypes of Acanthamoeba isolates were also characterised.ResultsEstimated prevalence of AK was 1.32%. The sensitivity of Acanthamoeba diagnostic PCRs (73.3% to 86.7%) did not differ significantly from that of culture (66.7%), or according to the target sequence or the technology. Sensitivity could be increased to 93.8% or 100% by combining two or three assays, respectively. PCR specificity (99.3% to 100%) differed between the assays. T4 was the predominant Acanthamoeba genotype (84.6%).ConclusionsCulture and a single PCR assay could lead to misdiagnosing AK. A combination of different PCR assays and improved sample quality could increase diagnosis sensitivity.

scholarly journals A rapid and automated sample-to-result Candida auris real-time PCR assay for high-throughput testing of surveillance samples with BD MAX™ open system

2019 ◽  
Author(s):  
L. Leach ◽  
A. Russell ◽  
Y. Zhu ◽  
S. Chaturvedi ◽  
V. Chaturvedi
Keyword(s):  
New York ◽  
Real Time ◽  
High Throughput ◽  
Open System ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Cross Reactivity ◽  
Pcr Assay ◽  
Candida Auris ◽  
Clinical Sensitivity

ABSTRACTThe multidrug-resistant yeast pathogen Candida auris continues to cause outbreaks and clusters of clinical cases worldwide. Previously, we developed a real-time PCR assay for the detection of C. auris from surveillance samples (Leach et al. JCM. 2018: 56, e01223-17). The assay played a crucial role in the ongoing investigations of C. auris outbreak in New York City. To ease the implementation of the assay in other laboratories, we developed an automated sample-to-result real-time C. auris PCR assay using BD MAX™ open system. We optimized sample extraction at three different temperatures and four incubation periods. Sensitivity was determined using eight pools of patient samples, and specificity was calculated using four clades of C. auris, and closely and distantly related yeasts. Three independent extractions and testing of two patient sample pools in the quadruplicate yielded assay precision. BD MAX™ optimum assay conditions were: DNA extraction at 75°C for 20 min, and the use of PerfeCTa Multiplex qPCR ToughMix. The limit of detection (LOD) of the assay was one C. auris CFU/PCR reaction. We detected all four clades of C. auris without cross-reactivity to other yeasts. Of the 110 patient surveillance samples tested, 50 were positive for C. auris using the BD MAX™ System with 96% clinical sensitivity and 94% accuracy compared to the manual assay. BD MAX™ assay allows high-throughput C. auris screening of 180 surveillance samples in a 12-hour workday.

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