Design and Production of a Chimeric Construct as a Genetically Positive Control for the Detection of Burkholderia and Yellow Fever Virus Using the Real-Time PCR

ABSTRACTDengue virus (DENV) is the cause of dengue / severe dengue and a virus of the Flaviviridae family, furthermore, dengue fever has rapidly spread in the world in recent decades. DENV is transmitted by female mosquitoes, mainly of the specie Aedes aegypti. The main method to control or prevent the transmission of DENV is to combat the mosquito vectors. Among these, one of important methods is to monitor the DENVs in the mosquito vectors.For the detection of DENV, nucleic acid amplification tests (NAAT) were recommended, of which criterion standard is real-time RT-PCR with highly sensitive and specific. However, it takes long time as to judge the result per a reaction, besides the necessity of the treatment of RNA in advance, example of extraction, concentration and purification.It was our object in this time to develop the method of real-time RT-PCR detecting DENVs in shorter time, moreover without especial treatment of RNA from the mosquito in advance. Besides, this work was performed with combing the mobile real-time PCR device with the one-step RT-PCR reagent.Firstly, we succeeded in shortening the time of real-time RT-PCR for the detection of DENV per one reaction, so that the judgement needed less than 20 minutes if genomic RNA treated in advance. Moreover, each value on the real-PCR device was quantitatively correlated with the positive control RNA from 1.0 × 10 ^ 3 copies to 1.0 × 10 ^ 0 copies per reaction (This correlation coefficient R2 > 0.95). Additionally, it made sure that this method could be applied to each DENV serotype.Secondly, we established the basis of procedure for the real-time RT-PCR without the treatment in advance so-called “direct”. As the result that the positive control RNA additive was utilized instead of the real DENV, spiked into the mosquito homogenized and sampled the supernatant without treatment, it was possible to detect on the real-time RT-PCR even if mosquitoes immediately after blood-feeding. For this reason, this method might be able to utilize in human sera, too.According to the results of this work, we could suggest the method is possible to detect DENV more quickly and more simply than heretofore. The Real-time “direct” RT-PCR, especially, could be performed with mobile real-time PCR PCR1100 device and one step RT-PCR reagent only. This method must help to detect some viruses other than DENV, too.

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Dengue Virus and Yellow Fever Virus Detection Using Reverse Transcription–Insulated Isothermal PCR and Comparison with Real-Time RT-PCR

American Journal of Tropical Medicine and Hygiene ◽

10.4269/ajtmh.19-0892 ◽

2020 ◽

Vol 103 (1) ◽

pp. 157-159

Author(s):

Victoria Stittleburg ◽

Alejandra Rojas ◽

Fátima Cardozo ◽

Flor M. Muñoz ◽

Edwin J. Asturias ◽

...

Keyword(s):

Dengue Virus ◽

Real Time ◽

Yellow Fever ◽

Reverse Transcription ◽

Yellow Fever Virus ◽

Virus Detection ◽

Fever Virus ◽

Rt Pcr ◽

Insulated Isothermal Pcr

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Yellow fever virus spread in Rio de Janeiro and Espírito Santo, 2016-2019: Phylodynamic assessment to improve intervention strategies

10.1101/711994 ◽

2019 ◽

Cited By ~ 1

Author(s):

Marta Giovanetti ◽

Marcos Cesar Lima de Mendonça ◽

Vagner Fonseca ◽

Maria Angélica Mares-Guia ◽

Allison Fabri ◽

...

Keyword(s):

Public Health ◽

Real Time ◽

Yellow Fever ◽

Rio De Janeiro ◽

Yellow Fever Virus ◽

Fever Virus ◽

Southeastern Brazil ◽

Espírito Santo ◽

Espirito Santo ◽

Southeastern Region

ABSTRACTThe recent re-emergence of yellow fever virus (YFV) in Brazil has raised serious concerns due to the virus’ rapid dissemination in the southeastern region. To better understand YFV genetic diversity and dynamics during the recent outbreak in southeastern Brazil we generated 18 complete and near-complete genomes from the peak of the epidemic curve from non-human primates (NHPs) and human infected cases across Espírito Santo and Rio de Janeiro states. Genomic sequencing of 18 YFV genomes revealed the timing, source and likely routes of yellow fever virus transmission and dispersion during the one of the largest outbreaks ever registered in Brazil. We showed that the recent YFV epidemic spillover southwards several times from Minas Gerais to Espírito Santo and Rio de Janeiro states in 2016 to 2019. The quick production and analysis of data from portable sequencing could identify the corridor of spread of YFV. These findings reinforce that real-time and continued genomic surveillance strategies can assist in the monitoring and public health responses of arbovirus epidemics.IMPORTANCEArbovirus infections in Brazil including Yellow Fever, Dengue, Zika and Chikungunya result in considerable morbidity and mortality and are pressing public health concerns. However, our understanding of these outbreaks is hampered by limited availability of real time genomic data. In this study, we investigated the genetic diversity and spatial distribution of YFV during the current outbreak in southeastern Brazil. To gain insights into the routes of YFV introduction and dispersion, we tracked the virus by sequencing YFV genomes sampled from non-human primates and infected patients from the southeastern region. Our study provides an understanding of how YFV initiates transmission in new Brazilian regions and illustrates that near-real time genomics in the field can augment traditional approaches to infectious disease surveillance and control.

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Development of a Real-Time Reverse Transcription-PCR Assay for Global Differentiation of Yellow Fever Virus Vaccine-Related Adverse Events from Natural Infections

Journal of Clinical Microbiology ◽

10.1128/jcm.00323-18 ◽

2018 ◽

Vol 56 (6) ◽

Cited By ~ 3

Author(s):

Holly R. Hughes ◽

Brandy J. Russell ◽

Eric C. Mossel ◽

John Kayiwa ◽

Julius Lutwama ◽

...

Keyword(s):

Adverse Events ◽

Real Time ◽

Yellow Fever ◽

Vaccine Strain ◽

Reverse Transcription ◽

Yellow Fever Virus ◽

Fever Virus ◽

Wild Type ◽

Pcr Assay ◽

Reverse Transcription Pcr

ABSTRACT Yellow fever (YF) is a reemerging public health threat, with frequent outbreaks prompting large vaccination campaigns in regions of endemicity in Africa and South America. Specific detection of vaccine-related adverse events is resource-intensive, time-consuming, and difficult to achieve during an outbreak. To address this, we have developed a highly transferable rapid yellow fever virus (YFV) vaccine-specific real-time reverse transcription-PCR (RT-PCR) assay that distinguishes vaccine from wild-type lineages. The assay utilizes a specific hydrolysis probe that includes locked nucleic acids to enhance specific discrimination of the YFV17D vaccine strain genome. Promisingly, sensitivity and specificity analyses reveal this assay to be highly specific to vaccine strain(s) when tested on clinical samples and YFV cell culture isolates of global origin. Taken together, our data suggest the utility of this assay for use in laboratories of varied capacity for the identification and differentiation of vaccine-related adverse events from wild-type infections of both African and South American origin.

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