scholarly journals Comparison of nine different commercially available molecular assays for detection of SARS-CoV-2 RNA

2021 ◽  
Author(s):  
Ute Eberle ◽  
◽  
Clara Wimmer ◽  
Ingrid Huber ◽  
Antonie Neubauer-Juric ◽  
...  
Keyword(s):  
Real Time ◽  
Rt Pcr ◽  
Diagnostic Assays ◽  
Molecular Assays ◽  
Pcr Assays ◽  
Laboratory Experiences

AbstractTo face the COVID-19 pandemic, the need for fast and reliable diagnostic assays for the detection of SARS-CoV-2 is immense. We describe our laboratory experiences evaluating nine commercially available real-time RT-PCR assays. We found that assays differed considerably in performance and validation before routine use is mandatory.

scholarly journals Mise au point de nouvelles techniques de diagnostic pour les maladies à orbivirus chez les animaux domestiques et sauvages

2009 ◽  
Vol 62 (2-4) ◽  
pp. 152
Author(s):  
S. Maan ◽  
N. Maan ◽  
K. Bankowska ◽  
A. Potgieter ◽  
K. Nomikou ◽  
...  
Keyword(s):  
Real Time ◽  
Genome Segment ◽  
Structural Proteins ◽  
Virus Species ◽  
Cross Contamination ◽  
Rt Pcr ◽  
Diagnostic Assays ◽  
Virus Core ◽  
Pcr Assays ◽  
Species Specific

The recent emergence and spread of bluetongue virus (BTV) across the whole of Europe suggests that other orbiviruses could also emerge to threaten livestock species and wildlife popula­tions in Europe and other parts of the world. The genus Orbivirus is the largest within the family Reoviridae, containing 22 virus species, as well as 14 unclassified orbiviruses. The orbiviruses are transmitted primarily by arthropod vectors (e.g. Culicoides, mosquitoes or ticks) and several are associated with severe and economically important diseases of livestock, including BTV in cattle and sheep, African horse sickness virus (AHSV), equine encephalosis virus (EEV) and Peruvian horse sickness virus (PHSV) in equids, as well as epizootic haemorrhagic disease virus (EHDV) in wild ungulates or cattle.  Recent incursions of BTV in Europe, Southeastern USA, Australia and Asia, EHDV in North Africa, the Middle East and the Mediterranean region, AHSV in sub-Saharan Africa, and EEV in Israel and Gambia, indicate a need for the development of faster, more sensitive and more reliable diagnostic assays. These are required to detect and identify rapidly the viruses and virus types involved, monitor their incidence and movement, and identify infected animals. The Orbivirus genome is composed of 10 linear segments of double-stranded ribonucleic acid (dsRNA), each segment coding for at least one viral protein. The outer capsid proteins VP2 and VP5 are situated on or near the surface of the virus particle and are more variable than components of the virus core, or the non-structural proteins. VP2 (encoded by Seg- 2) is the outermost of the BTV capsid proteins and represents the primary target antigen for neutralising antibodies, and hence Seg-2 is a target for the development of type-specific nucleic-acid-based diagnostic assays. In contrast, the genome segments coding for protein components of the virus core and/or the non-structural proteins can be used as targets for development of serogroup (virus-species) specific, reverse transcription - poly­merase chain reaction (RT-PCR) based diagnostic assays.  Virus species-specific and type-specific conventional (gel based) RT-PCR diagnostic assays, for the detection, identification and typing of some of these viruses (BTV, EHDV and AHSV), have been developed using the sequence data for segments 7 and 2, respectively. Initial evaluation studies indicate that these assays are reliable, specific, do not cross-react with related orbiviruses (group/species specific) or with related types (type specific). Although they are labour intensive, the results obtained can be confirmed by sequence analyses of the resulting complemen­tary deoxyribonucleic acid complementary (c) DNA amplicons, and phylogenetic comparisons to determine the strain of virus involved. However, conventional RT-PCR assays are prone to cross-contamination, potentially leading to false positive results.  The authors also describe group-specific real-time RT-PCR assays that use a ‘closed-tube’ format, which are therefore less suscep­tible to cross-contamination. These assays target the conserved genome segment 9, or genome segment 1, which can be used to detect all of the serotypes, as well as geographic variants (different topotypes) of BTV, EHDV, AHSV, EEV and PHSV. Type-specific real-time RT-PCR assays that target the most variable genome segment 2 can be used to differentiate 25 serotypes of BTV or the seven serotypes of EHDV. These diagnostic assays were found to be very sensitive, reproducible and suitable for rapid screening of field samples. Results will be presented from studies to optimise these RT-PCR assays.

scholarly journals Quantitation of replication of the HCV genome in human livers with end-stage cirrhosis by strand-specific real-time RT-PCR assays: Methods and clinical relevance

2009 ◽  
Vol 81 (9) ◽  
pp. 1569-1575 ◽  
Author(s):  
Lan Lin ◽  
Louis Libbrecht ◽  
Jannick Verbeeck ◽  
Chris Verslype ◽  
Tania Roskams ◽  
...  
Keyword(s):  
Real Time ◽  
Clinical Relevance ◽  
Rt Pcr ◽  
Pcr Assays ◽  
Human Livers ◽  
End Stage

Validation and comparison of different end point and real time RT-PCR assays for detection and genotyping of porcine reproductive and respiratory syndrome virus

2014 ◽  
Vol 201 ◽  
pp. 79-85 ◽  
Author(s):  
Michele Drigo ◽  
Giovanni Franzo ◽  
Ilaria Belfanti ◽  
Marco Martini ◽  
Alessandra Mondin ◽  
...  
Keyword(s):  
Real Time ◽  
Respiratory Syndrome Virus ◽  
Rt Pcr ◽  
End Point ◽  
Pcr Assays

scholarly journals Development of Reverse Transcription (RT)-PCR and Real-Time RT-PCR Assays for Rapid Detection and Quantification of Viable Yeasts and Molds Contaminating Yogurts and Pasteurized Food Products

2003 ◽  
Vol 69 (7) ◽  
pp. 4116-4122 ◽  
Author(s):  
Gianluca Bleve ◽  
Lucia Rizzotti ◽  
Franco Dellaglio ◽  
Sandra Torriani
Keyword(s):  
Real Time ◽  
Food Products ◽  
Fungal Species ◽  
Universal Primers ◽  
Rt Pcr ◽  
Specificity And Sensitivity ◽  
Actin Mrna ◽  
Pcr Assays ◽  
Food Commodities ◽  
Yeasts And Molds

ABSTRACT Reverse transcriptase PCR (RT-PCR) and real-time RT-PCR assays have been used to detect and quantify actin mRNA from yeasts and molds. Universal primers were designed based on the available fungal actin sequences, and by RT-PCR they amplified a specific 353-bp fragment from fungal species involved in food spoilage. From experiments on heat-treated cells, actin mRNA was a good indicator of cell viability: viable cells and cells in a nonculturable state were detected, while no signal was observed from dead cells. The optimized RT-PCR assay was able to detect 10 CFU of fungi ml−1 in pure culture and 103 and 102 CFU ml−1 in artificially contaminated yogurts and pasteurized fruit-derived products, respectively. Real-time RT-PCR, performed on a range of spoiled commercial food products, validated the suitability of actin mRNA detection for the quantification of naturally contaminating fungi. The specificity and sensitivity of the procedure, combined with its speed, its reliability, and the potential automation of the technique, offer several advantages to routine analysis programs that assess the presence and viability of fungi in food commodities.

scholarly journals Accurate and precise real-time RT-PCR assays for the identification of astrovirus associated encephalitis in cattle

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Ramona Lüthi ◽  
Céline L. Boujon ◽  
Ronja Kauer ◽  
Michel C. Koch ◽  
Ilias G. Bouzalas ◽  
...  
Keyword(s):  
Real Time ◽  
Rt Pcr ◽  
Pcr Assays

scholarly journals A New Highly Sensitive and Specific Real-Time PCR Assay Targeting the Malate Dehydrogenase Gene ofKingella kingaeand Application to 201 Pediatric Clinical Specimens

2018 ◽  
Vol 56 (8) ◽  
Author(s):  
Nawal El Houmami ◽  
Guillaume André Durand ◽  
Janek Bzdrenga ◽  
Anne Darmon ◽  
Philippe Minodier ◽  
...  
Keyword(s):  
Real Time ◽  
Malate Dehydrogenase ◽  
Real Time Pcr ◽  
Detection Sensitivity ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Content Type ◽  
Clinical Specimens ◽  
Specificity And Sensitivity ◽  
Pcr Assays

ABSTRACTKingella kingaeis a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium using culture and broad-range 16S rRNA gene PCR assays from clinical specimens have proven unsatisfactory; therefore, by the late 2000s, these were gradually phased out to explore the benefits of specific real-time PCR tests targeting thegroELgene and the RTX locus ofK. kingae. However, recent studies showed that real-time PCR (RT-PCR) assays targeting theKingellasp. RTX locus that are currently available for the diagnosis ofK. kingaeinfection lack specificity because they could not distinguish betweenK. kingaeand the recently describedKingella negevensisspecies. Furthermore,in silicoanalysis of thegroELgene from a large collection of 45K. kingaestrains showed that primers and probes fromK. kingaegroEL-based RT-PCR assays display a few mismatches withK. kingae groELvariations that may result in decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative togroEL- and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, aK. kingae-specific RT-PCR assay targeting the malate dehydrogenase (mdh) gene was developed for predicting no mismatch between primers and probe and 18 variants of theK. kingae mdhgene from 20 distinct sequence types ofK. kingae. This novelK. kingae-specific RT-PCR assay demonstrated high specificity and sensitivity and was successfully used to diagnoseK. kingaeinfections and carriage in 104 clinical specimens from children between 7 months and 7 years old.

scholarly journals Development and Evaluation of Novel and Highly Sensitive Single-Tube Nested Real-Time RT-PCR Assays for SARS-CoV-2 Detection

2020 ◽  
Vol 21 (16) ◽  
pp. 5674
Author(s):  
Cyril Chik-Yan Yip ◽  
Siddharth Sridhar ◽  
Kit-Hang Leung ◽  
Anthony Chin-Ki Ng ◽  
Kwok-Hung Chan ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Rt Pcr ◽  
Single Tube ◽  
Highly Sensitive ◽  
Testing Algorithms ◽  
Pcr Assays ◽  
Human Coronaviruses ◽  
Pooled Sample

Sensitive molecular assays are critical for coronavirus disease 2019 (COVID-19) diagnosis. Here, we designed and evaluated two single-tube nested (STN) real-time RT-PCR assays, targeting SARS-CoV-2 RdRp/Hel and N genes. Both STN assays had a low limit of detection and did not cross react with other human coronaviruses and respiratory viruses. Using 213 initial respiratory specimens from suspected COVID-19 patients, the sensitivity of both the STN COVID-19-RdRp/Hel and the STN COVID-19-N assays was 100% (99/99), while that of the comparator non-nested N assay was 95% (94/99). Among 108 follow-up specimens from confirmed COVID-19 patients who tested negative by the non-nested COVID-19-RdRp/Hel assay, 28 (25.9%) were positive for SARS-CoV-2 by the STN COVID-19-RdRp/Hel or the STN COVID-19-N assay. To evaluate the performance of our novel STN assays in pooled specimens, we created four sample pools, with each pool consisting of one low positive specimen and 49 negative specimens. While the non-nested COVID-19-RdRp/Hel assay was positive in only one of four sample pools (25%), both of the STN assays were positive in two of four samples pools (50%). In conclusion, the STN assays are highly sensitive and specific for SARS-CoV-2 detection. Their boosted sensitivity offers advantages in non-traditional COVID-19 testing algorithms such as saliva screening and pooled sample screening during massive screening.

Development of real-time and conventional RT-PCR assays for the detection of potato yellow vein virus (PYVV)

2006 ◽  
Vol 136 (1-2) ◽  
pp. 24-29 ◽  
Author(s):  
R. López ◽  
C. Asensio ◽  
M.M. Guzman ◽  
N. Boonham
Keyword(s):  
Real Time ◽  
Yellow Vein ◽  
Rt Pcr ◽  
Pcr Assays ◽  
Potato Yellow Vein Virus
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