Real-time molecular beacon NASBA for rapid and sensitive detection of norovirus GII in clinical samples

2009 ◽  
Vol 55 (12) ◽  
pp. 1375-1380 ◽  
Author(s):  
Safaa Lamhoujeb ◽  
Hugues Charest ◽  
Ismail Fliss ◽  
Solange Ngazoa ◽  
Julie Jean
Keyword(s):  
Real Time ◽  
Molecular Beacon ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Reading Frame ◽  
Viral Loads ◽  
Reverse Transcription Pcr ◽  
Highly Sensitive ◽  
Nasba Assay ◽  
Norovirus Gii

To improve the sensitivity and efficiency of the real-time nucleic acid sequence based amplification (NASBA) assay targeting the open reading frame 1–2 (ORF1–ORF2) junction of the norovirus (NoV) genome, a selection of clinical samples were analyzed. The assay results were compared with those of TaqMan and conventional reverse transcription PCR (RT-PCR) and a commercial enzyme-linked immunoassay (ELISA) for the specific detection of GII NoV in 96 fecal samples. Based on end-point dilution, the two real-time assays had similar sensitivities (0.01 particle detectable units), two log10cycles greater than that of conventional RT-PCR. GII NoV was detected in 88.54% of the samples by real-time NASBA, in 86.46% by TaqMan RT-PCR, in 81.25% by conventional RT-PCR, and in 65.7% by ELISA. The two real-time assays were in agreement for 88.5% of the samples. These results demonstrate that real-time NASBA with a molecular beacon probe is highly sensitive, accurate, and specific for NoV detection in clinical samples. Applying this technique to samples with complex matrix and low viral loads, such as food and environmental samples, could be useful for the detection of NoVs and will improve the prevention of NoV outbreaks.

scholarly journals A Multiallelic Molecular Beacon-Based Real-Time RT-PCR Assay for the Detection of SARS-CoV-2

Life ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1146
Author(s):  
Andreas C. Chrysostomou ◽  
Johana Hezka Rodosthenous ◽  
Cicek Topcu ◽  
Christina Papa ◽  
Antonia Aristokleous ◽  
...  
Keyword(s):  
Real Time ◽  
Molecular Beacon ◽  
Infectious Agent ◽  
Clinical Samples ◽  
Detection Accuracy ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Multiple Introductions ◽  
Detection Techniques ◽  
Reverse Transcription Pcr

Emerging infectious viruses have led to global advances in the development of specific and sensitive detection techniques. Viruses have an inherent potential to easily mutate, presenting major hurdles for diagnostics and requiring methods capable of detecting genetically diverse viral strains. One such infectious agent is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which emerged in December 2019 and has resulted in the global coronavirus disease 2019 (COVID-19) pandemic. This study presents a real-time reverse transcription PCR (RT-PCR) detection assay for SARS-CoV-2, taking into account its intrinsic polymorphic nature that arises due to genetic drift and recombination, as well as the possibility of continuous and multiple introductions of genetically nonidentical strains into the human population. This advance was achieved by using mismatch-tolerant molecular beacons designed to specifically detect the SARS-CoV-2 S, E, M, and N genes. These were applied to create a simple and reproducible real-time RT-PCR assay, which was validated using external quality control panels (QCMD: CVOP20, WHO: SARS-CoV-2-EQAP-01) and clinical samples. This assay was designed for high target detection accuracy and specificity and can also be readily adapted for the detection of other emerging and rapidly mutating pathogens.

scholarly journals Improved Detection of Rhinoviruses in Clinical Samples by Using a Newly Developed Nested Reverse Transcription-PCR Assay

1999 ◽  
Vol 37 (3) ◽  
pp. 524-530 ◽  
Author(s):  
Arno C. Andeweg ◽  
Theo M. Bestebroer ◽  
Martijn Huybreghs ◽  
Tjeerd G. Kimman ◽  
Jan C. de Jong
Keyword(s):  
Reverse Transcription ◽  
Rna Extraction ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Noncoding Regions ◽  
Reverse Transcription Pcr ◽  
Highly Sensitive ◽  
Virus Culture ◽  
Culture Positive

This paper describes the development and evaluation of a new nested reverse transcription (RT)-PCR for the detection of rhinovirus in clinical samples. The nucleotide sequences of the 5′ noncoding regions of 39 rhinoviruses were determined in order to map the most conserved subregions. We designed a set of rhinovirus-specific primers and probes directed to these subregions and developed a new nested RT-PCR. The new assay includes an optimal RNA extraction method and amplicon identification with probe hybridization to discriminate between rhinoviruses and the closely related enteroviruses. It proved to be highly sensitive and specific. When tested on a dilution series of cultured viruses, the new PCR protocol scored positive at 10- to 100-fold-higher dilutions than a previously used nested RT-PCR. When tested on a collection of clinical samples obtained from 1,070 acute respiratory disease patients who had consulted their general practitioners, the new assay demonstrated a rhinovirus in 24% of the specimens, including all culture-positive samples, whereas the previously used PCR assay or virus culture detected a rhinovirus in only 3.5 to 6% of the samples. This new assay should help determine the disease burden associated with rhinovirus infections.

scholarly journals Fast and low-cost detection of SARS-CoV-2 peptides by tandem mass spectrometry in clinical samples

2020 ◽  
Author(s):  
Karina Helena Morais Cardozo ◽  
Adriana Lebkuchen ◽  
Guilherme Goncalves Okai ◽  
Rodrigo Andrade Schuch ◽  
Luciana Godoy Viana ◽  
...  
Keyword(s):  
Real Time ◽  
Large Scale ◽  
Low Cost ◽  
Large Population ◽  
Virus Detection ◽  
Standard Test ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Fast Processing

Abstract The current outbreak of severe acute respiratory syndrome associated with coronavirus 2 (SARS-CoV-2) is pressing public health systems around the world, and large population testing is a key step to control this pandemic disease. Real-time reverse-transcription PCR (real-time RT-PCR) is the gold standard test for virus detection but the soaring demand for this test resulted in shortage of reagents and instruments, severely limiting its applicability to large-scale screening. To be used either as an alternative, or as a complement, to real-time RT-PCR testing, we developed a high-throughput targeted proteomics assay to detect SARS-CoV-2 proteins directly from clinical respiratory tract samples. Sample preparation was fully automated by using a modified magnetic particle-based proteomics approach implemented on a robotic liquid handler, enabling a fast processing of samples. The use of turbulent flow chromatography included four times multiplexed on-line sample cleanup and UPLC separation. MS/MS detection of three peptides from SARS-CoV-2 nucleoprotein and a 15N-labeled internal global standard was achieved within 2.5 min, enabling the analysis of more than 500 samples per day. The method was validated using 562 specimens previously analyzed by real-time RT-PCR and was able to detect over 83% of positive cases. No interference was found with samples from common respiratory viruses, including other coronaviruses (NL63, OC43, HKU1, and 229E). The strategy here presented has high sample stability and low cost and should be considered as an option to large population testing.

scholarly journals Fast and low-cost detection of SARS-CoV-2 peptides by tandem mass spectrometry in clinical samples

2020 ◽  
Author(s):  
Karina Helena Morais Cardozo ◽  
Adriana Lebkuchen ◽  
Guilherme Goncalves Okai ◽  
Rodrigo Andrade Schuch ◽  
Luciana Godoy Viana ◽  
...  
Keyword(s):  
Real Time ◽  
Large Scale ◽  
Low Cost ◽  
Large Population ◽  
Virus Detection ◽  
Standard Test ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Fast Processing

Abstract The current outbreak of severe acute respiratory syndrome associated with coronavirus 2 (SARS-CoV-2) is pressing public health systems around the world, and large population testing is a key step to control this pandemic disease. Real-time reverse-transcription PCR (real-time RT-PCR) is the gold standard test for virus detection but the soaring demand for this test resulted in shortage of reagents and instruments, severely limiting its applicability to large-scale screening. To be used either as an alternative, or as a complement, to real-time RT-PCR testing, we developed a high-throughput targeted proteomics assay to detect SARS-CoV-2 proteins directly from clinical respiratory tract samples. Sample preparation was fully automated by using a modified magnetic particle-based proteomics approach implemented on a robotic liquid handler, enabling a fast processing of samples. The use of turbulent flow chromatography included four times multiplexed on-line sample cleanup and UPLC separation. MS/MS detection of three peptides from SARS-CoV-2 nucleoprotein and a 15N-labeled internal global standard was achieved within 2.5 min, enabling the analysis of more than 500 samples per day. The method was validated using 562 specimens previously analyzed by real-time RT-PCR and was able to detect over 83% of positive cases. No interference was found with samples from common respiratory viruses, including other coronaviruses (NL63, OC43, HKU1, and 229E). The strategy here presented has high sample stability and low cost and should be considered as an option to large population testing.

scholarly journals Detection of SARS Coronavirus in Patients with Severe Acute Respiratory Syndrome by Conventional and Real-Time Quantitative Reverse Transcription-PCR Assays

2004 ◽  
Vol 50 (1) ◽  
pp. 67-72 ◽  
Author(s):  
Leo L M Poon ◽  
Kwok Hung Chan ◽  
On Kei Wong ◽  
Timothy K W Cheung ◽  
Iris Ng ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
N Gene ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
Copy Numbers ◽  
Stool Samples ◽  
Pcr Assays

Abstract Background: A novel coronavirus (CoV) was recently identified as the agent for severe acute respiratory syndrome (SARS). We compared the abilities of conventional and real-time reverse transcription-PCR (RT-PCR) assays to detect SARS CoV in clinical specimens. Methods: RNA samples isolated from nasopharyngeal aspirate (NPA; n = 170) and stool (n = 44) were reverse-transcribed and tested by our in-house conventional RT-PCR assay. We selected 98 NPA and 37 stool samples collected at different times after the onset of disease and tested them in a real-time quantitative RT-PCR specific for the open reading frame (ORF) 1b region of SARS CoV. Detection rates for the conventional and real-time quantitative RT-PCR assays were compared. To investigate the nature of viral RNA molecules in these clinical samples, we determined copy numbers of ORF 1b and nucleocapsid (N) gene sequences of SARS CoV. Results: The quantitative real-time RT-PCR assay was more sensitive than the conventional RT-PCR assay for detecting SARS CoV in samples collected early in the course of the disease. Real-time assays targeted at the ORF 1b region and the N gene revealed that copy numbers of ORF 1b and N gene sequences in clinical samples were similar. Conclusions: NPA and stool samples can be used for early diagnosis of SARS. The real-time quantitative RT-PCR assay for SARS CoV is potentially useful for early detection of SARS CoV. Our results suggest that genomic RNA is the predominant viral RNA species in clinical samples.

scholarly journals Sensitive Multiplex Real-Time Reverse Transcription-PCR Assay for the Detection of Human and Animal Noroviruses in Clinical and Environmental Samples

2007 ◽  
Vol 73 (17) ◽  
pp. 5464-5470 ◽  
Author(s):  
Sandro Wolf ◽  
Wendy M. Williamson ◽  
Joanne Hewitt ◽  
Malet Rivera-Aban ◽  
Susan Lin ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Wide Spectrum ◽  
Clinical Diagnostics ◽  
Target Sequence ◽  
Rt Pcr ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
Copy Numbers ◽  
Treated Drinking Water

ABSTRACT In this study, we developed a triplex real-time reverse transcription-PCR (RT-PCR)-based method that detects and distinguishs between noroviruses belonging to genogroups I, II, and III and that targets the junction between the regions of open reading frame 1 (ORF1) and ORF2. This is the first assay to include all three genogroups and the first real-time RT-PCR-based method developed for the detection of bovine noroviruses. The assay was shown to be broadly reactive against a wide spectrum of norovirus genotypes, including GI/1 through GI/7, GII/1 through GII/8, GII/10, GII/12, and GII/17, in different matrices (including fecal specimens, treated and raw sewage, source water, and treated drinking water). The assay is highly sensitive, detecting low copy numbers of plasmids that carry the target sequence. A new bovine norovirus, Bo/NLV/Norsewood/2006/NZL, was identified by this assay and was further genetically characterized. The results implicate a broad range of possible applications, including clinical diagnostics, tracing of fecal contaminants, and due to its sensitivity and broad reactivity, environmental studies.

Detection of a novel bunyavirus associated with fever, thrombocytopenia and leukopenia syndrome in Henan Province, China, using real-time reverse transcription PCR

2013 ◽  
Vol 62 (7) ◽  
pp. 1060-1064 ◽  
Author(s):  
Xueyong Huang ◽  
Licheng Liu ◽  
Yanhua Du ◽  
Hongxia Ma ◽  
Yujiao Mu ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Henan Province ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Serum Samples ◽  
Reverse Transcription Pcr

A novel bunyavirus associated with fever, thrombocytopenia and leukopenia syndrome (FTLS) was discovered in Henan Province, China. Here, we report the development of an assay for this novel bunyavirus based on real-time reverse transcription PCR (RT-PCR). The assay exhibited high sensitivity and specificity without cross-reactivity towards 13 other viruses that cause similar symptoms. To evaluate the performance of this assay in detecting clinical samples, we analysed 261 serum samples from patients in Henan Province between 2007 and 2010. Of these samples, 91.95 % were bunyavirus positive. Compared with serological assays, the real-time PCR assay was much more sensitive in identifying infected patients 1 to 7 days after the onset of symptoms.

scholarly journals Reducing COVID-19 quarantine with SARS-CoV-2 testing: a simulation study

BMJ Open ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. e050473
Author(s):  
Bo Peng ◽  
Wen Zhou ◽  
Rowland W Pettit ◽  
Patrick Yu ◽  
Peter G Matos ◽  
...  
Keyword(s):  
Optimal Strategies ◽  
Cost Effective ◽  
Transmission Risk ◽  
Sensitivity Analyses ◽  
Rt Pcr ◽  
Viral Loads ◽  
Reverse Transcription Pcr ◽  
Highly Sensitive ◽  
Antigen Tests ◽  
The Impact

ObjectiveTo evaluate the effectiveness of SARS-CoV-2 testing on shortening the duration of quarantines for COVID-19 and to identify the most effective choices of testing schedules.DesignWe performed extensive simulations to evaluate the performance of quarantine strategies when one or more SARS-CoV-2 tests were administered during the quarantine. Simulations were based on statistical models for the transmissibility and viral loads of SARS-CoV-2 infections and the sensitivities of available testing methods. Sensitivity analyses were performed to evaluate the impact of perturbations in model assumptions on the outcomes of optimal strategies.ResultsWe found that SARS-CoV-2 testing can effectively reduce the length of a quarantine without compromising safety. A single reverse transcription-PCR (RT-PCR) test performed before the end of quarantine can reduce quarantine duration to 10 days. Two tests can reduce the duration to 8 days, and three highly sensitive RT-PCR tests can justify a 6-day quarantine. More strategic testing schedules and longer quarantines are needed if tests are administered with less-sensitive RT-PCR tests or antigen tests. Shorter quarantines can be used for applications that tolerate a residual postquarantine transmission risk comparable to a 10-day quarantine.ConclusionsTesting could substantially reduce the length of isolation, reducing the physical and mental stress caused by lengthy quarantines. With increasing capacity and lowered costs of SARS-CoV-2 tests, test-assisted quarantines could be safer and more cost-effective than 14-day quarantines and warrant more widespread use.

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