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

2019 ◽  
Vol 57 (4) ◽  
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.

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

2011 ◽  
Vol 49 (7) ◽  
pp. 2620-2624 ◽  
Author(s):  
Susan Bennett ◽  
Heli Harvala ◽  
Jeroen Witteveldt ◽  
E. Carol McWilliam Leitch ◽  
Nigel McLeish ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Simultaneous Detection ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
One Step

scholarly journals Development of a Real-Time Reverse Transcription-PCR Assay for Global Differentiation of Yellow Fever Virus Vaccine-Related Adverse Events from Natural Infections

2018 ◽  
Vol 56 (6) ◽  
Author(s):  
Holly R. Hughes ◽  
Brandy J. Russell ◽  
Eric C. Mossel ◽  
John Kayiwa ◽  
Julius Lutwama ◽  
...  
Keyword(s):  
Adverse Events ◽  
Real Time ◽  
Yellow Fever ◽  
Vaccine Strain ◽  
Reverse Transcription ◽  
Yellow Fever Virus ◽  
Fever Virus ◽  
Wild Type ◽  
Pcr Assay ◽  
Reverse Transcription Pcr

ABSTRACT Yellow fever (YF) is a reemerging public health threat, with frequent outbreaks prompting large vaccination campaigns in regions of endemicity in Africa and South America. Specific detection of vaccine-related adverse events is resource-intensive, time-consuming, and difficult to achieve during an outbreak. To address this, we have developed a highly transferable rapid yellow fever virus (YFV) vaccine-specific real-time reverse transcription-PCR (RT-PCR) assay that distinguishes vaccine from wild-type lineages. The assay utilizes a specific hydrolysis probe that includes locked nucleic acids to enhance specific discrimination of the YFV17D vaccine strain genome. Promisingly, sensitivity and specificity analyses reveal this assay to be highly specific to vaccine strain(s) when tested on clinical samples and YFV cell culture isolates of global origin. Taken together, our data suggest the utility of this assay for use in laboratories of varied capacity for the identification and differentiation of vaccine-related adverse events from wild-type infections of both African and South American origin.

scholarly journals 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

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

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

2021 ◽  
pp. 104868
Author(s):  
Marielle BEDOTTO ◽  
Pierre-Edouard FOURNIER ◽  
Linda HOUHAMDI ◽  
Philippe COLSON ◽  
Didier RAOULT
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Pcr Assay ◽  
Reverse Transcription Pcr

scholarly journals Real-Time Reverse Transcription-PCR Assay for Comprehensive Detection of Human Rhinoviruses

2007 ◽  
Vol 46 (2) ◽  
pp. 533-539 ◽  
Author(s):  
X. Lu ◽  
B. Holloway ◽  
R. K. Dare ◽  
J. Kuypers ◽  
S. Yagi ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Pcr Assay ◽  
Reverse Transcription Pcr

Development of a real-time reverse transcription-PCR assay to detect and quantify group A rotavirus equine-like G3 strains

2021 ◽  
Author(s):  
Eric M. Katz ◽  
Mathew D. Esona ◽  
Rashi Gautam ◽  
Michael D. Bowen
Keyword(s):  
Viral Load ◽  
Real Time ◽  
Reverse Transcription ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Human Populations ◽  
Pcr Assay ◽  
Group A Rotavirus ◽  
Reverse Transcription Pcr ◽  
Group A

Since 2013, group A rotavirus strains characterized as novel DS-1-like inter-genogroup reassortant ‘equine-like G3’ strains have emerged and spread across five continents among human populations in at least 14 countries. Here we report a novel one-step TaqMan quantitative real-time reverse transcription-PCR assay developed to genotype and quantify the viral load for samples containing rotavirus equine-like G3 strains. Using a universal G forward primer and a newly designed reverse primer and TaqMan probe, we developed and validated an assay with a linear dynamic range of 2.3 × 10 9 – 227 copies per reaction and a limit of detection of 227 copies. The percent positive agreement, percent negative agreement, and precision of our assay were 100.00%, 99.63%, and 100.00%, respectively. This assay can simultaneously detect and quantify the viral load for samples containing DS-1-like inter-genogroup reassortant equine-like G3 strains with high sensitivity and specificity, faster turnaround time, and decreased cost and will be valuable for high-throughput screening of stool samples collected to monitor equine-like G3 strain prevalence and circulation among human populations throughout the world.

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