scholarly journals Development of a duplex real-time PCR assay for the differentiation of Blastomyces dermatitidis and B. gilchristii and a retrospective analysis of culture and primary specimens of blastomycosis cases from New York (2005-2019)

2020 ◽  
Author(s):  
Mitchell Kaplan ◽  
YanChun Zhu ◽  
Julianne V Kus ◽  
Lisa McTaggart ◽  
Vishnu Chaturvedi ◽  
...  
Keyword(s):  
New York ◽  
Retrospective Analysis ◽  
Real Time ◽  
Real Time Pcr ◽  
Blastomyces Dermatitidis ◽  
Pcr Assay ◽  
Clinical Specimens ◽  
Highly Sensitive ◽  
Predominant Pathogen ◽  
A Minor

Blastomycosis due to Blastomyces dermatitidis and B. gilchristii is a significant cause of respiratory mycoses in North America with occasionally reported outbreaks. We developed a highly sensitive, specific, and reproducible Taqman duplex real-time PCR assay for the differentiation of B. dermatitidis and B. gilchristii. The new assay permitted retrospective analysis of Blastomyces cultures (2005-2019) and primary clinical specimens from blastomycosis cases (2013-2019) from NY patients. We identified B. dermatitidis as the predominant pathogen in 38 cases of blastomycosis, while B. gilchristii was a minor pathogen involving five cases; these findings expand understanding of blastomycosis in New York. The duplex real-time PCR assay could be implemented in reference and public health laboratories to further understand the ecology and epidemiology of blastomycosis due to B. dermatitidis and B. gilchristii.

scholarly journals Development of a duplex real-time PCR assay for the differentiation of Blastomyces dermatitidis and B. gilchristii and a retrospective study of blastomycosis in New York (2005-2019)

2020 ◽  
Author(s):  
Mitchell Kaplan ◽  
YanChun Zhu ◽  
Julianne V Kus ◽  
Lisa McTaggart ◽  
Vishnu Chaturvedi ◽  
...  
Keyword(s):  
New York ◽  
Retrospective Study ◽  
North America ◽  
Real Time ◽  
Real Time Pcr ◽  
Blastomyces Dermatitidis ◽  
Pcr Assay ◽  
Rare Finding ◽  
Clinical Specimens ◽  
Highly Sensitive

Blastomycosis due to Blastomyces dermatitidis and B. gilchristii is a notable cause of respiratory mycoses in North America with recurrent outbreaks. We developed a highly sensitive, specific, and reproducible Taqman duplex real-time PCR assay for the differentiation of B. dermatitidis and B. gilchristii. The new assay permitted retrospective analysis of Blastomyces cultures (2005 to 2019), and primary clinical specimens (2013-2019) from NY patients. Blastomyces dermatitidis was the causal agent for the majority of 38 cases while B. gilchristii was implicated in five cases; a rare finding reported from New York. The duplex real-time PCR assay will be useful for further understanding of ecology and epidemiology of blastomycosis caused by B. dermatitidis and B. gilchristii.

A Real-Time PCR Assay for the Detection of Salmonella in a Wide Variety of Food and Food-Animal Matrices†

2007 ◽  
Vol 70 (5) ◽  
pp. 1080-1087 ◽  
Author(s):  
V. M. BOHAYCHUK ◽  
G. E. GENSLER ◽  
M. E. McFALL ◽  
R. K. KING ◽  
D. G. RENTER
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Culture Method ◽  
Pcr Assay ◽  
Culture Methods ◽  
The Real ◽  
Conventional Culture ◽  
Food Animal ◽  
Highly Sensitive ◽  
Field Samples

Conventional culture methods have traditionally been considered the “gold standards” for the isolation and identification of foodborne pathogens. However, culture methods are labor-intensive and time-consuming. We have developed a real-time PCR assay for the detection of Salmonella in a variety of food and food-animal matrices. The real-time PCR assay incorporates both primers and hybridization probes based on the sequence of the Salmonella invA gene and uses fluorescent resonance energy transfer technology to ensure highly sensitive and specific results. This method correctly classified 51 laboratory isolates of Salmonella and 28 non-Salmonella strains. The method was also validated with a large number of field samples that consisted of porcine feces and cecal contents, pork carcasses, bovine feces and beef carcasses, poultry cecal contents and carcasses, equine feces, animal feeds, and various food products. The samples (3,388) were preenriched in buffered peptone water and then selectively enriched in tetrathionate and Rappaport-Vassiliadis broths. Aliquots of the selective enrichment broths were combined for DNA extraction and analysis by the real-time PCR assay. When compared with the culture method, the diagnostic sensitivity of the PCR assay for the various matrices ranged from 97.1 to 100.0%, and the diagnostic specificity ranged from 91.3 to 100.0%. Kappa values ranged from 0.87 to 1.00, indicating excellent agreement of the real-time PCR assay to the culture method. The reduction in time and labor makes this highly sensitive and specific real-time PCR assay an excellent alternative to conventional culture methods for surveillance and research studies to improve food safety.

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 Evaluation of RealStar® Alpha Herpesvirus PCR Kit for Detection of HSV-1, HSV-2, and VZV in Clinical Specimens

2019 ◽  
Vol 2019 ◽  
pp. 1-6 ◽  
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.

scholarly journals Characterization of a universal screening approach for congenital CMV infection based on a highly-sensitive, quantitative, multiplex real-time PCR assay

PLoS ONE ◽  
2020 ◽  
Vol 15 (1) ◽  
pp. e0227143
Author(s):  
Angela Nagel ◽  
Emmanouela Dimitrakopoulou ◽  
Norbert Teig ◽  
Peter Kern ◽  
Thomas Lücke ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Universal Screening ◽  
Cmv Infection ◽  
Pcr Assay ◽  
Screening Approach ◽  
Highly Sensitive ◽  
Congenital Cmv Infection ◽  
Congenital Cmv

Paradigm for diagnosing mycobacterial disease: direct detection and differentiation of Mycobacterium tuberculosis complex and non-tuberculous mycobacteria in clinical specimens using multiplex real-time PCR

2018 ◽  
Vol 71 (9) ◽  
pp. 774-780 ◽  
Author(s):  
Jeong-Uk Kim ◽  
Dae-Shick Ryu ◽  
Choong-Hwan Cha ◽  
Seon-Hee Park
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Real Time ◽  
Real Time Pcr ◽  
Predictive Value ◽  
Direct Detection ◽  
Positive Culture ◽  
Nucleic Acid Amplification ◽  
Mycobacterial Disease ◽  
Pcr Assay ◽  
Clinical Specimens

AimsMycobacterium tuberculosis and non-tuberculous mycobacteria (NTM) are clinically different, and the rapid detection and differentiation of M. tuberculosis complex (MTBC) and NTM is crucial for patient management and infection control. Given the slow growth of most pathogenic mycobacteria, nucleic acid amplification assays are excellent tools for direct identification of mycobacteria in clinical specimens. Recently, a multiplex real-time PCR assay was developed that can directly detect 20 mycobacterial species in clinical specimens. Here, we evaluated the diagnostic performance of the assay for diagnosing mycobacterial disease under routine laboratory conditions.MethodsA total of 3334 specimens collected from 1437 patients suspected of tuberculosis infection were subjected to acid-fast bacilli staining, conventional culture and the multiplex real-time PCR assay. To evaluate the sensitivity and specificity of the assay, the overall diagnosis of tuberculosis was defined by positive culture plus medical history, and the 2007 American Thoracic Society and Infectious Disease Society of America diagnostic criteria for NTM disease were applied.ResultsThe sensitivity, specificity, positive predictive value and negative predictive value were 87.5%, 99.6%, 96.1% and 98.5%, respectively, for the detection of MTBC isolates and 53.3%, 99.9%, 95.2%, and 98.9%, respectively, for detecting NTM isolates.ConclusionsThus, the assay can correctly differentiate between MTBC and NTM isolates in clinical specimens and would be a useful tool for the rapid differentiation of tuberculosis and NTM disease, despite its limited sensitivity for the diagnosis of NTM disease.

scholarly journals Evaluation of a Multiplex Real-time PCR Assay for the Detection of Respiratory Viruses in Clinical Specimens

2012 ◽  
Vol 32 (6) ◽  
pp. 399-406 ◽  
Author(s):  
Insoo Rheem ◽  
Joowon Park ◽  
Tae-Hyun Kim ◽  
Jong Wan Kim
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Respiratory Viruses ◽  
Pcr Assay ◽  
Clinical Specimens
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