Rapid detection and differentiation of avian infectious bronchitis virus: an application of Mass genotype by melting temperature analysis in RT-qPCR using SYBR Green I

Okino, Cíntia Hiromi; Montassier, Maria de Fátima Silva; Oliveira, Andressa Peres de; Montassier, Hélio José

doi:10.1292/jvms.17-0566

Cited by 7 publications

(4 citation statements)

References 34 publications

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“…The genetic classification of IBV has relied on genotype-specific RT-qPCR assays, serotype-specific S1 RT-qPCR 32,40 and/or pan-IBV S1 RT-PCR assays coupled with Sanger sequencing. 8,31,49 Genotype-specific RT-qPCRs are limited to short fragments, which may miss important changes in the S1 (~1.6 kb) 44 outside the short target.…”

Section: Introductionmentioning

confidence: 99%

Real-time, MinION-based, amplicon sequencing for lineage typing of infectious bronchitis virus from upper respiratory samples

Butt

Erwood

Zhang

et al. 2019

Preprint

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Infectious bronchitis (IB) causes significant economic losses in the global poultry industry. Control of infectious bronchitis is hindered by the genetic diversity of the causative agent, infectious bronchitis virus (IBV), which has led to the emergence of several serotypes that lack complete serologic cross-protection. While serotyping by definition requires immunologic characterization, genotyping is an efficient means to identify IBVs detected in samples. Sanger sequencing of the S1 subunit of the spike gene is currently used to genotype IBV; however, the universal S1 PCR was created to work from cultured IBV and it is inefficient at detecting mixed isolates. This paper describes a MinION-based AmpSeq method that genetically typed IBV from clinical samples, including samples with multiple isolates. Total RNA was extracted from fifteen tracheal scrapings and choanal cleft swab samples, randomly reverse transcribed, and PCR amplified using modified S1-targeted primers. Amplicons were barcoded to allow for pooling of samples, processed per manufacturer's instructions into a 1D MinION sequencing library, and sequenced on the MinION. The AmpSeq method detected IBV in 13 of 14 IBV-positive samples.AmpSeq accurately detected and genotyped both IBV lineages in three of five samples containing two IBV lineages. Additionally, one sample contained three IBV lineages, and AmpSeq accurately detected two of the three. Strain identification, including detection of different strains from the same lineage, was also possible with this AmpSeq method. The results demonstrate the feasibility of using MinION-based AmpSeq for rapid and accurate identification and lineage typing of IBV from oral swab samples.Infectious bronchitis virus is an enveloped, pleomorphic gammacoronavirus with an unsegmented, single-stranded, positive-sense, 26-27.8 Kb, RNA genome that encodes the nonstructural polyprotein,1a and 1b, and several structural proteins: spike (S), envelop (E), membrane (M) and nucleocapsid (N). 38,41 In addition, two accessory genes, expressing 3a, 3b and 5a and 5b, respectively, have also been described. 6,14,38 The S protein is highly glycosylated and post-translational cleavage leads to two subunits: S1 and S2. 10,48 Besides acting as the viral attachment protein, the S1 protein is a major target of neutralizing antibodies. 7 As with many attachment proteins that are targets of virus-neutralizing antibodies, the S1 subunit is highly diverse with almost 50% of the amino acids differing among IBV serotypes. 2,21,38 Such variation leads to important biological differences between IBV serotypes and the emergence of novel variants. More than 60 serotypes of IBV have been reported, but the most common serotypes of All rights reserved. No reuse allowed without permission.Massachusetts. 16 This genetic diversity leads to emergence of new serotypes and a lack of complete cross-serotype protection by vaccines. 29 The correlation of IBV genotypes and serotypes of IBV has been reported 45 ; therefore, accurate genotypic identification o...

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Section: Introductionmentioning

confidence: 99%

Real-time, MinION-based, amplicon sequencing for lineage typing of infectious bronchitis virus from upper respiratory samples

Butt

Erwood

Zhang

et al. 2019

Preprint

View full text Add to dashboard Cite

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“…Several PCR options allow you to highly reliably determine the genotypic structure of the virus and attribute it to a specific strain. Most often, for the quantitative and qualitative determination of the incidence in birds caused by RNA-containing viruses, the real-time reverse transcription polymerase chain reaction (qRT-PCR or RT-PCR) method is used ( Shanmuganathan et al, 2017 ; Okino et al, 2018 ; Al-Jallad et al, 2020 ; Mo et al, 2020 ; Salem et al, 2020 ; Ongor et al, 2021 ; Worku et al, 2022 ) for the typing of the chicken IBV as well as for the isolation of the NDV. The difference between the methods of different researchers was using various primers and probes for typing viral strains.…”

Section: Introductionmentioning

confidence: 99%

The use of RT-PCR in the diagnosis and differentiation of vaccine strains of chicken infectious bronchitis and Newcastle disease

Assanov,

Bazarbayev,

Mussoyev

et al. 2023

Open Vet J

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Background: Infectious diseases of young and adult birds with respiratory syndrome are a significant deterrent to the development of industrial poultry farming due to a decrease in productivity and significant mortality. The only effective method of combating viral diseases is timely and targeted vaccination, which largely depends on laboratory diagnostic results. Aim: The aim of this article is to study the real-time reverse transcription polymerase chain reaction, which has the prospect of more effective diagnosis of vaccine strains of chicken infectious bronchitis and Newcastle disease. Methods: The fastest and most accurate method for the differential diagnosis of pathogens in an associative viral infection is reverse transcription polymerase chain reaction. The method proposed in the article for selecting primers for amplification made it possible to use this method for the simultaneous interspecies differential diagnosis of two or more viral agents, which significantly accelerated their diagnosis. Results: The correlation of the nucleotide sequence obtained as a result of sequencing to a specific strain of the virus is complicated by the lack of a single nomenclature mechanism for separating genetic groups. Conclusion: The results of this study will allow easy and fast typing of sequences into known and databased virus strains, and avoid further confusion in the nomenclature of genetic groups in the future.

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“…The genetic classification of IBV has relied on genotype-specific, reverse-transcription real-time PCR (RT-rtPCR) assays, serotype-specific S1 RT-rtPCR, 32,40 and/or pan-IBV S1 RT-PCR assays coupled with Sanger sequencing. 8,31,49 Genotype-specific RT-rtPCR assays are limited to short fragments, which may miss important changes in the S1 gene (~1.6 kb) 44 outside the short target.…”

Section: Introductionmentioning

confidence: 99%

Real-time, MinION-based, amplicon sequencing for lineage typing of infectious bronchitis virus from upper respiratory samples

et al. 2020

View full text Add to dashboard Cite

Infectious bronchitis (IB) causes significant economic losses in the global poultry industry. Control of IB is hindered by the genetic diversity of the causative agent, infectious bronchitis virus (IBV), which has led to the emergence of several serotypes that lack complete serologic cross-protection. Although serotyping requires immunologic characterization, genotyping is an efficient means to identify IBVs detected in samples. Sanger sequencing of the S1 subunit of the spike gene is currently used to genotype IBV; however, the universal S1 PCR was created to work from cultured IBV, and it is inefficient at detecting multiple viruses in a single sample. We describe herein a MinION-based, amplicon-based sequencing (AmpSeq) method that genetically categorized IBV from clinical samples, including samples with multiple IBVs. Total RNA was extracted from 15 tracheal scrapings and choanal cleft swab samples, randomly reverse transcribed, and PCR amplified using modified S1-targeted primers. Amplicons were barcoded to allow for pooling of samples, processed per manufacturer’s instructions into a 1D MinION sequencing library, and then sequenced on the MinION. The AmpSeq method detected IBV in 13 of 14 IBV-positive samples. AmpSeq accurately detected and genotyped both IBV lineages in 3 of 5 samples containing 2 IBV lineages. Additionally, 1 sample contained 3 IBV lineages, and AmpSeq accurately detected 2 of the 3 lineages. Strain identification, including detection of different IBVs from the same lineage, was also possible with this AmpSeq method. Our results demonstrate the feasibility of using MinION-based AmpSeq for rapid and accurate identification and lineage typing of IBV from oral swab samples.

show abstract

Rapid detection and differentiation of avian infectious bronchitis virus: an application of Mass genotype by melting temperature analysis in RT-qPCR using SYBR Green I

Cited by 7 publications

References 34 publications

Real-time, MinION-based, amplicon sequencing for lineage typing of infectious bronchitis virus from upper respiratory samples

Real-time, MinION-based, amplicon sequencing for lineage typing of infectious bronchitis virus from upper respiratory samples

The use of RT-PCR in the diagnosis and differentiation of vaccine strains of chicken infectious bronchitis and Newcastle disease

Real-time, MinION-based, amplicon sequencing for lineage typing of infectious bronchitis virus from upper respiratory samples

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