Long-term conservation of HCV RNA at 4°C using a new RNA stabilizing solution

Gonzalez-Perez, Idania; Cayarga, Anny Armas; Hernández, Yenitse Perea; Rosa, Iria García de la; González, Yaimé Josefina González; León, Carlos Silva; Alvarez, Rene

doi:10.1016/j.jviromet.2010.05.015

Cited by 7 publications

(5 citation statements)

References 28 publications

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“…However, extracted RNA stored in aqueous solutions, even at −70 °C, is prone to degradation [ 10 ]. Therefore, protecting RNA using various RNA safe solutions has been proposed [ 9 , 11 , 12 ]. The current study was designed to evaluate, different conditions used to effectively and safely store RNA for future downstream applications such as molecular diagnosis.…”

Section: Introductionmentioning

confidence: 99%

Impact of RNA Degradation on Viral Diagnosis: An Understated but Essential Step for the Successful Establishment of a Diagnosis Network

Relova

Rios

Acevedo

et al. 2018

Veterinary Sciences

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The current global conditions, which include intensive globalization, climate changes, and viral evolution among other factors, have led to an increased emergence of viruses and new viral diseases; RNA viruses are key drivers of this evolution. Laboratory networks that are linked to central reference laboratories are required to conduct both active and passive environmental surveillance of this complicated global viral environment. These tasks require a continuous exchange of strains or field samples between different diagnostic laboratories. The shipment of these samples on dry ice represents both a biological hazard and a general health risk. Moreover, the requirement to ship on dry ice could be hampered by high costs, particularly in underdeveloped countries or regions located far from each other. To solve these issues, the shipment of RNA isolated from viral suspensions or directly from field samples could be a useful way to share viral genetic material. However, extracted RNA stored in aqueous solutions, even at −70 °C, is highly prone to degradation. The current study evaluated different RNA storage conditions for safety and feasibility for future use in molecular diagnostics. The in vitro RNA-transcripts obtained from an inactivated highly pathogenic avian influenza (HPAI) H5N1 virus was used as a model. The role of secondary structures in the protection of the RNA was also explored. Of the conditions evaluated, the dry pellet matrix was best able to protect viral RNA under extreme storage conditions. This method is safe, cost-effective and assures the integrity of RNA samples for reliable molecular diagnosis. This study aligns with the globally significant “Global One Health” paradigm, especially with respect to the diagnosis of emerging diseases that require confirmation by reference laboratories.

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Section: Introductionmentioning

confidence: 99%

Impact of RNA Degradation on Viral Diagnosis: An Understated but Essential Step for the Successful Establishment of a Diagnosis Network

Relova

Rios

Acevedo

et al. 2018

Veterinary Sciences

View full text Add to dashboard Cite

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“…Additionally, a very strong correlation was observed between the number of viral reads generated through sequencing and the starting number of viral copies, indicating sequencing reads can be predicted by viral copies in a sample and vice versa, which has been confirmed by other studies as well [28]. Interestingly, the observation that the sensitivity of sequencing was higher from cell culture virus spiked into serum as opposed to clinical serum samples suggests that sample handling or perhaps the quality of the sample was an important factor for sequencing sensitivity [79], thus emphasizing the importance of careful handling, transporting, and storing of clinical samples to protect the viral RNA from degradation [80,81]. This also suggests that on-site sequencing of samples as opposed to a centralized diagnostic system may allow for higher sensitivity of detection due to the ability to immediately process samples after sampling.…”

Section: Discussionmentioning

confidence: 52%

Rapid, Unbiased PRRSV Strain Detection Using MinION Direct RNA Sequencing and Bioinformatics Tools

Tan

Dvorak

Murtaugh

2019

Viruses

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Prompt detection and effective control of porcine reproductive and respiratory syndrome virus (PRRSV) during outbreaks is important given its immense adverse impact on the swine industry. However, the diagnostic process can be challenging due to the high genetic diversity and high mutation rate of PRRSV. A diagnostic method that can provide more detailed genetic information about pathogens is urgently needed. In this study, we evaluated the ability of Oxford Nanopore MinION direct RNA sequencing to generate a PRRSV whole genome sequence and detect and discriminate virus at the strain-level. A nearly full length PRRSV genome was successfully generated from raw sequence reads, achieving an accuracy of 96% after consensus genome generation. Direct RNA sequencing reliably detected the PRRSV strain present with an accuracy of 99.9% using as few as 5 raw sequencing reads and successfully differentiated multiple co-infecting strains present in a sample. In addition, PRRSV strain information was obtained from clinical samples containing 10 4 to 10 6 viral copies or more within 6 hours of sequencing. Overall, direct viral RNA sequencing followed by bioinformatic analysis proves to be a promising approach for identification of the viral strain or strains involved in clinical infections, allowing for more precise prevention and control strategies during PRRSV outbreaks.2 of 18 against a new introduction from outside the farm indicates a need for enhanced biosecurity, while a re-break of a resident strain suggests better strategies for an elimination or vaccination program are needed. Another big challenge for PRRS control is that PRRSV vaccine is not completely effective at preventing and controlling infection due to the high genetic diversity of the virus, thus outbreaks still occur in vaccinated herds [13][14][15][16]. A diagnostic method which can provide genetic information about the strain causing infection would allow for identification of potential reasons for vaccination failure, such as limited cross-protection due to high genetic divergence from vaccine [17], or in the case of genetic similarity to vaccine, perhaps a reversion to virulence (escape mutation) of the vaccine itself [18]. In addition to the clinical challenges mentioned above, PRRSV has widely divergent genetic lineages and is a rapidly evolving pathogen with novel variants which seem to be more divergent and virulent than those in the past [10,[19][20][21]. The continuous emergence of new virulent strains causes unexpected devastating outbreaks, such as the severe outbreaks of HP-PRRSV in China and MN184 and NADC30 outbreaks in the United States [22][23][24][25]. The increasing incidence of co-infections of multiple strains further complicates PRRS diagnosis and control [26]. Hence, diagnostic tools that can provide more genetic information are extremely important for investigation, prevention, and control strategies for PRRSV outbreaks.Prompt detection of pathogens during an outbreak is essential for efficient disease control. Real-time quantitative...

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“…In summary, the HIV-1 and IC transcripts were obtained with the required quality to be used as standards for quantification purposes, considering also the advantage of their long-term stability when stored in the lysis reagent [20, 21]. It was obtained a high correlation between our assay and a commercial one.…”

Section: Resultsmentioning

confidence: 99%

“…The purified HIV-1 and IC transcripts were diluted 1 : 4 in a lysis reagent for preservation at 4°C [20, 21]. For the evaluation of the quality of the HIV-1 and IC transcripts, they were 10-fold serially diluted in lysis reagent from 1 : 4 × 10 3 to 1 : 4 × 10 9 .…”

Section: Methodsmentioning

confidence: 99%

Generation of HIV-1 and Internal Control Transcripts as Standards for an In-House Quantitative Competitive RT-PCR Assay to Determine HIV-1 Viral Load

Cayarga

Hernández

González

et al. 2011

Biotechnology Research International

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Human immunodeficiency virus type-1 (HIV-1) viral load is useful for monitoring disease progression in HIV-infected individuals. We generated RNA standards of HIV-1 and internal control (IC) by in vitro transcription and evaluated its performance in a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. HIV-1 and IC standards were obtained at high RNA concentrations, without DNA contamination. When these transcripts were included as standards in a qRT-PCR assay, it was obtained a good accuracy (±0.5 log10 unit of the expected results) in the quantification of the HIV-1 RNA international standard and controls. The lower limit detection achieved using these standards was 511.0 IU/mL. A high correlation (r = 0.925) was obtained between the in-house qRT-PCR assay and the NucliSens easyQ HIV-1 test (bioMerieux) for HIV-1 RNA quantitation with clinical samples (N = 14). HIV-1 and IC RNA transcripts, generated in this study, proved to be useful as standards in an in-house qRT-PCR assay for determination of HIV-1 viral load.

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Long-term conservation of HCV RNA at 4°C using a new RNA stabilizing solution

Cited by 7 publications

References 28 publications

Impact of RNA Degradation on Viral Diagnosis: An Understated but Essential Step for the Successful Establishment of a Diagnosis Network

Impact of RNA Degradation on Viral Diagnosis: An Understated but Essential Step for the Successful Establishment of a Diagnosis Network

Rapid, Unbiased PRRSV Strain Detection Using MinION Direct RNA Sequencing and Bioinformatics Tools

Generation of HIV-1 and Internal Control Transcripts as Standards for an In-House Quantitative Competitive RT-PCR Assay to Determine HIV-1 Viral Load

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