scholarly journals 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

2019 ◽  
Vol 57 (10) ◽  
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.

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.

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

2017 ◽  
Vol 56 (2) ◽  
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.

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.

scholarly journals Development and Validation of a Real-Time PCR Assay for Rapid Detection of Candida auris from Surveillance Samples

2017 ◽  
Vol 56 (2) ◽  
Author(s):  
L. Leach ◽  
Y. Zhu ◽  
S. Chaturvedi
Keyword(s):  
Health Care ◽  
Real Time ◽  
Real Time Pcr ◽  
Multidrug Resistant ◽  
Effective Control ◽  
Pcr Assay ◽  
Candida Auris ◽  
Content Type ◽  
The Real ◽  
Positive Results

ABSTRACT Candida auris is an emerging multidrug-resistant yeast causing invasive health care-associated infection with high mortality worldwide. Rapid identification of C. auris is of primary importance for the implementation of public health measures to control the spread of infection. To achieve these goals, we developed and validated a TaqMan-based real-time PCR assay targeting the internal transcribed spacer 2 (ITS2) region of the ribosomal gene. The assay was highly specific, reproducible, and sensitive, with the detection limit of 1 C. auris CFU/PCR. The performance of the C. auris real-time PCR assay was evaluated by using 623 surveillance samples, including 365 patient swabs and 258 environmental sponges. Real-time PCR yielded positive results from 49 swab and 58 sponge samples, with 89% and 100% clinical sensitivity with regard to their respective culture-positive results. The real-time PCR also detected C. auris DNA from 1% and 12% of swab and sponge samples with culture-negative results, indicating the presence of dead or culture-impaired C. auris. The real-time PCR yielded results within 4 h of sample processing, compared to 4 to 14 days for culture, reducing turnaround time significantly. The new real-time PCR assay allows for accurate and rapid screening of C. auris and can increase effective control and prevention of this emerging multidrug-resistant fungal pathogen in health care facilities.

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

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.

scholarly journals A TaqMan Probe-Based Real-Time PCR Assay for the Rapid Identification of the Emerging Multidrug-Resistant Pathogen Candida auris on the BD Max System

2019 ◽  
Vol 57 (7) ◽  
Author(s):  
Amorce Lima ◽  
Raymond Widen ◽  
Grant Vestal ◽  
Dominic Uy ◽  
Suzane Silbert
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Multidrug Resistant ◽  
Rapid Identification ◽  
Taqman Probe ◽  
Pcr Assay ◽  
Candida Auris ◽  
Closely Related Species ◽  
Content Type ◽  
Deep Wound

ABSTRACT Candida auris is an emerging multidrug-resistant fungal pathogen that has been associated with nosocomial bloodstream and deep wound infections causing a high mortality rate mainly in intensive care unit (ICU) patients. Laboratories currently rely on phenotypic testing using commercial automated systems for identification of yeasts; however, this technique has often led to misidentification of C. auris to other closely related species. We developed and validated a TaqMan-based real-time PCR assay on the BD Max platform targeting ribosomal DNA (rDNA) region nucleotide sequences to quickly and accurately test for C. auris infection from culture and clinical specimens. The assay is highly specific, reproducible, and sensitive, allowing detection of as low as 1 C. auris CFU per reaction within 3 h.

scholarly journals Validation of a Multiplex Real-Time PCR Assay for Detection of Mycobacterium spp., Mycobacterium tuberculosis Complex, and Mycobacterium avium Complex Directly from Clinical Samples by Use of the BD Max Open System

2016 ◽  
Vol 54 (6) ◽  
pp. 1644-1647 ◽  
Author(s):  
Talita T. Rocchetti ◽  
Suzane Silbert ◽  
Alicia Gostnell ◽  
Carly Kubasek ◽  
Raymond Widen
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Real Time ◽  
Mycobacterium Avium ◽  
Open System ◽  
Real Time Pcr ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Content Type ◽  
Multiplex Pcr Assay ◽  
Mycobacterium Spp

A multiplex real-time PCR was validated on the BD Max open system to detect differentMycobacterium tuberculosiscomplex,Mycobacterium aviumcomplex, andMycobacteriumspp. directly from clinical samples. The PCR results were compared to those with traditional cultures. The multiplex PCR assay was found to be a specific and sensitive method for the rapid detection of mycobacteria directly from clinical specimens.

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

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.

scholarly journals Comparison and Recommendations for Use of Dientamoeba fragilis Real-Time PCR Assays

2019 ◽  
Vol 57 (5) ◽  
Author(s):  
Rory Gough ◽  
John Ellis ◽  
Damien Stark
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Cross Reactivity ◽  
Content Type ◽  
Prevalence Studies ◽  
Dientamoeba Fragilis ◽  
Stool Samples ◽  
Pcr Method ◽  
Genetic Signatures

ABSTRACT Dientamoeba fragilis is a gastrointestinal trichomonad parasite whose pathogenicity is yet to be determined. The difficulty involved in microscopically diagnosing D. fragilis in feces led to the development of real-time PCR methodologies for the detection of D. fragilis in stool samples. Prevalence studies in Europe show much higher levels of infection where a laboratory-developed real-time assay is the predominant assay for the detection of Dientamoeba fragilis than in regions that use the EasyScreen assay for detection of gastrointestinal pathogens. The aim of this study was to compare a commercially available Dientamoeba fragilis assay (Genetic Signatures EasyScreen assay) to a widely used laboratory-developed real-time PCR method. Two hundred fifty fecal samples were screened using the laboratory-developed real-time assay on four real-time PCR platforms producing a number of discrepant results. Limit-of-detection studies were undertaken to attempt to resolve sensitivity for each platform tested. The presence or absence of Dientamoeba fragilis DNA in discrepant samples was shown using PCR amplicon next-generation sequencing. Eukaryotic 18S diversity profiling was conducted on discrepant samples to identify the presence or absence of additional protozoan species in samples that may be responsible for cross-reactivity seen in these samples. The results revealed the potential for multiple false-positive results when using the laboratory-developed real-time assay across multiple real-time platforms using manufacturer default settings. This report provides recommendations to resolve these issues where possible and suggestions for future prevalence studies, and it emphasizes the EasyScreen assay as the molecular method of choice as well as the need for standardization of detection assays across all nations screening for D. fragilis.

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