scholarly journals High-throughput multiplex real-time PCR assay for the simultaneous quantification of DNA and RNA viruses infecting cassava plants

2016 ◽  
Vol 120 (5) ◽  
pp. 1346-1356 ◽  
Author(s):  
G. Otti ◽  
S. Bouvaine ◽  
B. Kimata ◽  
G. Mkamillo ◽  
P.L. Kumar ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Rna Viruses ◽  
Pcr Assay ◽  
Simultaneous Quantification ◽  
Dna And Rna

scholarly journals Development of a High-Throughput Quantitative Assay for Detecting Herpes Simplex Virus DNA in Clinical Samples

1999 ◽  
Vol 37 (6) ◽  
pp. 1941-1947 ◽  
Author(s):  
Alexander J. Ryncarz ◽  
James Goddard ◽  
Anna Wald ◽  
Meei-Li Huang ◽  
Bernard Roizman ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Genital Tract ◽  
Clinical Samples ◽  
Pcr Assay ◽  
The Mean ◽  
Simplex Virus

We have developed a high-throughput, semiautomated, quantitative fluorescence-based PCR assay to detect and type herpes simplex virus (HSV) DNA in clinical samples. The detection assay, which uses primers to the type-common region of HSV glycoprotein B (gB), was linear from <10 to 108 copies of HSV DNA/20 μl of sample. Among duplicate samples in reproducibility runs, the assay showed less than 5% variability. We compared the fluorescence-based PCR assay with culture and gel-based liquid hybridization system with 335 genital tract specimens from HSV type 2 (HSV-2)-seropositive persons attending a research clinic and 380 consecutive cerebrospinal fluid (CSF) samples submitted to a diagnostic virology laboratory. Among the 162 culture-positive genital tract specimens, TaqMan PCR was positive for 157 (97%) specimens, whereas the quantitative-competitive PCR was positive for 144 (89%) specimens. Comparisons of the mean titer of HSV DNA detected by the two assays revealed that the mean titer detected by the gel-based system was slightly higher (median, 1 log). These differences in titers were in part related to the fivefold difference in the amount of HSV DNA used in the amplicon standards with the two assays. Among the 380 CSF samples, 42 were positive by both assays, 13 were positive only by the assay with the agarose gel, and 3 were positive only by the assay with the fluorescent probe. To define the subtype of HSV DNA detected in the screening assay, we also designed one set of primers which amplifies the gG regions of both types of HSV and probes which are specific to either HSV-1 (gG1) or HSV-2 (gG2). These probes were labeled with different fluorescent dyes (6-carboxyfluorescein for gG2 and 6-hexachlorofluorescein for gG1) to enable detection in a single PCR. In mixing experiments the probes discriminated the correct subtype in mixtures with up to a 7-log-higher concentration of the opposite subtype. The PCR typing results showed 100% concordance with the results obtained by assays with monoclonal antibodies against HSV-1 or HSV-2. Thus, while the real-time PCR is slightly less sensitive than the gel-based liquid hybridization system, the high throughput, the lack of contamination during processing, the better reproducibility, and the better ability to type the isolates rapidly make the real-time PCR a valuable tool for clinical investigation and diagnosis of HSV infection.

High-throughput two-step LNA real time PCR assay for the quantitative detection and genotyping of HPV prognostic-risk groups

2009 ◽  
Vol 45 (4) ◽  
pp. 304-310 ◽  
Author(s):  
Elisa Leo ◽  
Simona Venturoli ◽  
Monica Cricca ◽  
Monica Musiani ◽  
Marialuisa Zerbini
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Risk Groups ◽  
Quantitative Detection ◽  
Pcr Assay

A high-throughput microfluidic biochip to quantify bacterial adhesion to single host cells by real-time PCR assay

2013 ◽  
Vol 405 (12) ◽  
pp. 4277-4282 ◽  
Author(s):  
Rui Zhang ◽  
Hai-Qing Gong ◽  
Xu Dong Zeng ◽  
Chun Chau Sze
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Bacterial Adhesion ◽  
Host Cells ◽  
Pcr Assay ◽  
Single Host

Developmental validation of a real-time PCR assay for the simultaneous quantification of total human and male DNA

2008 ◽  
Vol 3 (1) ◽  
pp. 14-21 ◽  
Author(s):  
Benjamin E. Krenke ◽  
Nadine Nassif ◽  
Cynthia J. Sprecher ◽  
Curtis Knox ◽  
Melissa Schwandt ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Assay ◽  
Simultaneous Quantification ◽  
Developmental Validation

scholarly journals Quality control implementation for universal characterization of DNA and RNA viruses in clinical respiratory samples using single metagenomic next-generation sequencing workflow

2018 ◽  
Author(s):  
A. Bal ◽  
M. Pichon ◽  
C. Picard ◽  
JS. Casalegno ◽  
M. Valette ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Real Time ◽  
Real Time Pcr ◽  
Viral Genome ◽  
Viral Infections ◽  
Rna Viruses ◽  
Next Generation ◽  
Dna And Rna ◽  
Generation Sequencing

AbstractBackgroundIn recent years, metagenomic Next-Generation Sequencing (mNGS) has increasingly been used for an accurate assumption-free virological diagnosis. However, the systematic workflow evaluation on clinical respiratory samples and implementation of quality controls (QCs) is still lacking.MethodsA total of 3 QCs were implemented and processed through the whole mNGS workflow: a notemplate-control to evaluate contamination issues during the process; an internal and an external QC to check the integrity of the reagents, equipment, the presence of inhibitors, and to allow the validation of results for each sample. The workflow was then evaluated on 37 clinical respiratory samples from patients with acute respiratory infections previously tested for a broad panel of viruses using semi-quantitative real-time PCR assays (28 positive samples including 6 multiple viral infections; 9 negative samples). Selected specimens included nasopharyngeal swabs (n = 20), aspirates (n = 10), or sputums (n = 7).ResultsThe optimal spiking level of the internal QC was first determined in order to be sufficiently detected without overconsumption of sequencing reads. According to QC validation criteria, mNGS results were validated for 34/37 selected samples. For valid samples, viral genotypes were accurately determined for 36/36 viruses detected with PCR (viral genome coverage ranged from 0.6% to 100%, median = 67.7%). This mNGS workflow allowed the detection of DNA and RNA viruses up to a semi-quantitative PCR Ct value of 36. The six multiple viral infections involving 2 to 4 viruses were also fully characterized. A strong correlation between results of mNGS and real-time PCR was obtained for each type of viral genome (R2 ranged from 0.72 for linear single-stranded (ss) RNA viruses to 0.98 for linear ssDNA viruses).ConclusionsAlthough the potential of mNGS technology is very promising, further evaluation studies are urgently needed for its routine clinical use within a reasonable timeframe. The approach described herein is crucial to bring standardization and to ensure the quality of the generated sequences in clinical setting. We provide an easy-to-use single protocol successfully evaluated for the characterization of a broad and representative panel of DNA and RNA respiratory viruses in various types of clinical samples.

scholarly journals qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

10.3791/52941 ◽  
2015 ◽  
Author(s):  
Leslie A. Mitchell ◽  
Nick A. Phillips ◽  
Andrea Lafont ◽  
James A. Martin ◽  
Rupal Cutting ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Pcr Assay

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.

Real-time PCR assay for the simultaneous quantification of nitrifying and denitrifying bacteria in activated sludge

2007 ◽  
Vol 75 (1) ◽  
pp. 211-221 ◽  
Author(s):  
Joke Geets ◽  
Michaël de Cooman ◽  
Lieven Wittebolle ◽  
Kim Heylen ◽  
Bram Vanparys ◽  
...  
Keyword(s):  
Activated Sludge ◽  
Real Time ◽  
Real Time Pcr ◽  
Denitrifying Bacteria ◽  
Pcr Assay ◽  
Simultaneous Quantification
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