An Improved One-Step Real-Time Reverse Transcription-PCR Assay for Detection of Norovirus

ABSTRACTIntroductionThe SARS-CoV-2 pandemic has been associated with the occurrence since summer 2020 of several viral variants that overlapped or succeeded each other in time. Those of current concern harbor mutations within the spike receptor binding domain (RBD) that may be associated with viral escape to immune responses. In our geographical area a viral variant we named Marseille-4 harbors a S477N substitution in this RBD.Materials and methodsWe aimed to implement an in-house one-step real-time reverse transcription-PCR (qPCR) assay with a hydrolysis probe that specifically detects the SARS-CoV-2 Marseille-4 variant.ResultsAll 6 cDNA samples from Marseille-4 variant strains identified in our institute by genome next-generation sequencing (NGS) tested positive using our Marseille-4 specific qPCR, whereas all 32 cDNA samples from other variants tested negative. In addition, 39/42 (93%) respiratory samples identified by NGS as containing a Marseille-4 variant strain and 0/26 samples identified as containing non-Marseille-4 variant strains were positive. Finally, 1,585/2,889 patients SARS-CoV-2-diagnosed in our institute, 10/277 (3.6%) respiratory samples collected in Algeria, and none of 207 respiratory samples collected in Senegal, Morocco, or Lebanon tested positive using our Marseille-4 specific qPCR.DiscussionOur in-house qPCR system was found reliable to detect specifically the Marseille-4 variant and allowed estimating it is involved in more than half of our SARS-CoV-2 diagnoses since December 2020. Such approach allows the real-time surveillance of SARS-CoV-2 variants, which is warranted to monitor and assess their epidemiological and clinical characterics based on comprehensive sets of data.

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Sensitive and Specific Quantitative Detection of Rotavirus A by One-Step Real-Time Reverse Transcription-PCR Assay without Antecedent Double-Stranded-RNA Denaturation

Journal of Clinical Microbiology ◽

10.1128/jcm.01192-13 ◽

2013 ◽

Vol 51 (9) ◽

pp. 3047-3054 ◽

Cited By ~ 33

Author(s):

S. Mijatovic-Rustempasic ◽

K. I. Tam ◽

T. K. Kerin ◽

J. M. Lewis ◽

R. Gautam ◽

...

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Quantitative Detection ◽

Pcr Assay ◽

Double Stranded Rna ◽

Rotavirus A ◽

Reverse Transcription Pcr ◽

One Step

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Differentiation of PEDV Classical Attenuated Vaccine Strains from Wild-type Strains using One-Step Real-Time Fluorescent Reverse Transcription PCR Assay Targeting ORF1 Nucleotides Deletion Region

10.21203/rs.3.rs-529609/v1 ◽

2021 ◽

Author(s):

Zhilin Wang ◽

Xuerui Li ◽

Youjun Shang ◽

Jinyan Wu ◽

Zhen Dong ◽

...

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Economic Losses ◽

Wild Type ◽

Rt Pcr ◽

Pcr Assay ◽

Attenuated Vaccine ◽

Reverse Transcription Pcr ◽

One Step ◽

Type Strains

Abstract BackgroundPorcine epidemic diarrhea virus (PEDV) is a pathogen causing serious disease and resulting in severe economic losses in the swine industry. In recent years, although China has adopted a large-scale vaccine immunization strategy, many types of PEDV strains, including classical attenuated vaccine strains, have been discovered in the immunized pig herds. Therefore, monitoring the prevalence of different types of PEDV strains is particularly important for the production of pigs and the safety evaluation of related attenuated vaccines MethodsIn the study, a one-step real-time fluorescent reverse transcription PCR (one-step real-time RT-PCR) assay targeting 24-nucleotide deletion in the ORF1 region of three PEDV classical attenuated vaccine strains (derived from classical strains) was established, which could effectively distinguish PEDV classical attenuated vaccine strains and wild-type strains. ResultsIn our study, the RNA detection limits for PEDV wild-type strains and classical attenuated vaccine strains were 3.0×103 copies and 3.0×102 copies, respectively. This assay was highly specific for PEDV, with no cross-reactivity for other viruses, causing diarrheal disease. A total of 117 swine fecal samples were analysed by this established real-time RT-PCR assay, indicating that classical attenuated vaccine strains were present in the swine herds in Gansu province, China. Additionally, a pair of primers and two probes of the established assay can be placed in one reaction tube to distinguish PEDV classical attenuated vaccine strains and wild-type strains. ConclusionOur results provided an effective and cheap technology platform for clinical rapid identification testing and epidemiological investigations of PEDV wild-type strains and classical attenuated vaccine strains

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Implementation of an in-house real-time reverse transcription-PCR assay to detect the emerging SARS-CoV-2 N501Y variants

10.1101/2021.02.03.21250661 ◽

2021 ◽

Author(s):

Marielle Bedotto ◽

Pierre-Edouard Fournier ◽

Linda Houhamdi ◽

Philippe Colson ◽

Didier Raoult

Keyword(s):

Immune Responses ◽

Real Time ◽

Genome Sequencing ◽

Reverse Transcription ◽

Pcr Assay ◽

Reverse Transcription Pcr ◽

One Step ◽

Set Up ◽

Negative Controls ◽

Implementing Strategies

ABSTRACTThe SARS-CoV-2 pandemic has been associated with the emergence of several variants with a mutated spike glycoprotein that are of substantial concern regarding their transmissibility and ability to evade immune responses. This warrants implementing strategies for their detection and surveillance. We have set up an in-house one-step real-time reverse transcription-PCR (qPCR) assay that specifically detects SARS-CoV-2 spike N501Y variants. Our assay was positive for all 6 patients found spike N501Y-positive by genome sequencing. Ten cDNA samples for each of the 10 Marseille variants identified by genome sequencing and three nasopharyngeal samples of a spike N501Y-negative variant (Marseille-4) that predominates locally tested negative. All negative controls among which 5 SARS-CoV-2-negative nasopharyngeal samples tested negative. First use in the setting of diagnosis on 51 nasopharyngeal samples from SARS-CoV-2-positive but Marseille-4-negative patients showed positivity in 5 cases further confirmed by sequencing as from spike N501Y variant-infected patients. Thus, our in-house qPCR system was found reliable for the detection of the N501Y substitution and allowed estimating preliminarily that spike N501Y variant prevalence was 4% among SARS-CoV-2 diagnoses since January 2020.

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