Pre-Clinical Evaluation of a Real-Time PCR Assay on a Portable Instrument as a Possible Field Diagnostic Tool: Experiences from the Testing of Clinical Samples for African and Classical Swine Fever Viruses

2016 ◽  
Vol 64 (5) ◽  
pp. e31-e35 ◽  
Author(s):  
L. Liu ◽  
Y. Luo ◽  
F. Accensi ◽  
L. Ganges ◽  
F. Rodríguez ◽  
...  
Keyword(s):  
Real Time ◽  
Clinical Evaluation ◽  
Diagnostic Tool ◽  
Real Time Pcr ◽  
Classical Swine Fever ◽  
Clinical Samples ◽  
Portable Instrument ◽  
Pcr Assay

scholarly journals Clinical Evaluation of a Type III Secretion System Real-Time PCR Assay for Diagnosing Melioidosis

2006 ◽  
Vol 44 (8) ◽  
pp. 3028-3030 ◽  
Author(s):  
E. M. Meumann ◽  
R. T. Novak ◽  
D. Gal ◽  
M. E. Kaestli ◽  
M. Mayo ◽  
...  
Keyword(s):  
Real Time ◽  
Clinical Evaluation ◽  
Real Time Pcr ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Pcr Assay ◽  
Type Iii

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

2018 ◽  
Vol 56 (7) ◽  
pp. 1133-1139 ◽  
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.

scholarly journals A Real-Time PCR Assay for Simultaneous Detection and Differentiation of Four Common Entamoeba Species That Infect Humans

2020 ◽  
Vol 59 (1) ◽  
pp. e01986-20
Author(s):  
Ibne Karim M. Ali ◽  
Shantanu Roy
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Small Subunit ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Content Type ◽  
Rdna Sequences ◽  
Appropriate Treatment

ABSTRACTThere are over 40 species within the genus Entamoeba, eight of which infect humans. Of these, four species (Entamoeba histolytica, E. dispar, E. moshkovskii, and E. bangladeshi) are morphologically indistinguishable from each other, and yet differentiation is important for appropriate treatment decisions. Here, we developed a hydrolysis probe-based tetraplex real-time PCR assay that can simultaneously detect and differentiate these four species in clinical samples. In this assay, multicopy small-subunit (SSU) ribosomal DNA (rDNA) sequences were used as targets. We determined that the tetraplex real-time PCR can detect amebic DNA corresponding to as little as a 0.1 trophozoite equivalent of any of these species. We also determined that this assay can detect E. histolytica DNA in the presence of 10-fold more DNA from another Entamoeba species in mixed-infection scenarios. With a panel of more than 100 well-characterized clinical samples diagnosed and confirmed using a previously published duplex real-time PCR (capable of detecting E. histolytica and E. dispar), our tetraplex real-time PCR assay demonstrated levels of sensitivity and specificity comparable with those demonstrated by the duplex real-time PCR assay. The advantage of our assay over the duplex assay is that it can specifically detect two additional Entamoeba species and can be used in conventional PCR format. This newly developed assay will allow further characterization of the epidemiology and pathogenicity of the four morphologically identical Entamoeba species, especially in low-resource settings.

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.

Development of a LUX real-time PCR for the detection and quantification of human herpesvirus 7

2009 ◽  
Vol 55 (3) ◽  
pp. 319-325 ◽  
Author(s):  
Massimiliano Bergallo ◽  
Cristina Costa ◽  
Maria Elena Terlizzi ◽  
Francesca Sidoti ◽  
Samuela Margio ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Primary Infection ◽  
Latent Infection ◽  
Dynamic Range ◽  
Human Herpesvirus ◽  
Clinical Samples ◽  
House Light ◽  
Pcr Assay ◽  
Sensitivity Specificity

Human herpesvirus 7 is a highly seroprevalent β-herpesvirus that, following primary infection, remains latent in CD4+ T cells and determines a persistent rather than a latent infection in various tissues and organs, including the lung and skin. This paper describes the development of an in-house light upon extension real-time PCR assay for quantification of human herpesvirus 7 DNA in clinical samples. The efficiency, sensitivity, specificity, inter- and intra-assay variability, and dynamic range have been determined. Subsequently, the assay has been validated by evaluating the human herpesvirus 7 load in bronchoalveolar lavages and skin specimens, chosen as 2 persistency sites, from healthy and pathological individuals. The real-time PCR assay developed in this study could be a useful tool to detect and quantify human herpesvirus 7 DNA in different clinical specimens to elucidate its epidemiological and pathogenic roles.

scholarly journals Toward point-of-care diagnostics of Candida auris

2020 ◽  
Author(s):  
Geoffrey Mulberry ◽  
Sudha Chaturvedi ◽  
Vishnu Chaturvedi ◽  
Brian N. Kim
Keyword(s):  
New York ◽  
Real Time ◽  
Real Time Pcr ◽  
Positive Impact ◽  
Point Of Care ◽  
Low Cost ◽  
High Sensitivity ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Candida Auris

AbstractCandida auris is a multidrug-resistant yeast that presents global health threat for the hospitalized patients. Early diagnostic of C. auris is crucial in control, prevention, and treatment. Candida auris is difficult to identify with standard laboratory methods and often can be misidentified leading to inappropriate management. A newly-devised real-time PCR assay played an important role in the ongoing investigation of the C. auris outbreak in New York metropolitan area. The assay can rapidly detect C. auris DNA in surveillance and clinical samples with high sensitivity and specificity, and also useful for confirmation of C. auris cultures. Despite its positive impact, the real-time PCR assay is difficult to deploy at frontline laboratories due to high-complexity set-up and operation. Using a low-cost handheld real-time PCR device, we show that the C. auris can potentially be identified in a low-complexity assay without the need for high-cost equipment. An implementation of low-cost real-time PCR device in hospitals and healthcare facilities is likely to accelerate the diagnosis of C. auris and for control of the global epidemic.

scholarly journals Detection of Fusarium Species in Clinical Specimens by Probe-Based Real-Time PCR

2019 ◽  
Vol 5 (4) ◽  
pp. 105
Author(s):  
Jan Springer ◽  
Grit Walther ◽  
Volker Rickerts ◽  
Axel Hamprecht ◽  
Birgit Willinger ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Contact Lenses ◽  
Fusarium Species ◽  
Invasive Fungal Disease ◽  
Clinical Samples ◽  
Fusarium Spp ◽  
Pcr Assay ◽  
Clinical Specimens ◽  
Two Samples

The mold Fusarium is a ubiquitous fungus causing plant, animal and human infections. In humans, Fusarium spp. are the major cause of eye infections in patients wearing contact lenses or after local trauma. Systemic infections by Fusarium spp. mainly occur in immunosuppressed patients and can disseminate throughout the human body. Due to high levels of resistance to antifungals a fast identification of the causative agent is an urgent need. By using a probe-based real-time PCR assay specific for the genus Fusarium we analysed several different clinical specimens detecting Fusarium spp. commonly found in clinical samples in Germany. Also, a large collection of lung fluid samples of haematological patients was analysed (n = 243). In these, two samples (0.8%) were reproducibly positive, but only one could be confirmed by sequencing. For this case of probable invasive fungal disease (IFD) culture was positive for Fusarium species. Here we describe a rapid, probe-based real-time PCR assay to specifically detect DNA from a broad range of Fusarium species and its application to clinically relevant specimens.

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