For nasal swab-1 eluates, nucleic acid was extracted with a Roche Magnapure using a Roche Total Nucleic Acid Isolation Kit (Roche, Basel, Switzerland). Nucleic acid extraction and purification from nasal swab-2 eluates and bFLCs were performed using the QIAamp UltraSens Virus Kit (Qiagen, Manchester, UK) according to the manufacturer’s instructions [26 ] with the exception of the substitution of 5.6 µL of carrier RNA with 5.6 µL of a solution of 5 mg/mL linear acrylamide. Total nucleic acid was immediately extracted and purified from the swab eluates and bFLCs following the nuclease treatment.
Total nucleic acid isolation kit
Manufactured by Roche
Sourced in Germany, Canada
The Total Nucleic Acid Isolation Kit is a product designed for the extraction and purification of both DNA and RNA from a variety of sample types. The kit utilizes a spin column-based method to efficiently capture and isolate total nucleic acids, which can then be used for further downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Magna pure 96, by Roche (4 mentions)
Magna pure lc system, by Roche (4 mentions)
Magna pure lc, by Roche (4 mentions)
Quantstudio 6, by Thermo Fisher Scientific (3 mentions)
Magna pure, by Roche (2 mentions)
Magnapure platform, by Roche (2 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Uvt media, by BD (2 mentions)
Utm media, by Copan (2 mentions)
Abi prism 7000 sequence detection system, by Thermo Fisher Scientific (2 mentions)
Magna pure lc machine, by Roche (2 mentions)
Magna pure lc 2, by Roche (2 mentions)
Ribopure rna purification kit, by Thermo Fisher Scientific (2 mentions)
Taqman fast virus 1 step master mix, by Thermo Fisher Scientific (2 mentions)
Qiaamp ultrasens virus kit, by Qiagen (1 mentions)
Qiaamp dsp viral rna mini kit, by Qiagen (1 mentions)
Superscript 3 one step quantitative rt pcr kit, by Thermo Fisher Scientific (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Lightcycler 480 system, by Roche (1 mentions)
Nuclisens easymag system, by bioMérieux (1 mentions)
32 protocols using total nucleic acid isolation kit
1
Nucleic Acid Extraction from Nasal Swabs
2
Nucleic Acid Extraction from Centrifuged Samples
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200 μl of the centrifuged supernatant was aspirated, and extracted using Roche MagNA Pure LC 2.0 extractor with Roche Total Nucleic Acid Isolation Kit (Roche Applied Science, Switzerland) according to the manufacturer’s instructions.
3
SARS-CoV-2 RT-PCR Detection Protocol
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All patients had been diagnosed with SARS-CoV-2 with RT-PCR from the upper respiratory tract (pooled nasopharyngeal and throat swabs) during acute phase of the infection. Nucleic acid was extracted from clinical samples in a MagNA Pure 96 instrument using the Total Nucleic Acid isolation kit (Roche). RT-PCR targeting the RdRP region was performed in a QuantStudio 6 instrument (Applied Biosystems, Foster City, CA) using the primers and probe described [14 ]. Cycle threshold (Ct) values <38 were regarded as positive.
4
Quantification of HCV RNA in Cell Culture
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For determination of HCV RNA titers in culture supernatant, RNA was extracted from 200 µL supernatant using the Total Nucleic Acid Isolation Kit (Roche Applied Science); titers were determined by TaqMan real-time PCR as previously described47 (link).
5
HPV DNA Detection and Genotyping
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HPV laboratory testing was conducted similarly across all study years [13 (link)]. In brief, DNA was extracted using the MagnaPure platform (Total Nucleic Acid Isolation Kit, Roche, the Netherlands) and HPV DNA was amplified using SPF10 primer sets and detected using DNA enzyme-linked immunoassays (HPV-DEIA, DDL Diagnostics Laboratory, the Netherlands). Positive samples were genotyped with line-probe assay (HPV-LiPA25, DDL Diagnostics Laboratory, the Netherlands), which is able to detect hrHPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and lrHPV types 6, 11, 34, 40, 42, 43, 44, 53, 54, 66, 70, and 74. Also HPV-68, -73, and -97 can be detected, but these types cannot be distinguished from each other and are therefore classified as HPV-68.
6
Whole-Blood Nucleic Acid Extraction and qPCR
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Nucleic acids from 100 μl whole-blood samples stored in RNAprotect Cell Reagent were extracted using MagNAPure LC automatic extractor (Total Nucleic Acid Isolation Kit—High Performance, Roche Applied Science) and used for qPCR targeting
18S rRNA
41 (link). Genomic DNA from the same extraction was used to test for human haemoglobinopathies haemoglobin S and C
42 (link).
18S rRNA
41 (link). Genomic DNA from the same extraction was used to test for human haemoglobinopathies haemoglobin S and C
42 (link).
7
MERS-CoV Detection by rRT-PCR
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RNA was extracted from clinical specimens using either a QIAamp DSP Viral RNA Mini Kit (Catalog No. 61904, QIAGEN GmbH) or an automated MagNAPure 96 extraction instrument with a total nucleic acid isolation kit (Roche). The extraction was performed according to the manufacturers’ instructions and the extracted RNA was stored at −70°C.
MERS-CoVs were detected in specimens using real-time reverse transcription (rRT)-PCR. Extracted RNAs were screened by targeting the upE region, and positive results were confirmed by a subsequent amplification of orf1a using a PowerChek MERS Real-Time PCR kit (Kogene Biotech, Seoul, South Korea). All rRT-PCR reactions were performed using the 7500 Fast Real-Time PCR System (Applied Biosystems) with a total reaction volume of 20 μL (15 μL of PCR reaction mixture and 5 μL of template RNA). The thermocycling conditions included a reverse transcription reaction for 30 min at 50°C, followed by 10 min at 95°C, and then 40 cycles of 15 sec at 95°C and 60 sec at 60°C. A positive viral template control and a nontemplate control were included in each run. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was amplified simultaneously as an internal control to monitor PCR inhibition. A positive result was identified by a well-defined exponential fluorescence curve that crossed the defined threshold at ≤35 cycles in both the upE and ORF1a assays.
MERS-CoVs were detected in specimens using real-time reverse transcription (rRT)-PCR. Extracted RNAs were screened by targeting the upE region, and positive results were confirmed by a subsequent amplification of orf1a using a PowerChek MERS Real-Time PCR kit (Kogene Biotech, Seoul, South Korea). All rRT-PCR reactions were performed using the 7500 Fast Real-Time PCR System (Applied Biosystems) with a total reaction volume of 20 μL (15 μL of PCR reaction mixture and 5 μL of template RNA). The thermocycling conditions included a reverse transcription reaction for 30 min at 50°C, followed by 10 min at 95°C, and then 40 cycles of 15 sec at 95°C and 60 sec at 60°C. A positive viral template control and a nontemplate control were included in each run. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was amplified simultaneously as an internal control to monitor PCR inhibition. A positive result was identified by a well-defined exponential fluorescence curve that crossed the defined threshold at ≤35 cycles in both the upE and ORF1a assays.
8
Quantifying Mosquito-Borne Viral Transmission
9
Multiplex PCR for Respiratory Pathogens
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Nucleic acids extracted from 200 μL of the nasopharyngeal sample using a MagNA Pure LC instrument (Roche Diagnostics, Mannheim, Germany) and the Total Nucleic acid Isolation kit (Roche Diagnostic) were eluted in 100 μL elution buffer and were then stored at -20°C awaiting further analysis.
A previously described multiplex real-time PCR assay was used for detection of 15 different viruses (adenovirus, bocavirus, coronavirus 229E, HKU1, NL63 and OC43, enterovirus, influenza A and B, human metapneumovirus, parainfluenza 1–3, rhinovirus and respiratory syncytial virus (RSV)) and 5 different bacterial species (Bordetella pertussis, Chlamydia pneumoniae, H. influenzae, Mycoplasma pneumoniae and S. pneumoniae) [11 (link)]. A PCR Cycle threshold (Ct)-value of <35 was considered a positive result with values of Ct<30 indicating high levels of nucleic acids. Pneumococci were identified both by detection of the lytA gene included in the multiple pathogen panel, mentioned above and by occurrence of the cpsA gene included in the serotyping multiplex real-time PCR panel (see below). A sample was considered positive for pneumococci if one or both of these genes were detected. For analysis of pneumococci at high levels, either lytA or cpsA, or both, were detected at Ct levels below 30.
A previously described multiplex real-time PCR assay was used for detection of 15 different viruses (adenovirus, bocavirus, coronavirus 229E, HKU1, NL63 and OC43, enterovirus, influenza A and B, human metapneumovirus, parainfluenza 1–3, rhinovirus and respiratory syncytial virus (RSV)) and 5 different bacterial species (Bordetella pertussis, Chlamydia pneumoniae, H. influenzae, Mycoplasma pneumoniae and S. pneumoniae) [11 (link)]. A PCR Cycle threshold (Ct)-value of <35 was considered a positive result with values of Ct<30 indicating high levels of nucleic acids. Pneumococci were identified both by detection of the lytA gene included in the multiple pathogen panel, mentioned above and by occurrence of the cpsA gene included in the serotyping multiplex real-time PCR panel (see below). A sample was considered positive for pneumococci if one or both of these genes were detected. For analysis of pneumococci at high levels, either lytA or cpsA, or both, were detected at Ct levels below 30.
10
Influenza Virus Detection from Nasopharyngeal Swabs
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Example 1
Samples are taken from nasopharyngeal swabs (NPS) of patients presenting flu-like symptoms and the samples are each placed into approximately 3 ml of viral transport medium (for example M4, M4RT, M5, or M6 media (Remel), UVT media (Becton Dickinson), or UTM media (Copan)). Samples were refrigerated for transport at 2-8° C., and stored at that temperature for up to approximately 72 hours before processing. Samples which needed longer storage were stored at ≤−70° C. Nucleic acids were extracted from samples using standard laboratory methods to isolate nucleic acids (e.g., MagNA Pure LC System using the Total Nucleic Acid Isolation Kit (Roche) or the NucliSENS easyMAG System using the Automated Magnetic Extraction Reagents (bioMérieux)). A positive control sample is included which has a target sequence for each of the tested viral strains. That is, if the swine H1N1 influenza A virus is to be tested, a swine H1N1 influenza A virus target sequence is included. Here, the positive controls were pooled RNA transcript from a portion of an HA gene or of an NP gene for each of the subtypes of influenza. In addition, a negative control including the viral transport medium, but not including a target sequence, and Internal Controls were extracted alongside the samples.
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