SARS-CoV-2 variants in Paraguay: Detection and surveillance with a readily modifiable, multiplex real-time RT-PCR

Objectives The objective of the current study was to develop a lower-cost and scalable protocol to identify and monitor SARS-CoV-2 variants in Paraguay by pairing real-time RT-PCR detection of spike mutations with amplicon Sanger sequencing and whole-genome Nanopore sequencing. Methods 201 acute-phase nasopharyngeal samples from SARS-CoV-2-positive individuals were tested with two rRT-PCRs: 1) N2RP assay to confirm SARS-CoV-2 RNA detection (CDC N2 target), and 2) the Spike SNP assay to detect mutations in the spike receptor binding domain. The assay was performed with probes to identify mutations associated with the following variants: alpha (501Y), beta/gamma (417variant/484K/501Y), delta (452R/478K), and lambda (452Q/490S). Results All samples were positive for SARS-CoV-2 in the N2RP assay (mean Ct, 20.8; SD 5.6); 198/201 (98.5%) tested positive in the Spike SNP assay. The most common genotype was 417variant/484K/501Y, detected in 102/198 samples (51.5%) and most consistent with P.1 lineage (gamma variant) in Paraguay. No mutations (K417 only) were found in 64/198 (32.3%); and K417/484K was identified in 22/198 (11.1%), consistent with P.2 (zeta). Seven samples (3.5%) tested positive for 452R without 478K, and one sample with genotype K417/501Y was confirmed as B.1.1.7 (alpha). Results were confirmed by Sanger sequencing in 181/181 samples (100%) with high-quality amplicon sequences, and variant calls were consistent with Nanopore sequencing in 29/29 samples. Conclusions The Spike SNP assay provides accurate detection of mutations associated with SARS-CoV-2 variants. This can be implemented in laboratories performing rRT-PCR to improve population-level surveillance for these mutations and inform the judicious use of scarce sequencing resources.

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Rapid, sensitive, full genome sequencing of Severe Acute Respiratory Syndrome Virus Coronavirus 2 (SARS-CoV-2)

10.1101/2020.04.22.055897 ◽

2020 ◽

Cited By ~ 8

Author(s):

Clinton R Paden ◽

Ying Tao ◽

Krista Queen ◽

Jing Zhang ◽

Yan Li ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Sanger Sequencing ◽

Respiratory Syndrome Virus ◽

Full Genome ◽

Miseq Sequencing ◽

Genomic Information ◽

Rt Pcr ◽

Full Genome Sequencing ◽

High Quality ◽

Global Pandemic

AbstractSARS-CoV-2 recently emerged, resulting a global pandemic. Rapid genomic information is critical to understanding transmission and pathogenesis. Here, we describe validated protocols for generating high-quality full-length genomes from primary samples. The first employs multiplex RT-PCR followed by MinION or MiSeq sequencing. The second uses singleplex, nested RT-PCR and Sanger sequencing.

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A new dual-targeting real-time RT-PCR assay for hepatitis D virus RNA detection

Diagnostic Microbiology and Infectious Disease ◽

10.1016/j.diagmicrobio.2018.05.016 ◽

2018 ◽

Vol 92 (2) ◽

pp. 112-117 ◽

Cited By ~ 1

Author(s):

Yan Wang ◽

Jeffrey S. Glenn ◽

Mark A. Winters ◽

Li-ping Shen ◽

Ingrid Choong ◽

...

Keyword(s):

Real Time ◽

Rt Pcr ◽

Pcr Assay ◽

Rna Detection ◽

Hepatitis D ◽

Hepatitis D Virus ◽

Dual Targeting ◽

Virus Rna

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Development and Testing of a Method for Detecting Lujo Virus RNA by Reverse Transcription and Real Time Polymerase Chain Reaction

Problems of Particularly Dangerous Infections ◽

10.21055/0370-1069-2021-1-110-115 ◽

2021 ◽

pp. 110-115

Author(s):

E. V. Naidenova ◽

V. G. Dedkov ◽

D. A. Agafonov ◽

A. M. Senichkina ◽

M. V. Safonova ◽

...

Keyword(s):

Real Time ◽

Internal Control ◽

Biological Samples ◽

Control Sample ◽

Virus Genome ◽

Positive Control ◽

Control Panel ◽

Rt Pcr ◽

Rna Detection ◽

Virus Rna

The aim of the study was to develop and assess the efficacy of a method for Lujo virus RNA detection in clinical and biological samples using one-step real-time RT-PCR.Materials and methods. In order to select the conservative regions of the genome, we utilized the available in GenBank database Lujo virus sequences (https://www.ncbi. nlm.nih.gov/genbank) aligned in BioEdit 7.2.5 software package ( (IbisBiosciences, USA). To conduct one-round RTPCR, reverse transcriptase and TaqF-polimerase were used. Recombinant Escherichia coli strain, XL1-Blue, containing pGEM-T plasmid with inserted synthetically-generated fragment of the virus genome, was produced to make positive control sample (PCS). Constructed recombinant plasmids were used for creating RNA-containing PCS with protective protein shell of MS2-phage. Determination of specificity of the developed method was performed with the help of control panel of RNA and DNA of 23 viral strains related to 10 families; the sensitivity – the panel of biological samples artificially contaminated with PCS. Further testing was carried out at the premises of laboratory of the Russian-Guinean Center for Epidemiology and Prevention of Infectious Diseases (Kindia, Republic of Guinea) on 265 blood sera from practically healthy persons, 110 blood sera of cattle, 83 suspensions of ticks, and 165 suspensions of organs of small mammals collected in the territory of Guinea.Results and discussion. Two conservative polymerase gene fragments have been chosen as targets for Lujo virus RNA detection using RT-PCR. The combination of primers and probes has been experimentally selected, optimum composition of reaction mixture for PCR and mode of RT-PCR set-up established, as well as control samples C+, internal control, positive control sample developed. Sensitivity of the proposed method is 5·103 GE /ml, specificity – 100 %.

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Comparison of real-time RT-PCR assays for hepatitis E virus RNA detection

Journal of Clinical Virology ◽

10.1016/j.jcv.2013.06.038 ◽

2013 ◽

Vol 58 (1) ◽

pp. 36-40 ◽

Cited By ~ 34

Author(s):

Camelia Mokhtari ◽

Eric Marchadier ◽

Stephanie Haïm-Boukobza ◽

Asma Jeblaoui ◽

Sophie Tessé ◽

...

Keyword(s):

Real Time ◽

Hepatitis E Virus ◽

Hepatitis E ◽

Rt Pcr ◽

Rna Detection ◽

Virus Rna ◽

Pcr Assays

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Bluetongue Virus RNA Detection by Real-Time RT-PCR in Post-Vaccination Samples from Cattle

Transboundary and Emerging Diseases ◽

10.1111/tbed.12100 ◽

2013 ◽

Vol 62 (2) ◽

pp. 157-162 ◽

Cited By ~ 8

Author(s):

I. De Leeuw ◽

M. Garigliany ◽

G. Bertels ◽

T. Willems ◽

D. Desmecht ◽

...

Keyword(s):

Real Time ◽

Bluetongue Virus ◽

Rt Pcr ◽

Rna Detection ◽

Post Vaccination ◽

Virus Rna

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Improvement of Diagnostic System in a Real-Time RT-PCR “AmpliSens EBOV (ZAIRE)-FL” Format for Zaire ebolavirus RNA Detection

Problems of Particularly Dangerous Infections ◽

10.21055/0370-1069-2015-3-55-57 ◽

2015 ◽

pp. 55-57 ◽

Cited By ~ 1

Author(s):

V. G. Dedkov ◽

M. V. Safonova ◽

S. A. Bodnev ◽

A. S. Kabanov ◽

V. A. Safronov ◽

...

Keyword(s):

Real Time ◽

Diagnostic System ◽

Rt Pcr ◽

Rna Detection ◽

Zaire Ebolavirus

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Laboratory Methods Supporting Measles surveillance in Queensland, Australia, 2010 – 2017

10.1101/472175 ◽

2018 ◽

Cited By ~ 2

Author(s):

Jamie L. McMahon ◽

Judy Northill ◽

Mitchell Finger ◽

Michael Lyon ◽

Stephen B. Lambert ◽

...

Keyword(s):

Genetic Variation ◽

Real Time ◽

Sensitive Detection ◽

Rt Pcr ◽

High Quality ◽

Laboratory Methods ◽

Vaccine Preventable Diseases ◽

Global Travel ◽

Free Status ◽

Laboratory Workflow

ABSTRACTAustralia was officially recognised as having eliminated endemic measles circulation in 2014. Maintaining laboratory support for surveillance of vaccine-preventable diseases such as measles is an essential component of reaching and maintaining circulation-free status. Between 2010 and 2017 over 13,700 specimens were tested in our laboratory by real-time RT-PCR (RT-rPCR). Positive extracts from travellers were sequenced as required. Sequences were uploaded to GenBank and demonstrated the wide genetic variation expected from detection of MeV among virus introductions due to global travel. We describe the laboratory workflow employed in our laboratory between 2010 and 2017 for the sensitive detection of MeV infection, supporting high-quality surveillance.

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First Evidence of Powassan Virus (Flaviviridae) in Ixodes Scapularis in Appalachian Virginia, USA

American Journal of Tropical Medicine and Hygiene ◽

10.4269/ajtmh.21-0825 ◽

2021 ◽

Author(s):

Alexandra N. Cumbie ◽

Amanda M. Whitlow ◽

Gillian Eastwood

Keyword(s):

Real Time ◽

Adult Male ◽

Sanger Sequencing ◽

Ixodes Scapularis ◽

Rt Pcr ◽

Tick Vector ◽

Powassan Virus ◽

Floyd County

Here we report the first detection and confirmation of Powassan virus (POWV) (family: Flaviridae) in Ixodes scapularis ticks collected from Appalachian Virginia. Ixodes scapularis ticks were collected from vegetation across field sites in eight counties of western Virginia from June 2019 to April 2021. From these collections, one nymph and one adult male I. scapularis were determined to be positive for POWV using real-time RT-PCR and Sanger sequencing. Both positive ticks were collected from Floyd county, VA, at residential sites; the nymph in June 2020 and the adult male in April 2021. The presence of POWV in Virginia in its natural tick vector is crucial knowledge in beginning to understand the movement and transmission of this pathogen into new geographical areas and the risk it poses to medical and veterinary health.

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