Ez1 virus mini kit v2

Manufactured by Qiagen

Sourced in Germany, United States, France, United Kingdom, Spain

The EZ1 Virus Mini Kit v2.0 is a laboratory equipment product designed for the extraction and purification of viral nucleic acids from various sample types. The kit utilizes a magnetic bead-based technology to efficiently capture and isolate the target viral RNA or DNA. The isolated nucleic acids can then be used for downstream applications such as PCR or sequencing. The EZ1 Virus Mini Kit v2.0 is suitable for use with the EZ1 Advanced XL automated system.

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Close spelling variants of this product

Mini Kits (17 citations) EZ1 Virus Mini Kits v2.0 (2 citations)

122 protocols using ez1 virus mini kit v2

Avian Species Identification from Fecal Samples

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Feces were diluted with an equal amount of phosphate-buffered saline (PBS) to prepare a ~50% fecal suspension. Total nucleic acids (including host genomic DNA and viral RNA) were extracted from the fecal suspension, using the
Ambion Mag MAX-96 AI/ND Viral RNA Isolation Kit (Life Technologies, Carlsbad, CA, U.S.A.) or the EZ1 Virus Mini Kit v2.0 (Qiagen, Hilden, Germany). For the Mag MAX-96 AI/ND Viral RNA Isolation Kit, following overnight stationary
incubation to obtain a supernatant, 50 µl of fecal suspension supernatant was used to extract total nucleic acids according to the manufacturer’s instructions. For the EZ1 Virus Mini Kit v2.0, 250
µl of the fecal suspension was mixed with 750 µl of QIAzol lysis reagent (Qiagen). The solution was then mixed with 200 µl of chloroform by vortexing. Subsequent to
centrifugation at 12,000 ×g, 15 min, 4°C, 400 µl of the supernatant was used to extract total nucleic acids according to the manufacturer’s instructions. DNA concentration was measured using a
Qubit 3.0 Fluorometer (Life Technologies) and the Qubit dsDNA HS Assay Kit (Life Technologies) to confirm that the two types of total nucleic acid solutions were used as the DNA template for identification of host avian species.
Concentrations of 0.5 ng/µl and 1.1 ng/µl were obtained using the Mag MAX-96 AI/ND Viral RNA Isolation Kit and EZ1 Virus Mini Kit v2.0 solutions,
respectively.

ONUMA M., KAKOGAWA M., YANAGISAWA M., HAGA A., OKANO T., NEAGARI Y., OKANO T., GOKA K, & ASAKAWA M. (2017). Characterizing the temporal patterns of avian influenza virus introduction into Japan by migratory birds. The Journal of Veterinary Medical Science, 79(5), 943-951.

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Viral Genome Amplification Protocols

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To avoid DNA contamination, viral genome amplifications were performed in a molecular biology laboratory dedicated to clinical diagnosis which includes specific laboratories for each step of the process: nucleic acid extraction, mix preparation, RNA/cDNA manipulation, amplification and PCR product manipulation. In addition, complete genome amplification carried out at the same time was always performed in separate experiments.
Viral RNA was extracted from clarified supernatant medium using the EZ1 Virus Mini Kit v2 on the EZ1 Advanced XL Biorobot (both from Qiagen). Two complete genome amplification procedures were employed based on the sequencing method used. When Sanger sequencing was performed, a specific set of primers was used to generate amplicons covering the entire genome (excluding the first 18 nucleotides of the 5′UTR and the 22 nucleotides upstream of the polyA tail) with the Access RT-PCR System (Promega) as previously described^{15 (link)}. When Next-generation Sequencing (NGS) was performed, the complete viral genomes (excluding the first 18 nucleotides of the 5′UTR and the 88 nucleotides upstream of the polyA tail) were amplified in four fragments using specific sets of primers (Supplemental Table 6 in Supplemental Data) with the Superscript III One-Step RT-PCR Platinum TaqHifi kit (Life Technologies) following manufacturer’s instructions.

Mohamed Ali S., Amroun A., de Lamballerie X, & Nougairède A. (2018). Evolution of Chikungunya virus in mosquito cells. Scientific Reports, 8, 16175.

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SARS-CoV-2 RNA Detection by RT-PCR

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The native and processed sperm sample was centrifuged for 1 min at 3.500 rpm. RNA extraction was performed from 200 μL supernate with the use of the EZ1 Virus Mini Kit v2 (Qiagen) following the manufacturer’s instructions; 60 μL was eluted from the 200 μL starting material, and 5 μL of the eluate was tested by means of reverse-transcription polymerase chain reaction (RT-PCR) with the use of the TaqMan technique. A 113-base-pair amplicon in the E-gene of SARS-CoV-2 was amplified and detected, as previously described with minor modifications (11 ). RT-PCR was performed with the use of an ABI 7500 FAST sequence detector system (PE Applied Biosystems). The thermal protocol described was shortened to 40 cycles of 95°C. We used the LightMix Modular SARS and Wuhan CoV E-gene (cat. no. 53-0776-96) and the LightMix Modular EAV RNA Extraction Control. Moreover, we used the AgPath-ID One-Step RT-PCR Kit (Applied Biosystems cat. no. 4387391; DNA-standard plasmid pEX-A128-nCoV2019-E-gene).

Holtmann N., Edimiris P., Andree M., Doehmen C., Baston-Buest D., Adams O., Kruessel J.S, & Bielfeld A.P. (2020). Assessment of SARS-CoV-2 in human semen—a cohort study. Fertility and Sterility, 114(2), 233-238.

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Competitive Passage of Chikungunya Virus Variants

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As described previously for chikungunya virus WT_IC virus was competed with NS5_Reenc_IC virus [27 (link)]: five initial TCID50 ratios (WT_IC/NS5_Reenc_IC virus: 1/99, 20/80, 50/50, 80/20, 99/1) were used to infect a 25cm² culture flask of confluent BHK21 cells at a calculated moi of 0.5. Cells were washed twice with HBSS and then incubated for 48h after addition of 7mL of medium. Recovered infectious cell supernatant was then sequentially passaged 10 times in the same manner with the clarified cell supernatant medium from the previous passage. At each passage, a calculated moi of 1 was used. Aliquots of cell supernatant from each passage were clarified by centrifugation and stored at -80°C. Viral RNA was extracted from clarified culture supernatant medium using the EZ1 Virus Mini Kit v2 on the EZ1 Biorobot (both from Qiagen). Using two specific quantitative real time RT-PCR assays targeting the re-encoded NS5 coding region (see the quantitative real time RT_PCR assays section for more details), the amount of viral RNA was assessed for each virus (WT_IC and NS5_Reenc_IC) and the ratio of the two values (WT_IC/NS5_Reenc_IC) was calculated.

de Fabritus L., Nougairède A., Aubry F., Gould E.A, & de Lamballerie X. (2015). Attenuation of Tick-Borne Encephalitis Virus Using Large-Scale Random Codon Re-encoding. PLoS Pathogens, 11(3), e1004738.

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Complete CHIKV Genome Sequencing Protocol

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A complete genome sequence (excluding the first 18 nucleotides of the 5'UTR and the 20 nucleotides upstream the polyA tail) was produced from the first passage of a CHIKV strain isolated from a 3-year old boy presenting with fever and dermatological signs and who was living in the district of Makélékélé, Brazzaville. Nucleic acids were extracted from cell culture supernatants using the EZ1 Virus Mini Kit v2 and the EZ1 Biorobot (Qiagen). Primers previously designed to sequence the LR2006 OPY1 CHIKV strain were used to generate PCR products with the Access RT-PCR System (Promega). Amplicons were purified and sequenced using the Sanger method and the BigDye Terminator v3.1 Cycle Sequencing Kit on an ABI Prism 31310X Genetic Analyser sequencer (both from Life technologies). Analysis of sequencing chromatograms was performed using the Sequencher 4.9 software (Gene Codes Corporation).

Moyen N., Thiberville S.D., Pastorino B., Nougairede A., Thirion L., Mombouli J.V., Dimi Y., Leparc-Goffart I., Capobianchi M.R., Lepfoundzou A.D, & de Lamballerie X. (2014). First Reported Chikungunya Fever Outbreak in the Republic of Congo, 2011. PLoS ONE, 9(12), e115938.

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SARS-CoV-2 and Omicron Detection

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Viral nucleic acid extraction was performed using EZ1 Virus Mini Kit v2 (Qiagen, Hilden, Germany) on the EasyOne DNA isolation system (Qiagen). Bio-Speedy SARS-CoV-2 + Omicron RT-qPCR (Bioeksen R&D Technologies, Istanbul, Turkey) was employed for the detection of SARS-CoV-2 ORF1ab + N genes and NSP6 LSG105-107del mutation; PCR amplification and analysis were performed on a Bio-Rad CFX96 Real-time System (BioRad, Hercules, CA, USA). The samples with the cycle threshold (Ct) value < 33 for SARS-CoV-2 ORF1ab + N genes were considered positive for SARS-CoV-2 RNA. For the SARS-CoV RNA positive samples, if the difference between Ct values of NSP6 105-107/106-108del and Ct ofORF1ab + N genes is < 6, the sample is considered as an Omicron variant according to manufacturer instructions.

Keske Ş., Güney-Esken G., Vatansever C., Beşli Y., Kuloğlu Z.E., Nergiz Z., Barlas T., Şencanlı Ö., Kuşkucu M.A., Palaoğlu E., Can F, & Önder E.r.g.ö.n.ü.l. (2022). Duration of infectious shedding of SARS-CoV-2 Omicron variant and its relation with symptoms. Clinical Microbiology and Infection, 29(2), 221-224.

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MERS-CoV Diagnosis from Clinical Samples

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NPAs, oropharyngeal swabs, tracheal aspirates, and throat swabs were collected from MERS-CoV-positive patients (45 to 74 years old) admitted to different hospitals within Saudi Arabia. MERS-CoV diagnosis was confirmed by reverse transcriptase PCR (RT-PCR) (bioMérieux Diagnostics). For this study, the NPAs with a confirmed MERS-CoV diagnosis from the Ministry of Health (MOH) Saudi Arabia, had no identifying information. The sampling NPAs, oropharyngeal swabs, tracheal aspirates, and throat swabs was carried out as per the MOH’s guidelines. Samples were stored at −80°C until used. RNA from the NPAs was extracted using an EZ1 virus minikit v2 (955134; Qiagen). The RNA concentration was measured by the Qubit RNA broad-range (BR) assay (32852; Qiagen). Information on samples from patients used in this study is included in Table 4. This information includes sex, age, hospital, specimen type, threshold cycle (C_T) value of the E gene and ORF1AB, comorbidities, outcome, and whether the patient was in an intensive care unit.

Aljabr W., Alruwaili M., Penrice-Randal R., Alrezaihi A., Harrison A.J., Ryan Y., Bentley E., Jones B., Alhatlani B.Y., AlShahrani D., Mahmood Z., Rickett N.Y., Alosaimi B., Naeem A., Alamri S., Alsran H., Hamed M.E., Dong X., Assiri A.M., Alrasheed A.R., Hamza M., Carroll M.W., Gemmell M., Darby A., Donovan-Banfield I., Stewart J.P., Matthews D.A., Davidson A.D, & Hiscox J.A. (2021). Amplicon and Metagenomic Analysis of Middle East Respiratory Syndrome (MERS) Coronavirus and the Microbiome in Patients with Severe MERS. mSphere, 6(4), e00219-21.

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Multiplex Viral Detection from Serum

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Nucleic acids were extracted from serum samples using the EZ1 Virus Mini Kit v2 and an EZ1 advanced XL Biorobot workstation (Qiagen). Detection of viral genomes was performed using a one-step TaqMan real time RT-PCR procedure and the kit SuperScript III Platinum One-Step Quantitative RT-PCR System with ROX (Life Technologies) for the CHIKV and the dengue virus (DENV), as previously described [21] (link), [22] (link), [23] (link). In addition, plasma samples were tested for the presence of Plasmodium falciparum genomes using a previously reported real time PCR procedure targeting the P. falciparum aquaglyceroporin gene [21] (link), [22] (link), [23] (link) and the Platinum Quantitative PCR SuperMix-UDG (Life Technologies).

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Microbial Identification and SARS-CoV-2 Detection

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In our hospital, automated blood culture system (BacT/ALERT 3D system, bioMérieux, France) is used. For identification of the bacteria, conventional laboratory methods and Matrix-Assisted Laser Desorption Ionization Time-of-Flight (VITEK^® MS system, bioMérieux, France) were used. Antimicrobial susceptibility test results were interpreted based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations. Besides, for defined cases that clinically requires an urgent result, lower respiratory tract samples (sputum, endotracheal aspirate specimens), were enrolled with BioFire^® FilmArray^® Pneumonia plus (bioMérieux, France).
As for SARS-CoV-2 RNA RT-PCR, nucleic acid extraction was performed using EZ1 Virus Mini Kit v2 (Qiagen, Hilden, Germany) on the EasyOne DNA isolation system (Qiagen, Hilden, Germany). Bio-Speedy SARS-CoV-2 þ Omicron RT-qPCR (Bioeksen R&D Technologies, Istanbul, Turkey) was used for the detection of SARS-CoV-2 ORF1ab&N genes; PCR amplification and analysis were performed on a Bio-Rad CFX96 Real-time System (BioRad, Hercules, CA, USA). The samples with the cycle threshold (Ct) value < 33 were considered positive for SARS-CoV-2 RNA.

Kaçmaz B., Keske Ş., Sişman U., Ateş S.T., Güldan M., Beşli Y., Palaoğlu E., Çakar N, & Ergönül Ö. (2023). COVID-19 associated bacterial infections in intensive care unit: a case control study. Scientific Reports, 13, 13345.

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SARS-CoV-2 RT-PCR Detection Protocol

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RT–PCR used targets in the open reading flame 1a (ORF1a) and spike (S) of SARS-CoV-2 according to the guideline [24 (link)] from the National Institute of Infectious Diseases in Japan. Total RNA was extracted using an EZ1 Virus Mini Kit v2.0 (Qiagen, Tokyo, Japan) according to the manufacturer’s instructions. RT-PCR was performed using a QuantiTect Probe RT-PCR Kit (Qiagen) and an N2 primer set.

Mitsumura T., Okamoto T., Shirai T., Iijima Y., Sakakibara R., Honda T., Ishizuka M., Aiboshi J., Tateishi T., Tamaoka M., Shigemitsu H., Arai H., Otomo Y., Tohda S., Anzai T., Takahashi K., Yasuda S, & Miyazaki Y. (2021). Predictors associated with clinical improvement of SARS-CoV-2 pneumonia. Journal of Infection and Chemotherapy, 27(6), 857-863.

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