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Article: Fast detection of SARS-CoV-2 variants including Omicron using one-step RT-PCR and Sanger sequencing

Bloemen, Mandy / Rector, Annabel / Swinnen, Jill / Ranst, Marc Van / Maes, Piet / Vanmechelen, Bert / Wollants, Elke

Journal of virological methods. 2022 June, v. 304

2022

Abstract: SARS-CoV-2 has kept the world in suspense for almost 2 years now. The virus spread rapidly worldwide and several variants of concern have emerged: Alpha, Beta, Gamma, Delta and recently Omicron. A rapid method to detect key mutations is needed because ... ...

Abstract	SARS-CoV-2 has kept the world in suspense for almost 2 years now. The virus spread rapidly worldwide and several variants of concern have emerged: Alpha, Beta, Gamma, Delta and recently Omicron. A rapid method to detect key mutations is needed because these variants may jeopardize the effectiveness of immune protection following vaccination or past infection. This article describes an easy, cheap and fast method for the detection of mutations in the spike protein that are indicative for specific variants. This method can easily distinguish Omicron from other variants.
Keywords	Severe acute respiratory syndrome coronavirus 2 ; rapid methods ; vaccination ; viruses
Language	English
Dates of publication	2022-06
Publishing place	Elsevier B.V.
Document type	Article
ZDB-ID	8013-5
ISSN	1879-0984 ; 0166-0934
ISSN (online)	1879-0984
ISSN	0166-0934
DOI	10.1016/j.jviromet.2022.114512
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Fast detection of SARS-CoV-2 variants including Omicron using one-step RT-PCR and Sanger sequencing.

Bloemen, Mandy / Rector, Annabel / Swinnen, Jill / Ranst, Marc Van / Maes, Piet / Vanmechelen, Bert / Wollants, Elke

Journal of virological methods

2022 Volume 304, Page(s) 114512

Abstract	SARS-CoV-2 has kept the world in suspense for almost 2 years now. The virus spread rapidly worldwide and several variants of concern have emerged: Alpha, Beta, Gamma, Delta and recently Omicron. A rapid method to detect key mutations is needed because these variants may jeopardize the effectiveness of immune protection following vaccination or past infection. This article describes an easy, cheap and fast method for the detection of mutations in the spike protein that are indicative for specific variants. This method can easily distinguish Omicron from other variants.
MeSH term(s)	COVID-19/diagnosis ; Humans ; Mutation ; Reverse Transcriptase Polymerase Chain Reaction ; SARS-CoV-2/genetics
Language	English
Publishing date	2022-03-04
Publishing country	Netherlands
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	8013-5
ISSN	1879-0984 ; 0166-0934
ISSN (online)	1879-0984
ISSN	0166-0934
DOI	10.1016/j.jviromet.2022.114512
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Article ; Online: Phylogenetic analysis of open reading frame of 11 gene segments of novel human-bovine reassortant RVA G6P[1] strain in Pakistan.

Tamim, Sana / Heylen, Elisabeth / Zeller, Mark / Ranst, Marc Van / Matthijnssens, Jelle / Salman, Muhammad / Aamir, Uzma Bashir / Sharif, Salman / Ikram, Aamer / Hasan, Fariha

Journal of medical virology

2020 Volume 92, Issue 12, Page(s) 3179–3186

Abstract: Multiple Rotavirus A (RVA) strains are linked with gastrointestinal infections in children that fall in age bracket of 0 to 60 months. However, the problem is augmented with emergence of unique strains that reassort with RVA strains of animal origin. The ...

Abstract	Multiple Rotavirus A (RVA) strains are linked with gastrointestinal infections in children that fall in age bracket of 0 to 60 months. However, the problem is augmented with emergence of unique strains that reassort with RVA strains of animal origin. The study describes the sequence analysis of a rare G6P[1] rotavirus strain isolated from a less than 1 year old child, during rotavirus surveillance in Rawalpindi district, Pakistan in 2010. Extracted RNA from fecal specimen was subjected to high throughput RT-PCR for structural and nonstructural gene segments. The complete rotavirus genome of one isolate RVA/Human-wt/PAK/PAK99/2010/G6P[1] was sequenced for phylogenetic analysis to elucidate the evolutionary linkages and origin. Full genome examination of novel strain RVA/Human-wt/PAK/PAK99/2010/G6P[1] revealed the unique genotype assemblage: G6-P[1]-I2-R2-C2-M2-A3-N2-T6-E2-H1. The evolutionary analyses of VP7, VP4, NSP1 and NSP3 gene segments revealed that PAK99 clustered with bovine, or cattle-like rotavirus strains from other closely related species, in the genotypes G6, P[1], A3 and T6 respectively. Gene segments VP6, VP1, VP2, VP3, NSP2 and NSP4 all possessed the DS-1-like bovine genotype 2 and bovine (-like) RVA strains instead of RVA strains having human origin. However, the NSP5 gene was found to cluster closely with contemporary human Wa-like rotavirus strains of H1 genotype. This is the first report on bovine-human (Wa-like reassortant) genotype constellation of G6P[1] strain from a human case in Pakistan (and the second description worldwide). Our results emphasize the significance of incessant monitoring of circulating RVA strains in humans and animals for better understanding of RV evolution.
MeSH term(s)	Pakistan ; Rotavirus/genetics ; Rotavirus/classification ; Rotavirus/isolation & purification ; Humans ; Phylogeny ; Rotavirus Infections/virology ; Animals ; Reassortant Viruses/genetics ; Reassortant Viruses/classification ; Reassortant Viruses/isolation & purification ; Genotype ; Infant ; Cattle ; Genome, Viral ; Feces/virology ; RNA, Viral/genetics ; Open Reading Frames ; Sequence Analysis, DNA
Chemical Substances	RNA, Viral
Language	English
Publishing date	2020-02-21
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	752392-0
ISSN	1096-9071 ; 0146-6615
ISSN (online)	1096-9071
ISSN	0146-6615
DOI	10.1002/jmv.25625
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Article ; Online: Early experiences with antibody testing in a Flemish nursing home during an acute COVID-19 outbreak: a retrospective cohort study

Buntinx, Frank / Claes, Peter / Gulikers, Marjo / Verbakel, Jan Y / Lepeleire, Jan De / Elst, Michael Van der / Ranst, Marc Van / Vermeersch, Pieter

Abstract: objectives: to assess the prevalence of COVID-19 (PCR-test) in residents and staff of a nursing home. To examine the presence of IgM and IgG antibodies in the sample and the relation between PCR and antibody test results. design: cross-sectional and ( ... ...

Abstract	objectives: to assess the prevalence of COVID-19 (PCR-test) in residents and staff of a nursing home. To examine the presence of IgM and IgG antibodies in the sample and the relation between PCR and antibody test results. design: cross-sectional and (retrospective) cohort study setting: a nursing home for the elderly Bessemerberg in Lanaken (Belgium) with up to 130 beds. Lanaken is situated in the Belgian province with the highest COVID-19 prevalence. participants: residents (N=108) and staff members (N=93) of the nursing home outcomes: PCR, IgM and IgG results: the prevalence of COVID-19, based on PCR test was 34% (N=40) for residents and 13% (N=11) for staff members, respectively. Of the residents, 13% showed positive IgM results and 15% positive IgG results. In 17% of the residents, at least one of the antibodies was positive. In total 13% of the staff members had positive IgM and 16% had a positive IgG. In 20% of the staff members at least one of these antibody tests was positive. In PCR positive residents, the percentage of IgM positive, IgG positive, and at least one of both was 28%, 34%, and 41%. In PCR positive staff, we found 30%, 60%, and 60%. Additional antibody tests were performed in nine residents between day 11 and 14 after the positive PCR test. Of those, 7 (78%) tested positive on at least one antibody. When retesting three weeks later, all remaining residents also tested positive. conclusions: Recently it was reported that in Belgium antibodies are present in 3-4% of the general population. Although, the prevalence in our residents is higher, the number is largely insufficient for herd immunity. In staff members of the regional hospital the prevalence of antibodies was 6%. The higher prevalence in nursing home staff (21%) may be related to the complete absence of good quality protection in the first weeks of the outbreak.
Keywords	covid19
Publisher	MedRxiv; WHO
Document type	Article ; Online
Note	WHO #Covidence: #20105874
DOI	10.1101/2020.05.18.20105874
Database	COVID19

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Article ; Online: Erratum: Emergence of human G2P[4] rotaviruses containing animal derived gene segments in the post-vaccine era.

Zeller, Mark / Nuyts, Valerie / Heylen, Elisabeth / Coster, Sarah De / Conceição-Neto, Nádia / Ranst, Marc Van / Matthijnssens, Jelle

Scientific reports

2017 Volume 7, Page(s) 39436

Language	English
Publishing date	2017-01-03
Publishing country	England
Document type	Journal Article
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/srep39436
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Molecular Methods in Food Safety Microbiology: Interpretation and Implications of Nucleic Acid Detection

Ceuppens, Siele / Li, Dan / Uyttendaele, Mieke / Renault, Pierre / Ross, Paul / Ranst, Marc Van / Cocolin, Luca / Donaghy, John

Comprehensive reviews in food science and food safety. 2014 July, v. 13, no. 4

2014

Abstract: Because of increasing demand for rapid results, molecular techniques are now applied for the detection of microorganisms in foodstuffs. However, interpretation problems can arise for the results generated by molecular methods in relation to the ... ...

Abstract	Because of increasing demand for rapid results, molecular techniques are now applied for the detection of microorganisms in foodstuffs. However, interpretation problems can arise for the results generated by molecular methods in relation to the associated public health risk. Discrepancies between results obtained by molecular and conventional culture methods stem from the difference in target, namely nucleic acids instead of actively growing microorganisms. Nucleic acids constitute 5% to 15% of the dry weight of all living cells and are relatively stable, even after cell death, so they may be present in a food matrix after the foodborne microorganisms have been inactivated. Therefore, interpretation of the public health significance of positive results generated by nucleic acid detection methods warrants some additional consideration. This review discusses the stability of nucleic acids in general and highlights the persistence of microbial nucleic acids after diverse food‐processing techniques based on data from the scientific literature. Considerable amounts of DNA and RNA (intact or fragmented) persist after inactivation of bacteria and viruses by most of the commonly applied treatments in the food industry. An overview of the existing adaptations for molecular assays to cope with these problems is provided, including large fragment amplification, flotation, (enzymatic) pretreatment, and various binding assays. Finally, the negligible risks of ingesting free microbial nucleic acids are discussed and this review ends with the future perspectives of molecular methods such as next‐generation sequencing in diagnostic and source attribution food microbiology.
Keywords	DNA ; RNA ; bacteria ; cell death ; food industry ; food matrix ; food microbiology ; food processing ; food safety ; foods ; high-throughput nucleotide sequencing ; public health ; risk ; source attribution ; viruses
Language	English
Dates of publication	2014-07
Size	p. 551-577.
Publishing place	Institute of Food Technologists
Document type	Article
ISSN	1541-4337
DOI	10.1111/1541-4337.12072
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Accuracy and reproducibility of low-dose submillisievert chest CT for the diagnosis of COVID-19

Dangis, Anthony / Gieraerts, Christopher / Bruecker, Yves De / Janssen, Lode / Valgaeren, Hanne / Obbels, Dagmar / Gillis, Marc / Ranst, Marc Van / Frans, Johan / Demeyere, Annick / Symons, Rolf

Radiol Cardiothorac Imaging

Abstract: PURPOSE: To demonstrate the accuracy and reproducibility of low-dose submillisievert chest CT for the diagnosis of COVID-19 infection in emergency room (ER) patients. MATERIALS AND METHODS: This was a HIPAA-compliant, institutional review board-approved ... ...

Abstract	PURPOSE: To demonstrate the accuracy and reproducibility of low-dose submillisievert chest CT for the diagnosis of COVID-19 infection in emergency room (ER) patients. MATERIALS AND METHODS: This was a HIPAA-compliant, institutional review board-approved retrospective study. From March 14(th) to March 24(th) 2020, 192 ER patients with symptoms suggestive of COVID-19 infection were studied with low-dose chest CT and real time polymerase chain reaction (RT-PCR). Image analysis included likelihood of COVID-19 infection and semi-quantitative extent of lung involvement. CT images were analyzed by 2 radiologists blinded to RT-PCR results. Reproducibility was assessed with McNemar test and intra-class correlation coefficient (ICC). Time between CT acquisition and report was measured. RESULTS: When compared to RT-PCR, low-dose submillisievert chest CT demonstrated excellent sensitivity, specificity, positive predictive value, negative predictive value, and accuracy for diagnosis of COVID-19 (86.7%, 93.6%, 91.1%, 90.3%, and 90.2%, respectively), in particular in patients with clinical symptoms for >48h (95.6%, 93.2%, 91.5%, 96.5%, and 94.4%, respectively). In patients with a positive CT, likelihood of disease increased from 43.2% (pre-test probability) to 91.1% or 91.4% (post-test probability), while in patients with a negative CT, likelihood of disease decreased to 9.6% or 3.7% for all patients or those with clinical symptoms for >48h, respectively. The prevalence of alternative diagnoses based on chest CT in patients without COVID-19 infection was 17.6%. Mean effective radiation dose was 0.56±0.25 mSv (SD). Median time between CT acquisition and report was 25 minutes (IQR: 13-49 minutes). Intra- and interreader reproducibility of CT was excellent (all ICC□0.95) without significant bias in Bland-Altman analysis. CONCLUSION: Low-dose submillisievert chest CT allows for rapid, accurate and reproducible assessment of COVID-19 infection in ER patients, in particular in patients with symptoms lasting longer than 48 hours. Chest CT has the additional advantage of offering alternative diagnoses in a significant subset of patients.
Keywords	covid19
Publisher	PMC
Document type	Article ; Online
DOI	10.1148/ryct.2020200196
Database	COVID19

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Article: Automated Extraction of Viral-Pathogen RNA and DNA for High-Throughput Quantitative Real-Time PCR

Beuselinck, Kurt / Ranst, Marc van / Eldere, J. van

Journal of clinical microbiology. 2005 Nov., v. 43, no. 11

2005

Abstract: The performance of the m1000 system (Abbott Laboratories, Illinois) as a front-end extraction system for high-throughput "in-house" quantitative real-time PCR assays was analyzed and compared to that of manual extraction of plasma and serum samples ( ... ...

Abstract	The performance of the m1000 system (Abbott Laboratories, Illinois) as a front-end extraction system for high-throughput "in-house" quantitative real-time PCR assays was analyzed and compared to that of manual extraction of plasma and serum samples (hepatitis C virus [HCV] and hepatitis B virus [HBV]) and EDTA-blood samples (cytomegalovirus [CMV] and Epstein-Barr virus [EBV]). Linearity of extraction was tested on dilution series of HCV and HBV reference materials. The correlation coefficient for standard curves based on repeated extraction runs was 0.97 ± 0.06 for HCV and 0.97 ± 0.03 for HBV, indicating a linear extraction from 100 to 1.0 x 10⁵ HCV IU/ml and from 100 to 1.0 x 10⁶ HBV IU/ml. Intra- and interrun variability was below 0.23 log₁₀ IU/ml for 2.98 to 5.28 log₁₀ HCV IU/ml and 2.70 to 5.20 log₁₀ HBV IU/ml. Correlation between automated and manual extraction was very good. For HCV, the correlation coefficient was 0.91 and the mean difference in viral load was 0.13 log₁₀ HCV IU/ml. For HBV, the correlation coefficient was 0.98 and the mean difference in viral load 0.61 log₁₀ HBV IU/ml. For CMV and EBV, the correlation coefficient was 0.98 and the mean difference in viral load 0.33 log₁₀ copies/ml. Accuracy was confirmed with a reference panel (QCMD, Glasgow, Scotland) for all four assays. No cross-contamination was observed when extracting strongly positive polyomavirus samples (8.10 log₁₀ copies/ml) interspersed with polyomavirus-negative samples. Automated extraction via the m1000 system offers a high reliability of extraction and resulted in a strong reduction of the required extraction hands-on time for high-throughput PCR compared to manual extraction protocols.
Language	English
Dates of publication	2005-11
Size	p. 5541-5546.
Document type	Article
ZDB-ID	390499-4
ISSN	1098-660X ; 0095-1137
ISSN (online)	1098-660X
ISSN	0095-1137
Database	NAL-Catalogue (AGRICOLA)

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Article: Isolation and cloning of two variant papillomaviruses from domestic pigs: Sus scrofa papillomaviruses type 1 variants a and b

Stevens, Hans / Rector, Annabel / Kroght, Kees van der / Ranst, Marc van

Journal of general virology. 2008 Oct., v. 89, pt. 10

2008

Abstract: The healthy skin of two female domestic pigs (Sus scrofa domestica) was sampled with cotton-tipped swabs. Total genomic DNA was extracted from the samples and subjected to PCR with degenerate papillomavirus (PV)-specific primers. Similarity searches ... ...

Abstract	The healthy skin of two female domestic pigs (Sus scrofa domestica) was sampled with cotton-tipped swabs. Total genomic DNA was extracted from the samples and subjected to PCR with degenerate papillomavirus (PV)-specific primers. Similarity searches performed with BLASTN showed that partial E1 and L1 sequences of two novel PVs were amplified. Subsequently, the complete genomes of these Sus scrofa papillomaviruses (SsPVs) were amplified by long-template PCR, cloned and sequenced using a transposon insertion method. They contained the typical PV open reading frames (ORFs) E1, E2, E4, E6, L1 and L2, but the E7 ORF was absent in both viruses. Pairwise nucleotide sequence alignment of the L1 ORFs of the SsPVs showed 98.5 % similarity, classifying these viruses as SsPV type 1 'variants' (SsPV-1a and -1b). Based on a concatenated alignment of the E1, E2, L1 and L2 ORFs of SsPV-1 variants a and b, and 81 other human and animal PV type species, a neighbour-joining phylogenetic tree was constructed. This phylogenetic analysis showed that the SsPV-1a and -1b variants did not cluster with the other PVs of artiodactyls (cloven-hoofed) host species, but clustered on the edge of the genus Alphapapillomavirus, very near to the root of this genus.
Keywords	Papillomaviridae ; swine ; genome ; DNA ; nucleotide sequences ; open reading frames ; sequence alignment ; phylogeny ; molecular cloning
Language	English
Dates of publication	2008-10
Size	p. 2475-2481.
Document type	Article
ZDB-ID	219316-4
ISSN	1465-2099 ; 0022-1317
ISSN (online)	1465-2099
ISSN	0022-1317
Database	NAL-Catalogue (AGRICOLA)

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Article: Development of One-Step, Real-Time, Quantitative Reverse Transcriptase PCR Assays for Absolute Quantitation of Human Coronaviruses OC43 and 229E

Vijgen, Leen / Keyaerts, Els / Moe̊s, Elien / Maes, Piet / Duson, Griet / Ranst, Marc van

Journal of clinical microbiology. 2005 Nov., v. 43, no. 11

2005

Abstract: The clinical significance of human coronaviruses in more severe respiratory illnesses has recently been shown to be higher than was previously assumed. Rapid and reliable diagnosis of human coronavirus infections therefore becomes indispensable in a ... ...

Abstract	The clinical significance of human coronaviruses in more severe respiratory illnesses has recently been shown to be higher than was previously assumed. Rapid and reliable diagnosis of human coronavirus infections therefore becomes indispensable in a routine clinical setting. In this study, we present a very sensitive and specific TaqMan-based, real-time quantitative reverse transcriptase PCR (qRT-PCR) for the rapid detection and quantitation of human coronaviruses (HCoVs) OC43 and 229E. Absolute viral load measurement in clinical samples was achieved through the construction of in-house HCoV OC43 and 229E cRNA standards for the generation of a standard curve. The HCoV OC43 assay allows quantitation over a range from 20 to 2 x 10⁸ RNA copies per reaction mixture (5 [micro]l RNA extract). When this is extrapolated to clinical samples, this corresponds to a detection range of 10³ to 10¹⁰ viral genome equivalents per ml. By using the HCoV 229E qRT-PCR assay, viral RNA copies ranging from 200 to 2 x 10⁹ per reaction mixture can be detected, which corresponds to 10⁴ to 10¹¹ viral genome equivalents per ml sample. A total of 100 respiratory samples screened for the presence of HCoVs OC43 and 229E by using conventional RT-PCR were assessed in parallel by the qRT-PCR assays. By use of the real-time qRT-PCR techniques, the detection rate of HCoVs OC43 and 229E increased from 2.0% to 3.1% and from 0.3% to 2.5%, respectively. The real-time qRT-PCR assays described here allow the rapid, specific, and sensitive laboratory detection and quantitation of human coronaviruses OC43 and 229E.
Language	English
Dates of publication	2005-11
Size	p. 5452-5456.
Document type	Article
ZDB-ID	390499-4
ISSN	1098-660X ; 0095-1137
ISSN (online)	1098-660X
ISSN	0095-1137
Database	NAL-Catalogue (AGRICOLA)

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