Development and validation of a real-time TaqMan PCR assay for the detection of betanodavirus in 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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Evaluation of RealStar® Alpha Herpesvirus PCR Kit for Detection of HSV-1, HSV-2, and VZV in Clinical Specimens

BioMed Research International ◽

10.1155/2019/5715180 ◽

2019 ◽

Vol 2019 ◽

pp. 1-6 ◽

Cited By ~ 2

Author(s):

Cyril C. Y. Yip ◽

Siddharth Sridhar ◽

Kit-Hang Leung ◽

Andrew K. W. Cheng ◽

Kwok-Hung Chan ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Test Performance ◽

Analytical Sensitivity ◽

Pcr Assay ◽

Clinical Specimens ◽

Varicella Zoster ◽

Simplex Virus ◽

Hsv 1

Several commercial PCR kits are available for detection of herpes simplex virus (HSV) and varicella zoster virus (VZV), but the test performance of one CE-marked in vitro diagnostic kit—RealStar® alpha Herpesvirus PCR Kit—has not been well studied. This study evaluated the performance of RealStar® alpha Herpesvirus PCR Kit 1.0 on the LightCycler® 480 Instrument II for detection and differentiation of HSV-1, HSV-2, and VZV in human clinical specimens. We evaluated the analytical sensitivity of the RealStar® and in-house multiplex real-time PCR assays using serial dilutions of nucleic acids extracted from clinical specimens. The analytical sensitivity of the RealStar® assay was 10, 32, and 100 copies/reaction for HSV-1, HSV-2, and VZV, respectively, which was slightly higher than that of the in-house multiplex real-time PCR assay. Reproducibility of the cycle threshold (Cp) values for each viral target was satisfactory with the intra- and interassay coefficient of variation values below 5% for both assays. One-hundred and fifty-three clinical specimens and 15 proficiency testing samples were used to evaluate the diagnostic performance of RealStar® alpha Herpesvirus PCR Kit against the in-house multiplex real-time PCR assay. The RealStar® assay showed 100% sensitivity and specificity when compared to the in-house assay. Cp values of the RealStar® and in-house assays showed excellent correlation. RealStar® alpha Herpesvirus PCR is a sensitive, specific, and reliable assay for the detection of HSV-1, HSV-2, and VZV, with less extensive verification requirements compared to a laboratory developed assay.

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Development and validation of a SYBR Green real-time PCR assay for rapid and quantitative detection of goose interferons and proinflammatory cytokines

Poultry Science ◽

10.3382/ps/pev241 ◽

2015 ◽

Vol 94 (10) ◽

pp. 2382-2387 ◽

Cited By ~ 5

Author(s):

Hao Zhou ◽

Shun Chen ◽

Yulin Qi ◽

Mingshu Wang ◽

Renyong Jia ◽

...

Keyword(s):

Real Time ◽

Proinflammatory Cytokines ◽

Real Time Pcr ◽

Sybr Green ◽

Quantitative Detection ◽

Pcr Assay ◽

Development And Validation

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Correction for Hilbert et al., Development and Validation of a Highly Accurate Quantitative Real-Time PCR Assay for Diagnosis of Bacterial Vaginosis

Journal of Clinical Microbiology ◽

10.1128/jcm.00831-16 ◽

2016 ◽

Vol 54 (7) ◽

pp. 1930-1930 ◽

Cited By ~ 1

Author(s):

David W. Hilbert ◽

William L. Smith ◽

Sean G. Chadwick ◽

Geoffrey Toner ◽

Eli Mordechai ◽

...

Keyword(s):

Bacterial Vaginosis ◽

Real Time ◽

Real Time Pcr ◽

Quantitative Real Time Pcr ◽

Pcr Assay ◽

Development And Validation

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Real-time TaqMan PCR for rapid detection and typing of genes encoding CTX-M extended-spectrum β-lactamases

Journal of Medical Microbiology ◽

10.1099/jmm.0.46909-0 ◽

2007 ◽

Vol 56 (1) ◽

pp. 52-55 ◽

Cited By ~ 94

Author(s):

Christopher I. Birkett ◽

Hugo A. Ludlam ◽

Neil Woodford ◽

Derek F. J. Brown ◽

Nicholas M. Brown ◽

...

Keyword(s):

Real Time ◽

Taqman Assay ◽

Esbl Production ◽

Pcr Assay ◽

Extended Spectrum ◽

Genes Encoding ◽

Taqman Pcr ◽

Assay Time ◽

Phenotypic Methods ◽

Single Reaction

The prevalence of CTX-M-producing members of the Enterobacteriaceae is increasing worldwide. A novel, multiplex, real-time TaqMan PCR assay to detect and type bla CTX-M genes is described which is an improvement on previously described techniques with respect to reduced assay time, elimination of the need for protracted post-PCR processing and the convenience of a single reaction vessel. Based on β-lactam antibiogram and MIC data, 478 of 1279 Enterobacteriaceae isolates from clinical blood and urine culture specimens were selected and tested for extended-spectrum β-lactamase (ESBL) production using phenotypic methods. The new TaqMan assay detected and typed bla CTX-M genes in 21 of 28 ESBL-producing isolates.

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