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.

scholarly journals Development and Evaluation of Novel Real-Time Reverse Transcription-PCR Assays with Locked Nucleic Acid Probes Targeting Leader Sequences of Human-Pathogenic Coronaviruses

2015 ◽  
Vol 53 (8) ◽  
pp. 2722-2726 ◽  
Author(s):  
Jasper Fuk-Woo Chan ◽  
Garnet Kwan-Yue Choi ◽  
Alan Ka-Lun Tsang ◽  
Kah-Meng Tee ◽  
Ho-Yin Lam ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Reverse Transcription ◽  
Locked Nucleic Acid ◽  
Small Rna Sequencing ◽  
Nucleic Acid Probes ◽  
Reverse Transcription Pcr ◽  
Highly Sensitive ◽  
Pcr Assays ◽  
Human Coronaviruses

Based on findings in small RNA-sequencing (Seq) data analysis, we developed highly sensitive and specific real-time reverse transcription (RT)-PCR assays with locked nucleic acid probes targeting the abundantly expressed leader sequences of Middle East respiratory syndrome coronavirus (MERS-CoV) and other human coronaviruses. Analytical and clinical evaluations showed their noninferiority to a commercial multiplex PCR test for the detection of these coronaviruses.

scholarly journals Detection of Sesame Allergen Traces with Two PCR Assays - The Challenge to Protect Food-Allergic Consumers

2015 ◽  
Vol 3 (4) ◽  
pp. 210
Author(s):  
Dimitra Panagiotis Houhoula ◽  
Vasilios Belsis ◽  
Leonidas Georgopoulos ◽  
Virginia Giannou ◽  
Vasiliki R. Kyrana ◽  
...  
Keyword(s):  
Food Allergy ◽  
Real Time ◽  
Real Time Pcr ◽  
Food Industry ◽  
Food Samples ◽  
Rt Pcr ◽  
Highly Sensitive ◽  
Pcr Method ◽  
Pcr Assays ◽  
Positive Results

The purpose of this study was to investigate the possible presence of sesame in commercial foods normally carrying no warning for the allergen, but which may have been subjected to contamination during processing. One hundred units of widely consumed goods with high potential to contain allergenic substances deriving from nuts were analyzed, using sensitive and capable PCR (C-PCR) and Real Time PCR (RT-PCR) methodologies. Of the products examined, 15 (15.0%) declared the presence of sesame, 36 (36.0%) carried no food allergy label, 44 (44.0%) were marked by the phrase “may contain traces of nuts” and 5 (5.0%) carried the indication “may contain sesame traces”. The sesame-positive products detected using the C-PCR method were 15 (100%), 12 (33.3%), 14 (31.8%) and 3 (60%), respectively. Using the RT-PCR technique, positive results were obtained for 15 (100%), 18 (50.0%), 18 (20.5%) and 5 (100%) samples, respectively. The results indicate that the PCR methods applied are highly sensitive and selective, which makes them suitable for the detection of sesame traces in food samples. In addition, they can be useful for monitoring the effectiveness of cleaning processes in the production units of the food industry.

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 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

Performance Comparison of Multiplexed Fluorescent Resonance Emission Transfer Hybridization Probes Across Roche LightCycler® Real-Time PCR Systems for the Detection of Bartonella species

2021 ◽  
Vol 156 (Supplement_1) ◽  
pp. S134-S135
Author(s):  
T Berent ◽  
T Rothstein ◽  
S Buckwalter ◽  
R Patel
Keyword(s):  
Real Time ◽  
Whole Blood ◽  
New Technologies ◽  
Limit Of Detection ◽  
Rt Pcr ◽  
Bartonella Species ◽  
Pcr Assay ◽  
Fixed Tissue ◽  
Hybridization Probes ◽  
Ffpe Samples

Abstract Introduction/Objective Molecular assays for Bartonella species are important in diagnosing infection and expediting patient treatment. Real time polymerase chain reaction (RT-PCR) using fluorescent resonance energy transfer (FRET) hybridization probes can be used to detect Bartonella species in blood and fresh/fixed tissue biopsies in RT-PCR instruments. Over time, new technologies and reagents are introduced and existing PCR primers and FRET probes must be re-validated on new platforms. This study aimed to compare the performance of a Bartonella RT-PCR assay using the sunsetting Roche LightCycler® 2.0 (Roche Diagnostics, Indianapolis, IN) and newer LightCycler® 480 RT- PCR instruments. Methods/Case Report DNA was extracted from 132 historically positive, whole organism spiked, and historically negative whole blood and formalin fixed paraffin embedded (FFPE) samples. Samples were run on the LightCycler® 2.0 using instrument specific LightCycler® FastStart DNA Master HybProbe enzyme and compared to results generated using the LightCycler® 480 and its instrument specific LightCycler® 480 Genotyping Master enzyme. During optimization, MgCl2 concentrations and thermocycling profiles were adjusted. Accuracy, specificity, inclusivity, and limit of detection studies were performed. Crossing point (Cp), melting temperature (Tm), fluorescent peak and fluorescent background values were compared between the two instruments. Results (if a Case Study enter NA) The agreement in accuracy between the LightCycler® 2.0 and the LightCycler® 480 was 100% for whole blood samples. For historically positive FFPE samples, LightCycler® 2.0 sensitivity and LightCycler® 480 sensitivity were 86% and 100%, respectively. Specificity and inclusivity of the assay were identical between the two instruments. The limit of detection in whole blood was 5-fold lower on the LightCycler® 480 (50 copies/µL) compared to the LightCycler® 2.0 (250 copies/µL). Mean Cp and fluorescent peak intensity values increased by 5.1% and 65-fold, respectively. Conclusion The study demonstrates similar performance and improved limit of detection for the Bartonella FRET hybridization probe RT-PCR assay on the LightCycler® 480 compared to the LightCycler® 2.0.

A novel and highly sensitive real-time nested RT-PCR assay in a single closed tube for detection of enterovirus

2018 ◽  
Vol 90 (3) ◽  
pp. 181-185 ◽  
Author(s):  
Xin-xin Shen ◽  
Fang-zhou Qiu ◽  
Huai-long Zhao ◽  
Meng-jie Yang ◽  
Liu Hong ◽  
...  
Keyword(s):  
Real Time ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Highly Sensitive ◽  
Closed Tube

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.

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