scholarly journals Development of the kit for diagnostics of COVID-19 by real time RT-PCR

2020 ◽  
Author(s):  
AK Shuryaeva ◽  
TV Malova ◽  
EE Davydova ◽  
YuA Savochkina ◽  
EV Bogoslovskaya ◽  
...  
Keyword(s):  
Real Time ◽  
Diagnostic System ◽  
High Specificity ◽  
Test System ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
The Russian Federation ◽  
Diagnostic Sensitivity And Specificity ◽  
Respiratory Coronavirus ◽  
The City

Late in December 2019, an outbreak of an unknown coronavirus, later identified as SARS-CoV- 2, emerged in the city of Wuhan, China. It causes a dangerous respiratory coronavirus disease in humans - COVID-19. Objective. To detect cases of the disease and prevent its spread across the Russian Federation it is necessary to create an effective diagnostic test system. Material and methods. Based on the analysis of the alignment of the SARS-CoV-2 nucleotide sequences, primers and a probe for RT-PCR were selected, and the analysis conditions were optimized. Results. The diagnostic system was developed and registered in the shortest possible time in real-time RT-PCR format for detecting SARS-CoV-2 coronavirus RNA in smears from the nasopharynx and oropharynx, sputum and feces. The high specificity of the system was verified on a representative set of viruses and microorganisms, the analytical sensitivity was 1x103 copies / ml in smears from the mucous membrane of the nasopharynx and oropharynx and sputum, 5x104 copies / ml in fecal samples. Diagnostic sensitivity and specificity established during clinical trials on samples from patients with confirmed COVID-19 infection, from patients with a different etiology of a disease and clinically healthy people were to 100% (range 94.2-100% with a confidence level of 95%).

scholarly journals Comparison of SARS-CoV-2 detection in Saliva by real-time RT-PCR and RT-PCR/MALDI-TOF Methods

2021 ◽  
Author(s):  
Matthew M. Hernandez ◽  
Radhika Banu ◽  
Paras Shrestha ◽  
Armi Patel ◽  
Feng Chen ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
High Agreement ◽  
Diagnostic Assays ◽  
Specimen Collection ◽  
Diagnostic Sensitivity And Specificity ◽  
Analytical Sensitivities ◽  
Respiratory Specimens

The coronavirus disease 2019 (COVID-19) pandemic has accelerated the need for rapid implementation of diagnostic assays for detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in respiratory specimens. While multiple molecular methods utilize nasopharyngeal specimens, supply chain constraints and need for easier and safer specimen collection warrant alternative specimen types, particularly saliva. Although saliva has been found to be a comparable clinical matrix for detection of SARS-CoV-2, evaluations of diagnostic and analytic performance across platforms for this specimen type are limited. Here, we compared two methods for SARS-CoV-2 detection in saliva: the Roche cobas® 6800/8800 SARS-CoV-2 real-time RT-PCR Test and the Agena Biosciences MassARRAY® SARS-CoV-2 Panel/MassARRAY® System. Overall, both systems had high agreement with one another, and both demonstrated high diagnostic sensitivity and specificity when compared to matched patient upper respiratory specimens. We also evaluated the analytical sensitivity of each platform and determined the limit of detection of the Roche assay was four times lower than that of Agena for saliva specimens (390.6 v. 1,562.5 copies/mL). Furthermore, across individual target components of each assay, T2 and N2 targets had the lowest limits of detection for each platform, respectively. Together, we demonstrate that saliva represents an appropriate specimen for SARS-CoV-2 detection in two technologies that have high agreement and differ in analytical sensitivities overall and across individual component targets. The addition of saliva as an acceptable specimen and understanding the sensitivity for testing on these platforms can further inform public health measures for screening and detection to combat the COVID-19 pandemic.

531: Quantitative Real-Time RT-PCR for AMACR Distinguishes Prostate Cancer with High Specificity From Benign Lesions

2005 ◽  
Vol 173 (4S) ◽  
pp. 145-145 ◽  
Author(s):  
Martin Schostak ◽  
Hans Krause ◽  
Jens Köllermann ◽  
Mark Schrader ◽  
Bernd Straub ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Real Time ◽  
High Specificity ◽  
Rt Pcr ◽  
Benign Lesions

scholarly journals Development of a Diagnostic System for Novel Influenza A(H7N9) Virus Using a Real-Time RT-PCR Assay in Japan

2015 ◽  
Vol 68 (2) ◽  
pp. 113-118 ◽  
Author(s):  
Ikuyo Takayama ◽  
Hitoshi Takahashi ◽  
Mina Nakauchi ◽  
Shiho Nagata ◽  
Masato Tashiro ◽  
...  
Keyword(s):  
Real Time ◽  
Influenza A ◽  
Diagnostic System ◽  
H7n9 Virus ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Novel Influenza A

scholarly journals Validation of a Non-Specific Dye Real-Time PCR Assay for Porcine Adulteration in Meatball Using ND5 Primer

2017 ◽  
Vol 17 (2) ◽  
pp. 167 ◽  
Author(s):  
Tri Joko Raharjo ◽  
Ery Nourika Alfiraza ◽  
Esti Enjelina ◽  
Deni Pranowo
Keyword(s):  
Real Time ◽  
Annealing Temperature ◽  
Limit Of Detection ◽  
Melting Curve ◽  
High Specificity ◽  
Optimization Method ◽  
Amyl Alcohol ◽  
Melting Curve Analysis ◽  
Rt Pcr ◽  
Temperature Optimization

Porcine adulteration in meatball samples were analyzed using real-time polymerase chain reaction (RT-PCR), based on the ND5 primer obtained by previous study. This work consisted of three stages which were annealing temperature optimization, method validation, and application. DNA template was extracted using phenol-CIAA (chloroform-iso amyl alcohol) method. The optimum annealing temperature for ND5 primers (forward primer 5'-CATTCGCCTCACTCACATTAACC-3' and reverse primer 5'-AAGAGAGAGTTCTACGGTCTGTAG-3') was 58.0 °C, obtained after testing annealing at 50.5 to 59.5 °C gradient temperature with 5 °C interval. Melting curve analysis was done at 65.0 to 95.0 °C, with increasing temperature for 0.5 °C per 2 sec. Method was validated for its specificity, precision and limit of detection. RT-PCR method with ND5 primers produced 227 bp DNA fragment with 78.50 °C Tm value. From eight commercial meatball samples, one was detected containing porcine. The methods showed high specificity and precision, with experimentally determined limits for porcine were no less than 1%.

scholarly journals Simultaneous Detection of North American and European Porcine Reproductive and Respiratory Syndrome Virus Using Real-Time Quantitative Reverse Transcriptase–PCR

2005 ◽  
Vol 17 (2) ◽  
pp. 165-170 ◽  
Author(s):  
Steven B. Kleiboeker ◽  
Susan K. Schommer ◽  
Sang-Myeong Lee ◽  
Sandy Watkins ◽  
Wayne Chittick ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
North American ◽  
Simultaneous Detection ◽  
Viral Rna ◽  
Respiratory Syndrome Virus ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Field Isolates ◽  
Pcr Assays

Porcine reproductive and respiratory syndrome (PRRS) is 1 of the most economically important diseases of swine. Detection of the etiologic agent, PRRS virus (PRRSV), represents a diagnostic challenge due to the heterogeneity of field isolates as well as the propensity for swine to develop persistent infection in which virus is difficult to detect. Recently European (EU) lineage PRRSV isolates, which are genetically divergent from North American (NA) isolates, have been introduced into NA swine further complicating efforts to diagnose this disease. In this study, real-time ( TaqMan) reverse transcriptase (RT)–PCR assays were developed for multiplex detection, differentiation, and quantification of NA and EU PRRSV field isolates. Oligonucleotide primers and dual-labeled probes were selected from conserved regions of open-reading frame 7 and the 3'-untranslated region. The real-time RT-PCR assays described for the NA or EU genotype of PRRSV detected viral RNA from 83/83 strains (74 NA; 9 EU) previously isolated by cell culture between 1992 and 2003. The analytical sensitivity of both assays was consistently found to be less than a single TCID50, which corresponded to 5–10 RNA molecules, and was not significantly reduced when the reactions were performed in a multiplex format. When performing multiplex reactions, sensitive detection was possible even when 1 viral RNA concentration was up to 5,000-fold higher than the second. The diagnostic sensitivity and specificity of the multiplex reaction was found to be at a minimum equivalent to that of both nested RT-PCR and virus isolation.

scholarly journals Identification of Salmonella spp and serovars Typhimurium, Enteritidis by qPCR

2020 ◽  
pp. 21-31
Author(s):  
N. Rublenko
Keyword(s):  
Real Time ◽  
High Sensitivity ◽  
High Specificity ◽  
Analytical Sensitivity ◽  
Reaction Products ◽  
Salmonella Spp ◽  
Bacterial Dna ◽  
E Coli ◽  
Polymerase Chain ◽  
Primer Sets

This article presents the results of the identification of the Salmonella genus as well as serovars Enteritidis and Typhimurium by a real-time polymerase chain reaction. We constructed three pairs of primers and fluorescent probes to simultaneously identify the Salmonella genus, serovars Enteritidis and Typhimurium in a qPCR. The specificity of the primers was evaluated on Salmonella strains of different serovars from the National Center for Strains of Microorganisms (UNCMS) strains of the State Scientific Control Institute of Biotechnology and Strains of Microorganisms (SSCIBSM) and 46 Salmonella strains isolated from poultry. E. coli ATCC 25922, Bacillus cereus ATCC 11778, Listeria monocytogenes ATCC 19112 from UNCMS collection were used to check the specificity of the primers as heterologous samples. Bacterial DNA was extracted using a DNA Sorb B (Amplisens) kit, and realtime PCR was accomplished with the "Real-time PCR kit" (Syntol) on Bio-rad CFX. A series of 10-fold S. Typhimurium and S. Enteritidis DNA dilutions were studied to evaluate the sensitivity of the primers: 10-1-10-5. The analytical sensitivity of primers for detection of the genus Salmonella is: for S. Typhimurium - 0.25 ng/sample (Typhimurium) and S. Enteritidis - 0.27 ng/ sample (Enteritidis). The results of the studies confirmed the specificity of the primer set and the high sensitivity. No hybridization of primers with DNA samples of other bacteria found, in particular, the nonspecific reaction products were absent. The primer sets for the detection of DNA of Enteritidis and Typhimurium serovars also has high specificity. If necessary, this set of primers can be used to perform a multiplex qPCR, that can simultaneously identify bacteria of the Salmonella genus and differentiate Enteritidis and Typhimurium serovars. Keywords: Salmonella, bacteria, polymerasechainreaction, DNA, qPCR.

ASSESSMENT OF THE POSSIBILITY OF APPLICATION IN LABORATORY PRACTICE OF REAGENT KIT FOR DIAGNOSIS OF GLANDERS AND MELIOIDOSIS BY REAL-TIME POLYMERASE CHAIN REACTION

2019 ◽  
Vol 64 (11) ◽  
pp. 700-704
Author(s):  
L. V. Lemasova ◽  
G. A. Tkachenko ◽  
E. V. Prokhvatilova ◽  
L. I. Belitskaya ◽  
D. V. Viktorov ◽  
...  
Keyword(s):  
Real Time ◽  
Diagnostic Value ◽  
Medical Laboratory ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Laboratory Practice ◽  
Environmental Objects ◽  
Polymerase Chain ◽  
State Examination

The reagent kit AmpligenBurk-mallei/pseudomallei-RT PCR is designed for detecting in vitro diagnostics and differentiate the DNA of glanders and melioidosis pathogens by real-time multiplex PCR in biological (clinical) material and cultures of microorganisms, as well as environmental objects and solid food products (rice). During clinical testing diagnostic value of reagent kit AmpligenBurk-mallei/pseudomallei-RT PCR has been studied. Based on the results obtained, a high analytical sensitivity (1×103 microbe cells/ml) and specificity (100%) of PCR-RT with the developed reagent kit were established, regardless of the type of material being studied. The diagnostic sensitivity of PCR-RT using a set of reagents was at least 98.0% and specificity at least 99%. The stages of state examination have been completed, a registration certificate has been obtained at Roszdravnadzor, production, sale and use of reagent kit in medical laboratory practice have been permitted.

scholarly journals Detection and Characterization of Diphtheria Toxin Gene-Bearing Corynebacterium Species through a New Real-Time PCR Assay

2020 ◽  
Vol 58 (10) ◽  
Author(s):  
Margaret M. Williams ◽  
Jessica L. Waller ◽  
Janessa S. Aneke ◽  
Michael R. Weigand ◽  
Maureen H. Diaz ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Toxin Production ◽  
Toxin Gene ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Culture Methods ◽  
Content Type ◽  
Corynebacterium Ulcerans

ABSTRACT Respiratory diphtheria, characterized by a firmly adherent pseudomembrane, is caused by toxin-producing strains of Corynebacterium diphtheriae, with similar illness produced occasionally by toxigenic Corynebacterium ulcerans or, rarely, Corynebacterium pseudotuberculosis. While diphtheria laboratory confirmation requires culture methods to determine toxigenicity, real-time PCR (RT-PCR) provides a faster method to detect the toxin gene (tox). Nontoxigenic tox-bearing (NTTB) Corynebacterium isolates have been described, but impact of these isolates on the accuracy of molecular diagnostics is not well characterized. Here, we describe a new triplex RT-PCR assay to detect tox and distinguish C. diphtheriae from the closely related species C. ulcerans and C. pseudotuberculosis. Analytical sensitivity and specificity of the assay were assessed in comparison to culture using 690 previously characterized microbial isolates. The new triplex assay characterized Corynebacterium isolates accurately, with 100% analytical sensitivity for all targets. Analytical specificity with isolates was 94.1%, 100%, and 99.5% for tox, Diph_rpoB, and CUP_rpoB targets, respectively. Twenty-nine NTTB Corynebacterium isolates, representing 5.9% of 494 nontoxigenic isolates tested, were detected by RT-PCR. Whole-genome sequencing of NTTB isolates revealed varied mutations putatively underlying their lack of toxin production, as well as eight isolates with no mutation in tox or the promoter region. This new Corynebacterium RT-PCR method provides a rapid tool to screen isolates and identify probable diphtheria cases directly from specimens. However, the sporadic occurrence of NTTB isolates reinforces the viewpoint that diphtheria culture diagnostics continue to provide the most accurate case confirmation.

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