Improved Molecular Typing Assay for Rhinovirus Species A, B, and C

Bochkov, Yury A.; Grindle, Kristine A.; Vang, Fue; Evans, Michael D.; Gern, James E.

doi:10.1128/jcm.00075-14

Cited by 89 publications

(80 citation statements)

References 38 publications

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“…Other genotype sequence groups with more mismatches than genotype group N did not show amplification inhibition by RT-dPCR. It is likely that the effect of a sequence mismatch on amplification is highly context dependent, given that the 5= NCR region of HRV harbors significant RNA secondary structure (20). Nonetheless, dPCR is a better alternative to qPCR on templates known to have significant sequence diversity that cannot be avoided during primer and probe design.…”

Section: Discussionmentioning

confidence: 99%

Superiority of Digital Reverse Transcription-PCR (RT-PCR) over Real-Time RT-PCR for Quantitation of Highly Divergent Human Rhinoviruses

et al. 2017

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Human rhinoviruses (HRV) comprise 3 species representing more than 150 genotypes. As an important human respiratory pathogen, molecular detection is an indispensable tool for diagnosis and surveillance. However, the sequence diversity of HRV genotypes poses challenges for developing robust molecular methods that detect all genotypes with equal efficiencies. This study compares the accuracies of reverse transcription-quantitative PCR (RT-qPCR) and reverse transcription-digital PCR (RT-dPCR) for quantifying HRV RNA using genotype-specific primers and probes and a consensus primer/probe set targeting the 5= noncoding region of HRV. When using consensus primers and probes for the quantification of HRV, RT-dPCR outperformed RT-qPCR by consistently and accurately quantifying HRV RNAs across more genotype groups, despite the presence of up to 2 target-sequence mismatches within the primer or probe binding region. Because it does not rely on amplification efficiency, which can be affected by sequence mismatches in primer/probe binding regions, RT-dPCR may be the optimal molecular method for future HRV quantification studies and for quantitating other viruses with high sequence diversity.KEYWORDS human rhinovirus, digital PCR, qPCR H uman rhinoviruses (HRV) are important human respiratory pathogens that are small positive-sense RNA viruses within the family Picornaviridae. There are more than 150 genotypes of HRV that have been recognized within species A, B, and C of the genus Enterovirus (1). Molecular assays, such as reverse-transcription PCR (RT-PCR), are the most useful methods for detecting HRV in clinical samples (2-4). Most HRV RT-PCR assays target the conserved 5= noncoding region (NCR), which exhibits the greatest sequence homology among the HRV genotypes. However, even in the 5= NCR, consensus primer and probe sets must be designed with degenerate and modified bases or multiple oligonucleotides to amplify all HRV genotypes (5-7).Although qualitative detection of HRV by RT-PCR is currently sufficient for determining HRV infection, accurate quantification of HRV RNA in clinical samples is needed for studies associating HRV viral load with viral transmission and with patient symptoms and outcomes. Viral load studies of other respiratory viruses have shown that a correlation exists between viral load and disease severity (8-10). Accurate quantification of HRV will also be required for evaluating the performances of future antiviral drugs. Real-time RT-PCR assays, when accompanied by the amplification of standard curves (RT-qPCR), can be used to quantify the number of viral copies in a sample. However, RT-qPCR assays using quantification with a consensus HRV primer and probe set may not give accurate results for all genotypes due to amplification inefficiencies caused by base mismatches between the primer and probe sequences and the specific

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Section: Discussionmentioning

confidence: 99%

Superiority of Digital Reverse Transcription-PCR (RT-PCR) over Real-Time RT-PCR for Quantitation of Highly Divergent Human Rhinoviruses

et al. 2017

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“…In our laboratory, RV was examined in respiratory samples collected at recruitment and retrieved from the PMH Department of microbiology, using a two-step PCR amplification of the RV 5’ non-coding region as previously described [2, 6, 24]. If positive, PCR products were DNA sequenced and RV species were determined by alignment with the RV sequence of 101 classical serotypes and the 53 recently identified types using phylogenetic tree reconstruction with Clustal X Software.…”

Section: Methodsmentioning

confidence: 99%

Recurrent Rhinovirus Detections in Children Following a Rhinovirus-Induced Wheezing Exacerbation: A Retrospective Study

Zhang

et al. 2015

Int. J. Pediatr. Child Health

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Introduction It is unclear if children with a rhinovirus (RV)-induced wheezing exacerbation are more susceptible to viruses longitudinally, and whether a parental history of asthma and/or allergy impacts their susceptibility. The objective of this study was to determine if RV, RV-A and RV-C related wheezing exacerbations in children were associated with prior or subsequent viral detections and investigate the role of parental history of asthma and allergy. Materials and methods Children presenting to hospital with acute wheeze were prospectively recruited and tested for respiratory viruses. Data on viruses detected in other respiratory samples (May 1997 to December 2012) were collected from hospital microbiology records and additional RV testing was performed on stored hospital respiratory samples (September 2009 to December 2012). A positive parental history was defined as either parent with self-reported asthma and/or allergy. Results At recruitment, RV was detected in 69.2% of samples from children with an acute wheezing episode (n=373, 0–16 years of age), with RV-C the most common virus (65.5%). Children with a history of parental asthma and/or allergy and RV at recruitment had a 14-fold increased incidence rate ratio (IRR) of subsequent RV detection (IRR 14.0, 95% CI 1.9–104.1; p=0.01) compared with children without RV at recruitment. Children without this parental history had a reduced incident rate ratio for samples assessed during this time (IRR 0.5, 95% CI 0.3–0.9; p=0.03). Conclusion Children with a parental history of asthma and/or allergy may become more susceptible to recurrent symptomatic RV infections.

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“…В семействе Paramyxoviridae в 80% случаев выделялись че-ловеческие респираторно-синцитиальные ви-русы (hRSV), в семействе Picornaviridae доми-нировали риновирусы А (65%) и риновирусы С (35%), а в семействе Orthomyxoviridae 96% кон-тиг были гомологичны геному вируса гриппа А [34]. Представленные результаты согласуются с данными других исследователей, посвящен-ными вирусной составляющей метагенома ды-хательных путей человека [35,36].…”

Section: Fundamental and Clinical Medicineunclassified

“…В арсенале хи-рурга есть МРТ-холангиография и операцион-ная холангиография [35,36]. Лечение Хирургическое лечение синдрома Мириззи: при I типе -холецистэктомия, ревизия желч-ных протоков с дренированием Т-образной трубкой; при II, III типах -холецистэктомия, удаление конкрементов, ушивание свищевого отверстия атравматическим рассасывающим-ся шовным материалом или пластика дефекта оставленной частью культи желчного пузыря, дренирование Т-образной трубкой; при IV типе -предпочтительнее гепатикоеюностомия с пет-лёй тощей кишки по Ру, так как стенка обще-го печеночного протока полностью разрушена.…”

Section: рак желчного пузыряunclassified

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Improved Molecular Typing Assay for Rhinovirus Species A, B, and C

Cited by 89 publications

References 38 publications

Superiority of Digital Reverse Transcription-PCR (RT-PCR) over Real-Time RT-PCR for Quantitation of Highly Divergent Human Rhinoviruses

Superiority of Digital Reverse Transcription-PCR (RT-PCR) over Real-Time RT-PCR for Quantitation of Highly Divergent Human Rhinoviruses

Recurrent Rhinovirus Detections in Children Following a Rhinovirus-Induced Wheezing Exacerbation: A Retrospective Study

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