Real-Time PCR Detection of Parapoxvirus DNA,

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.

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

Foods ◽

10.3390/foods9030332 ◽

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.

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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

10.1101/608190 ◽

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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Real-time PCR assay for detection and differentiation of Coccidioides immitis and Coccidioides posadasii from culture and clinical specimens

10.1101/2021.04.08.21254780 ◽

2021 ◽

Author(s):

Sudha Chaturvedi ◽

Tanya R Victor ◽

Anuradha Marathe ◽

Ketevan Sidamonidze ◽

Kelly L Crucillo ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Cross Reactivity ◽

Coccidioides Immitis ◽

Coccidioides Posadasii ◽

Pcr Assay ◽

Valley Fever ◽

Clinical Specimens ◽

Wide Range

Coccidioidomycosis (Valley Fever) is a pulmonary and systemic fungal disease with increasing incidence and expanding endemic areas. The differentiation of etiologic agents Coccidioides immitis and C. posadasii remains problematic in the clinical laboratories as conventional PCR and satellite typing schemes are not facile. Therefore, we developed Cy5- and FAM-labeled TaqMan-probes for duplex real-time PCR assay for rapid differentiation of C. immitis and C. posadasii from culture and clinical specimens. The RRA2 gene encoding proline-rich antigen 2, specific for Coccidioides genus, was the source for the first set of primers and probe. Coccidioides immitis contig 2.2 (GenBank: AAEC02000002.1) was used to design the second set of primers and probe. The second primers/probe did not amplify the corresponding C. posadasii DNA, because of an 86-bp deletion in the contig. The assay was highly sensitive with limit of detection of 0.1 pg gDNA/PCR reaction, which was equivalent to approximately ten genome copies of C. immitis or C. posadasii. The assay was highly specific with no cross-reactivity to the wide range of fungal and bacterial pathogens. Retrospective analysis of fungal isolates and primary specimens submitted from 1995 to 2020 confirmed 129 isolates and three primary specimens as C. posadasii and 23 isolates as C. immitis from human coccidioidomycosis cases, while all eight primary samples from two animals were confirmed as C. posadasii. A preliminary analysis of cerebrospinal fluid (CSF) and pleural fluid samples showed positive correlation between serology tests and real-time PCR for two of the 15 samples. The Coccidioides spp. duplex real-time PCR will allow rapid differentiation of C. immitis and C. posadasii from clinical specimens and further augment the surveillance of coccidioidomycosis.

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Real-time PCR assay for detection and differentiation of Coccidioides immitis and Coccidioides posadasii from culture and clinical specimens

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0009765 ◽

2021 ◽

Vol 15 (9) ◽

pp. e0009765

Author(s):

Sudha Chaturvedi ◽

Tanya R. Victor ◽

Anuradha Marathe ◽

Ketevan Sidamonidze ◽

Kelly L. Crucillo ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Cross Reactivity ◽

Coccidioides Immitis ◽

Coccidioides Posadasii ◽

Pcr Assay ◽

Valley Fever ◽

Clinical Specimens ◽

Wide Range

Coccidioidomycosis (Valley Fever) is a pulmonary and systemic fungal disease with increasing incidence and expanding endemic areas. The differentiation of etiologic agents Coccidioides immitis and C. posadasii remains problematic in the clinical laboratories as conventional PCR and satellite typing schemes are not facile. Therefore, we developed Cy5- and FAM-labeled TaqMan-probes for duplex real-time PCR assay for rapid differentiation of C. immitis and C. posadasii from culture and clinical specimens. The RRA2 gene encoding proline-rich antigen 2, specific for Coccidioides genus, was the source for the first set of primers and probe. Coccidioides immitis contig 2.2 (GenBank: AAEC02000002.1) was used to design the second set of primers and probe. The second primers/probe did not amplify the corresponding C. posadasii DNA, because of an 86-bp deletion in the contig. The assay was highly sensitive with limit of detection of 0.1 pg gDNA/PCR reaction, which was equivalent to approximately ten genome copies of C. immitis or C. posadasii. The assay was highly specific with no cross-reactivity to the wide range of fungal and bacterial pathogens. Retrospective analysis of fungal isolates and primary specimens submitted from 1995 to 2020 confirmed 168 isolates and four primary specimens as C. posadasii and 30 isolates as C. immitis from human coccidioidomycosis cases, while all eight primary samples from two animals (rhesus monkey and rhinoceros) were confirmed as C. posadasii. A preliminary analysis of cerebrospinal fluid (CSF) and pleural fluid samples showed positive correlation between serology tests and real-time PCR for two of the 15 samples. The Coccidioides spp. duplex real-time PCR will allow rapid differentiation of C. immitis and C. posadasii from clinical specimens and further augment the treatment and surveillance of coccidioidomycosis.

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A Rapid and Automated Sample-to-Result Candida auris Real-Time PCR Assay for High-Throughput Testing of Surveillance Samples with the BD Max Open System

Journal of Clinical Microbiology ◽

10.1128/jcm.00630-19 ◽

2019 ◽

Vol 57 (10) ◽

Cited By ~ 6

Author(s):

L. Leach ◽

A. Russell ◽

Y. Zhu ◽

S. Chaturvedi ◽

V. Chaturvedi

Keyword(s):

Real Time ◽

High Throughput ◽

Open System ◽

Real Time Pcr ◽

Limit Of Detection ◽

Cross Reactivity ◽

Pcr Assay ◽

Candida Auris ◽

Content Type ◽

Clinical Sensitivity

ABSTRACT The 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 (L. Leach, Y. Zhu, and S. Chaturvedi, J Clin Microbiol 56:e01223-17, 2018, https://doi.org/10.1128/JCM.01223-17). The assay played a crucial role in the ongoing investigations of the 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 the 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 quadruplicate yielded assay precision. BD Max optimum assay conditions were as follows: DNA extraction at 75°C for 20 min and the use of PerfeCTa multiplex quantitative PCR (qPCR) ToughMix. The limit of detection (LOD) of the assay was one C. auris CFU/PCR. 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 results of the manual assay. The BD Max assay allows high-throughput C. auris screening of 180 surveillance samples in a 12-h workday.

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Performance evaluation of cobas HBV real-time PCR assay on Roche cobas 4800 System in comparison with COBAS AmpliPrep/COBAS TaqMan HBV Test

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2017-1133 ◽

2018 ◽

Vol 56 (7) ◽

pp. 1133-1139 ◽

Cited By ~ 7

Author(s):

Hanah Kim ◽

Mina Hur ◽

Eunsin Bae ◽

Kyung-A Lee ◽

Woo-In Lee

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Nucleic Acid Amplification ◽

Coefficient Of Determination ◽

Clinical Samples ◽

Pcr Assay ◽

Cobas Taqman ◽

Limit Of Quantitation ◽

Two Systems

Abstract Background: Hepatitis B virus (HBV) nucleic acid amplification testing (NAAT) is important for the diagnosis and management of HBV infection. We evaluated the analytical performance of the cobas HBV NAAT (Roche Diagnostics GmbH, Mannheim, Germany) on the cobas 4800 System in comparison with COBAS AmpliPrep/COBAS TaqMan HBV Test (CAP/CTM HBV). Methods: Precision was evaluated using three levels of cobas HBV/HCV/HIV-1 Control Kit, and linearity was evaluated across the anticipated measuring range (10.0–1.0×109 IU/mL) at seven levels using clinical samples. Detection capability, including limit of blank (LOB), limit of detection (LOD) and limit of quantitation (LOQ), was verified using the 4th WHO International Standard for HBV DNA for NAT (NIBSC code: 10/266). Correlation between the two systems was compared using 205 clinical samples (102 sera and 103 EDTA plasma). Results: Repeatability and total imprecision (coefficient of variation) ranged from 0.5% to 3.8% and from 0.5% to 3.5%, respectively. Linearity (coefficient of determination, R2) was 0.999. LOB, LOD and LOQ were all acceptable within the observed proportion rate (85%). Correlation was very high between the two systems in both serum and plasma samples (correlation coefficient [r]=0.995). Conclusions: The new cobas HBV real-time PCR assay on the cobas 4800 System showed reliable analytical performances.

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Real-Time PCR Assay for Detection of a New Simulant for Poxvirus Biothreat Agents

Applied and Environmental Microbiology ◽

10.1128/aem.02120-08 ◽

2009 ◽

Vol 75 (6) ◽

pp. 1614-1620 ◽

Cited By ~ 9

Author(s):

Laurence Garnier ◽

Jean-Christophe Gaudin ◽

Paul Bensadoun ◽

Isabelle Rebillat ◽

Yannick Morel

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Rna Virus ◽

Cydia Pomonella ◽

Aerosol Sampling ◽

Pcr Assay ◽

Double Stranded Dna ◽

Technological Developments ◽

Virus Model

ABSTRACT Research and financial efforts spent on biodefense technologies highlight the current concern for biothreat event preparedness. Nonhazardous but relevant “simulant” microorganisms are typically used to simplify technological developments, testing, and staff training. The bacteriophage MS2, a small RNA virus, is classically used as the reference simulant for biothreat viruses within the biodefense community. However, variola virus, considered a major threat, displays very different features (size, envelope, and double-stranded DNA genome). The size parameter is critical for aerosol sampling, detection, and protection/filtration technologies. Therefore, a panel of relevant simulants should be used to cover the diversity of biothreat agents. Thus, we investigated a new virus model, the Cydia pomonella granulovirus (baculovirus), which is currently used as a biopesticide. It displays a size similar to that of poxviruses, is enveloped, and contains double-stranded DNA. To provide a molecular tool to detect and quantify this model virus, we developed an assay based on real-time PCR, with a limit of detection ranging from roughly 10 to a few tens of target copies per μl according to the sample matrix. The specificity of the assay against a large panel of potential cross-reactive microorganisms was checked, and the suitability of the assay for environmental samples, especially aerosol studies, was determined. In conclusion, we suggest that our PCR assay allows Cydia pomonella granulovirus to be used as a simulant for poxviruses. This assay may also be useful for environmental or crop treatment studies.

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

Journal of Medical Microbiology ◽

10.1099/jmm.0.47707-0 ◽

2009 ◽

Vol 58 (1) ◽

pp. 65-68 ◽

Cited By ~ 56

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.

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Multicenter Clinical Validation of a Cartridge-Based Real-Time PCR System for Detection of Coccidioides spp. in Lower Respiratory Specimens

Journal of Clinical Microbiology ◽

10.1128/jcm.01277-17 ◽

2017 ◽

Vol 56 (2) ◽

Cited By ~ 9

Author(s):

Michael A. Saubolle ◽

Bette R. Wojack ◽

Anne M. Wertheimer ◽

Atehkeng Z. Fuayagem ◽

Stephen Young ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Fungal Species ◽

Cross Reactivity ◽

Clinical Test ◽

Target Sequence ◽

Fungal Culture ◽

Content Type ◽

Bronchial Wash

ABSTRACT Available methods for the diagnosis of coccidioidomycosis have significant shortcomings relative to accuracy and timeliness. We retrospectively and prospectively evaluated the diagnostic performance and reproducibility of a new cartridge-based real-time PCR assay for Coccidioides spp. directly in lower respiratory secretions and compared them to today's “gold standard,” fungal culture. The GeneSTAT Coccidioides assay uses a 106-bp target sequence repeated multiple times (∼60×) per genome, thus lowering the limit of detection (LOD) for extracted DNA to 10 genome equivalents/ml. A total of 332 prospective and retrospective individual patient specimens were tested. The retrospective samples consisted of 100 bronchoalveolar lavage or bronchial wash (BAL/BW) (51 positive and 49 negative by culture) specimens that had been collected previously and stored at −70°C. These samples were tested by the GeneSTAT Coccidioides assay across three clinical test sites. The sensitivity was 100%, and the specificity ranged between 93.8% and 100%. There was minimal variance in the percent agreement across the three sites, 95.6% to 100%. Additionally, a total of 232 fresh (prospective) deidentified BAL/BW specimens were tested across the three clinical sites, which included a number of specimens from Southern California to provide a diversity of isolates. Specimens were tested by fungal culture, with any isolates of Coccidioides, except for one, being confirmed by molecular means (AccuProbe). The sensitivity of the GeneSTAT Coccidioides assay across the three sites was 100% (4/4) for positive fresh specimens, and the overall specificity of the assay was 99.6% (227/228), ranging from 98.1% to 100%. In testing for cross-reactivity, the assay was 100% specific when screened against 47 different bacterial, viral, and fungal species.

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

Journal of Clinical Microbiology ◽

10.1128/jcm.02217-20 ◽

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.

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