Virus inactivation by nucleic acid extraction reagents

Blow, Jamie A.; Dohm, David J.; Negley, Diane L.; Mores, Christopher N.

doi:10.1016/j.jviromet.2004.03.015

Cited by 142 publications

(109 citation statements)

References 3 publications

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“…The previous study tested filoviruses or filovirus tissue samples and not the more representative, in terms of a practical diagnostic sample type, whole blood. It is possible that blood extends a protective effect to suspended virions against inactivation methods, which was not tested by Blow et al (15). It may also be that the formulation of Buffer AVL has changed in the period between the two studies.…”

Section: Discussionmentioning

confidence: 98%

“…Buffer AVL and/or ethanol were mixed by vortexing with blood or serum samples for 10 min at a ratio of 4:1 (vol/vol) reagent/sample, retaining the ratio of reagent to sample as defined in the viral RNA minikit instructions (14) and as tested in the previous Buffer AVL study (15). Typically, 560 l Buffer AVL or ethanol was added to 140-l EBOVKikwit blood samples (14).…”

Section: Methodsmentioning

confidence: 99%

“…Buffer AVL contains a chaotropic salt (guanidine isothiocyanate) and has previously been reported to inactivate EBOV (15), and it is the first reagent used in common RNA extraction techniques (14). In this study, we extended the findings of previous research to evaluate the EBOV inactivation efficacies of ethanol and heat in whole blood.…”

Section: A N Outbreak Of Ebola Virus Disease (Evd) Occurred In West Amentioning

confidence: 99%

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Buffer AVL Alone Does Not Inactivate Ebola Virus in a Representative Clinical Sample Type

et al. 2015

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Rapid inactivation of Ebola virus (EBOV) is crucial for high-throughput testing of clinical samples in low-. In response to this outbreak, the international community has deployed an increasing number of Ebola diagnostic laboratories into the main West African countries affected (Guinea, Liberia, and Sierra Leone). Rapid diagnosis of EVD in humans is critical in the management of this disease in outbreak situations, as it allows prompt isolation and the chance to provide the best supportive care to patients, which helps reduce the overall infection rate and break the transmission chain.The preferred clinical sample for testing for Ebola virus (EBOV), an enveloped negative-sense single-strand RNA virus, is EDTA-blood, serum, or plasma with the primary diagnostic technology being real-time PCR (2). Other sample types, such as swabs or urine, may also be received by a laboratory. EBOV is designated in the United Kingdom by the Advisory Committee on Dangerous Pathogens (ACDP) as a hazard group 4 pathogen that must be handled under containment level (CL) 4 standards (biosafety level 4 [BSL4] in other countries). As such stringent laboratory infrastructure and containment procedures are required to handle viable EBOV material, only a few laboratories in Europe and elsewhere are suitably equipped (3). Within the timelines and budgets available, it has been impractical to create this laboratory infrastructure in West Africa, and therefore diagnostic laboratories have relied on methods that rapidly inactivate EBOV prior to routine processing and testing of samples by PCR.Laboratory methods of EBOV inactivation include gamma irradiation (4), nanoemulsion (5), photoinducible alkylating agents (6), and UV radiation (7), but these methods are primarily used for research purposes and may not be practicable in an outbreak situation that is likely to involve a high number of samples but reduced capability for handling and manipulation. In this context, any inactivation method must also be compatible with the EBOV PCR diagnostic approach.The CDC recommends Triton X-100 and heat treatment for 1 h for diagnostic samples containing hemorrhagic fever viruses (8), and this method has been adopted by many laboratories for handling of samples that may contain EBOV (9). Heating (alone or with acetic acid) for 1 h at 60°C has also been shown to reduce the titer of EBOV (10). Other guidelines can be nonspecific, specifying only the need for inactivation but not suggesting how (11) or suggesting generic use of denaturing/lysis buffers and/or heat (12). In the United Kingdom, the Advisory Committee on Dangerous Pathogens guidelines state that samples from confirmed cases may be processed in a containment level 2 laboratory using routine autoanalyzers if a containment level 4 laboratory is not available and provided specific procedures are followed (13). Within these guidelines, which encompass the application of multiple clinical tests, there is no specific requirement to inactivate EBOV (or other viral hemorrhagic fever agents) within a sa...

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Section: Discussionmentioning

confidence: 98%

Section: Methodsmentioning

confidence: 99%

Section: A N Outbreak Of Ebola Virus Disease (Evd) Occurred In West Amentioning

confidence: 99%

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Buffer AVL Alone Does Not Inactivate Ebola Virus in a Representative Clinical Sample Type

et al. 2015

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“…The use of commercially available NA extraction reagents for EBOV inactivation is well known (13), and these reagents are currently used (15). A recent report by Smither et al, in 2015, showed that the frequently used AVL buffer alone did not inactivate EBOV.…”

Section: Discussionmentioning

confidence: 99%

“…Presently, laboratory EBOV inactivation is accomplished by gamma irradiation (8), UV radiation (9), nanoemulsion (10), and photoinducible alkylating agents (11), but these methods are not applicable in outbreak situations or as bedside inactivation methods. Other EBOV inactivation methods, such as acetic acid (12), heat (12), AVL buffer (13), TRIzol (13) or the combination of heat and Triton X-100 (14), are more applicable in outbreak situations and are currently used in field laboratories. Unfortunately, all of these methods require hands-on handling and manipulation of the sample before EBOV is inactivated.…”

mentioning

confidence: 99%

Rapid Bedside Inactivation of Ebola Virus for Safe Nucleic Acid Tests

et al. 2016

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Rapid bedside inactivation of Ebola virus would be a solution for the safety of medical and technical staff, risk containment, sample transport, and high-throughput or rapid diagnostic testing during an outbreak. We show that the commercially available Magna Pure lysis/binding buffer used for nucleic acid extraction inactivates Ebola virus. A rapid bedside inactivation method for nucleic acid tests is obtained by simply adding Magna Pure lysis/binding buffer directly into vacuum blood collection EDTA tubes using a thin needle and syringe prior to sampling. The ready-to-use inactivation vacuum tubes are stable for more than 4 months, and Ebola virus RNA is preserved in the Magna Pure lysis/binding buffer for at least 5 weeks independent of the storage temperature. We also show that Ebola virus RNA can be manually extracted from Magna Pure lysis/binding buffer-inactivated samples using the QIAamp viral RNA minikit. We present an easy and convenient method for bedside inactivation using available blood collection vacuum tubes and reagents. We propose to use this simple method for fast, safe, and easy bedside inactivation of Ebola virus for safe transport and routine nucleic acid detection. The most recent Ebola virus disease (EVD) outbreak began in West Africa in December 2013. As of March 2016, the number of confirmed, probable, and suspected EVD cases reported worldwide was 28,646. Guinea, Liberia, and Sierra Leone were the most affected countries with 3,804, 10,666 and 14,122 cases, respectively (1).Ebola virus (EBOV) is classified as a risk group 4 pathogen that requires handling under biosafety level 4 (BSL-4) conditions. To meet this requirement, several mobile BSL-4 facilities were used during the recent West Africa outbreak (1, 2). However, extensive safety precautions and training of medical and technical staff are needed to ensure personal safety (2-6). As of August 2015, 880 health care workers had been diagnosed with EVD, and 512 had died from the disease (7). Rapid bedside inactivation of EBOV would be a solution for the safety of medical and technical staff, risk containment, and easier transport of samples without requiring expensive category A shipping. Additionally, this process removes the need for sample handling under high-containment environments and facilitates high-throughput and rapid testing under nonbiosafety laboratory conditions and, thus, a rapid diagnosis of the disease.There is a need for a simple, efficient, and safe bedside inactivation method for EBOV. Presently, laboratory EBOV inactivation is accomplished by gamma irradiation (8), UV radiation (9), nanoemulsion (10), and photoinducible alkylating agents (11), but these methods are not applicable in outbreak situations or as bedside inactivation methods. Other EBOV inactivation methods, such as acetic acid (12), heat (12), AVL buffer (13), TRIzol (13) or the combination of heat and Triton X-100 (14), are more applicable in outbreak situations and are currently used in field laboratories. Unfortunately, all of these methods require...

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Single‐Step Method of Total RNA Isolation by Guanidine–Phenol Extraction

Chomczyński¹,

Wilfinger²,

Mackey³

2013

Encyclopedia of Life Sciences

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The original single‐step method is the first procedure to isolate purified total ribonucleic acid (RNA) from a variety of sources including tissues and cells from human, animal, plant, yeast, bacterial and viral origins, without the requirement of high‐speed ultracentrifugation. The method is based on liquid‐phase separation resulting in sequestration of pure RNA into the aqueous phase. RNA is precipitated from the aqueous phase, dissolved, reprecipitated and washed with alcohol before the final solubilisation step. The entire procedure can be completed in less than 4 h and it provides RNA that is suitable for many sensitive downstream applications such as RNase protection assays, northern blotting, sequencing studies and reverse transcription‐polymerase chain reaction. This pioneering methodology has served as the impetus for the development of newer and improved RNA extraction methodology that now enable investigators to extract and purify RNA in less than 60 min. Key Concepts: Enzymes within living cells rapidly degrade RNA after a tissue sample is removed from the donor. To prevent RNA degradation, tissue samples that cannot be immediately processed must be rapidly frozen with dry ice or liquid nitrogen and stored frozen at −80 °C until the RNA can be extracted. At the time of RNA extraction, frozen cells or tissues must be immersed in the denaturing solution and rapidly homogenised before the tissue thaws in order to inactivate RNAse and avoid RNA degradation. The quality of the recovered RNA is dependent on a delicate balance of salt concentration and optimal pH. Overloading the extraction solution with too much tissue or diluting the denaturing solution beyond what is specified in the protocol will impact the quality of the resulting RNA. The extraction of RNA from samples that have a high buffering capacity, such as blood, plasma or tissue culture medium, requires greater care in order to maintain optimal salt balance and pH control. Overdrying of the RNA pellets will impede RNA solubilisation. RNA should be solubilised at a concentration that will be appropriate for meaningful spectrophotometric quantitation as well as subsequent downstream molecular biology applications. Enzymes that are involved in RNA degradation are ubiquitous and special care must be taken to avoid RNAse contamination during RNA solubilisation and storage.

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Virus inactivation by nucleic acid extraction reagents

Cited by 142 publications

References 3 publications

Buffer AVL Alone Does Not Inactivate Ebola Virus in a Representative Clinical Sample Type

Buffer AVL Alone Does Not Inactivate Ebola Virus in a Representative Clinical Sample Type

Rapid Bedside Inactivation of Ebola Virus for Safe Nucleic Acid Tests

Single‐Step Method of Total RNA Isolation by Guanidine–Phenol Extraction

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