Rapid Simultaneous Detection of Enterovirus and Parechovirus RNAs in Clinical Samples by One-Step Real-Time Reverse Transcription-PCR Assay

The early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is required to identify and isolate contagious patients to prevent further transmission of SARS-CoV-2. In this study, we present a multitarget real-time TaqMan reverse transcription PCR (rRT-PCR) assay for the quantitative detection of SARS-CoV-2 and some of its circulating variants harboring mutations that give the virus a selective advantage. Seven different primer-probe sets that included probes containing locked nucleic acid (LNA) nucleotides were designed to amplify specific wild-type and mutant sequences in Orf1ab, Envelope (E), Spike (S), and Nucleocapsid (N) genes. Furthermore, a newly developed primer-probe set targeted human β2-microglobulin (B2M) as a highly sensitive internal control for RT efficacy. All singleplex and fourplex assays detected £ 14 copies/reaction of quantified synthetic RNA transcripts, with a linear amplification range of nine logarithmic orders. Primer-probe sets for detection of SARS-CoV-2 exhibited no false-positive amplifications with other common respiratory pathogens, including human coronaviruses NL63, 229E, OC43, and HKU-1. Fourplex assays were evaluated using 160 clinical samples positive for SARS-CoV-2. Results showed that SARS-CoV-2 viral RNA was detected in all samples, including viral strains harboring mutations in the Spike coding sequence that became dominant in the pandemic. Given the emergence of SARS-CoV-2 variants and their rapid spread in some populations, fourplex rRT-PCR assay containing four primer-probe sets represents a reliable approach to allow quicker detection of circulating relevant variants in a single reaction.

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Development of Group- and Serotype-Specific One-Step SYBR Green I-Based Real-Time Reverse Transcription-PCR Assay for Dengue Virus

Journal of Clinical Microbiology ◽

10.1128/jcm.41.6.2408-2416.2003 ◽

2003 ◽

Vol 41 (6) ◽

pp. 2408-2416 ◽

Cited By ~ 125

Author(s):

P.-Y. Shu ◽

S.-F. Chang ◽

Y.-C. Kuo ◽

Y.-Y. Yueh ◽

L.-J. Chien ◽

...

Keyword(s):

Dengue Virus ◽

Real Time ◽

Reverse Transcription ◽

Sybr Green ◽

Sybr Green I ◽

Pcr Assay ◽

Reverse Transcription Pcr ◽

One Step

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Simultaneous Detection and Differentiation between Wild-Type and Vaccine Measles Viruses by a Multiplex Real-Time Reverse Transcription-PCR Assay

Journal of Clinical Microbiology ◽

10.1128/jcm.01828-18 ◽

2019 ◽

Vol 57 (4) ◽

Cited By ~ 2

Author(s):

Kanti Pabbaraju ◽

Kara Gill ◽

Anita A. Wong ◽

Graham A. Tipples ◽

Joanne Hiebert ◽

...

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Measles Virus ◽

Respiratory Infections ◽

Simultaneous Detection ◽

Contact Tracing ◽

Wild Type ◽

Pcr Assay ◽

Live Attenuated Vaccine ◽

Reverse Transcription Pcr

ABSTRACT Measles is one of the most contagious viral respiratory infections and was declared to be eliminated from Canada in 1998; however, measles cases and outbreaks still occur every year through reintroduction from other parts of the world. Laboratory confirmation of measles virus (MV) RNA by real-time PCR provides a definitive diagnosis, and molecular analysis to determine the genotype is the only way to distinguish between wild-type and vaccine strains. This distinction is important since live attenuated vaccine strains are able to replicate in the patient and can be associated with rash and fever but are poorly transmissible, if at all. Prompt reporting of measles cases to local authorities, including differentiation between wild-type and vaccine strains, allows for optimal management and contact tracing. The development and validation of a multiplex real-time reverse transcription-PCR (rtRT-PCR) assay for the simultaneous detection and differentiation of the Moraten and Schwarz vaccine strains from presumptive wild-type MV in a format that can be easily implemented for high-throughput testing of patient samples are reported here. This assay is sensitive, specific, reproducible, and 100% accurate in comparison with the gold standard comparator assay.

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

Journal of Medical Microbiology ◽

10.1099/jmm.0.049577-0 ◽

2013 ◽

Vol 62 (7) ◽

pp. 1060-1064 ◽

Cited By ~ 8

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.

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Implementation of an in-house real-time reverse transcription-PCR assay for the rapid detection of the SARS-CoV-2 Marseille-4 variant

10.1101/2021.02.03.21250823 ◽

2021 ◽

Author(s):

Marielle Bedotto ◽

Pierre-Edouard Fournier ◽

Linda Houhamdi ◽

Anthony Levasseur ◽

Jeremy Delerce ◽

...

Keyword(s):

Immune Responses ◽

Real Time ◽

Reverse Transcription ◽

Geographical Area ◽

Viral Escape ◽

Pcr Assay ◽

Reverse Transcription Pcr ◽

One Step ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

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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