A new real-time PCR method to overcome significant quantitative inaccuracy due to slight amplification inhibition

Guescini, Michele; Sisti, Davide; Rocchi, Marco; Stocchi, Laura; Stocchi, Vilberto

doi:10.1186/1471-2105-9-326

Cited by 154 publications

(177 citation statements)

References 36 publications

(47 reference statements)

Supporting

Mentioning

170

Contrasting

Unclassified

Order By: Relevance

“…In this study, centrifugation was used to clarify fecal samples prior to RT-qRT-PCR, which reduces inhibition [33]. Potential inhibition in both water and fecal samples was also addressed in this study by quantifying using the Cy 0 method [37]. Conversely, RT-PCR may detect fragments from virus particles that have lost infectivity, over-estimating the potential for subsequent transmission of influenza at the site.…”

Section: Discussionmentioning

confidence: 99%

“…Influenza RNA in environmental samples was quantified using a modified standard curve-based method, the Cy 0 method [37]. Unlike the cycle-threshold ( Ct ) method, the Cy 0 method does not require the assumption of uniform reaction efficiency between standards and unknowns, making it ideal for the quantification of environmental samples that are likely to contain PCR-inhibiting compounds [37].…”

Section: Methodsmentioning

confidence: 99%

“…Unlike the cycle-threshold ( Ct ) method, the Cy 0 method does not require the assumption of uniform reaction efficiency between standards and unknowns, making it ideal for the quantification of environmental samples that are likely to contain PCR-inhibiting compounds [37]. The Cy 0 value is the intersection point between the abscissa axis and tangent of the inflection point of the Richards curve obtained by the non-linear regression of raw data [37]. As with Ct , a smaller Cy 0 indicates a higher number of genome copies, or target virus, in the sample.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Detection of Influenza A viruses at migratory bird stopover sites in Michigan, USA

Lickfett

Clark

Gehring

et al. 2018

Infection Ecology & Epidemiology

View full text Add to dashboard Cite

Introduction: Influenza A viruses have the potential to cause devastating illness in humans and domestic poultry. Wild birds are the natural reservoirs of Influenza A viruses and migratory birds are implicated in their global dissemination. High concentrations of this virus are excreted in the faeces of infected birds and faecal contamination of shared aquatic habitats can lead to indirect transmission among birds via the faecal-oral route. The role of migratory birds in the spread of avian influenza has led to large-scale surveillance efforts of circulating avian influenza viruses through direct sampling of live and dead wild birds. Environmental monitoring of bird habitats using molecular detection methods may provide additional information on the persistence of influenza virus at migratory stopover sites distributed across large spatial scales. Materials and methods: In the current study, faecal and water samples were collected at migratory stopover sites and evaluated for Influenza A by real-time quantitative reverse transcriptase PCR. Results and Discussion: This study found that Influenza A was detected at 53% of the evaluated stopover sites, and 7% and 4.8% of the faecal and water samples, respectively, tested positive for Influenza A virus. Conclusion: Environmental monitoring detected Influenza A at stopover sites used by migratory birds.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Detection of Influenza A viruses at migratory bird stopover sites in Michigan, USA

Lickfett

Clark

Gehring

et al. 2018

Infection Ecology & Epidemiology

View full text Add to dashboard Cite

show abstract

“…Реакция была оптимизирована для выполнения анализа на ампли-фикаторе iCycler iQ5 («Bio-Rad», США). Пороговый выход кривых амплификации ПЦР-РВ (Сq) опре-деляли методом Cy 0 [8]. Выделение ДНК из лейко-цитов цельной крови проводили с использовани-ем реагента «ДНК-экспресс-кровь» (ООО «НПФ Литех»), а также с использованием набора «ДНК-Сорб-В» (ФБУН «Центральный НИИ эпидемиоло-гии»).…”

Section: материалы и методыunclassified

Developing method for allelic somatic mutations burden of Janus kinase 2 (V617F JAK2) detection in pools of venous blood samples

Gorbenko¹,

Stolyar²,

Субботина³

et al. 2016

Lab. sluzh.

View full text Add to dashboard Cite

“…Esse programa foi desenvolvido seguindo as orientações presentes no artigo de Guescini et al (2008). A programadora Carine de Godoy…”

Section: Programa Para Cálculo Do Cy0unclassified

Extração de adenovírus em sistemas micelares de duas fases aquosas

Molino¹

View full text Add to dashboard Cite

Palavras-chave:Extração líquido-líquido. Adenovirus. Sistema micelar de duas fases aquosas. Biopartículas. ABSTRACTBiotechnological processes depend significantly on separation and purification techniques used to maintain cost-effective industrial-scale production of biotechnological products for commercial, industrial and therapeutic uses. The application of the aqueous two-phase micelar system (ATPMS) is proposed as an alternative for purification, since it allows the separation and analysis of biomolecules /bioparticles, often without loses of activity or their properties. This allows to perform a selective partition that enables high yields. This work aims to study the use of this methodology in the extraction and purification of adenovirus in micelle aqueous twophase formed by TritonX-114/Viral suspension. All assays were performed in 5 g systems following a full factorial design (2 3 ) with four central points. The studied factors were extraction temperature, pH of the viral suspension and concentration of the surfactant. Systems containing masses of 3g, 10g and 40g were evaluated.Extraction procedure effects over integrity and infectivity of adenovirus were also evaluated. Some of the parameters evaluated in the viral recovery process were viral potency (R Pv ) and recovery of viral specific potency (R Pvφ ). These two parameters measure the inactivation of Adenovirus by the extraction process and both showed improvement when compared with the viral suspension for some of the systems studied (i.e RPv: 341% and RPvφ 1466%). These results show that ATPMS selectively partition the infectious viral particles. According to the results of the experimental design is possible to increase, even further, these results controlling the surfactant concentration, viral suspension pH and temperature of extraction.

show abstract

A new real-time PCR method to overcome significant quantitative inaccuracy due to slight amplification inhibition

Cited by 154 publications

References 36 publications

Detection of Influenza A viruses at migratory bird stopover sites in Michigan, USA

Detection of Influenza A viruses at migratory bird stopover sites in Michigan, USA

Developing method for allelic somatic mutations burden of Janus kinase 2 (V617F JAK2) detection in pools of venous blood samples

Extração de adenovírus em sistemas micelares de duas fases aquosas

Contact Info

Product

Resources

About