Specimens aliquots of 150 µL of plasma and approximately 2 - 3 × 106 PBMCs were thawed, and then pellets resuspended in 150 µL of phosphate-buffered saline (PBS) were used for DNA extraction and processed by NucleoSpin® Dx Virus kit (Macherey-Nagel, Duren, Germany), following the manufacturer’s instructions.
Nucleospin dx virus kit
Manufactured by Macherey-Nagel
Sourced in Germany
The NucleoSpin® Dx Virus kit is a product from Macherey-Nagel designed for the extraction and purification of viral nucleic acids. It provides a reliable and efficient method for isolating viral RNA and DNA from various sample types.
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6 protocols using nucleospin dx virus kit
1
DNA extraction from plasma and PBMCs
2
Evaluating SARS-CoV-2 Variant Detection
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Amplification efficiency of the designed primer sets was evaluated using viral RNA extracts from two sequenced SARS-CoV-2 strains: the original Wuhan strain 210207 (GISAID EPI_ISL_437689) and VOC B.1.1.7 strain (GISAID EPI_ISL_683466) in the Pasteur Institute . Viral RNA was extracted from infected cell culture supernatants using the NucleoSpin Dx Virus kit (Macherey-Nagel), following the manufacturers’ protocol. Viral RNA extracts (5 µL) were analyzed either using the IP2/IP4 dualplex real-time reverse-transcriptase (RT)-PCR assay, developed by following the Pasteur Institute and targeting conserved regions of the SARS-CoV-2 RdRP gene31 , or primer set B.1.1.7-1, using the LightCycler EvoScript RNA SYBR Green I Master kit (Roche). Both RT-PCR assays were conducted on a LightCycler® 480 System (Roche), using the thermal cycling program described in the Pasteur Institute protocol31 .
3
Quantitative RT-PCR for Luciferase Encapsidated RNA
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Luciferase encapsidated RNA was extracted using Nucleospin Dx Virus kit (Macherey-Nagel) from 150 µL of pseudotype suspension, according to the manufacturer's instructions. Quantitative RT-PCR targeting the luciferase gene was carried out using the following primers [forward 5′-ACACCCCAACATCTTCGAC-3′ , Reverse 5′-TCGCGGTTGTTACTTGACTG-3′] and probe [5′-FAM-TTGGAGCACGGAAAGACGATGAC-BHQ1-3′] (BHQ1: black hole quencher 1) with a LightCycler 480 instrument (Roche) and a SuperScript III Platinum OneStep RT-PCR kit (Invitrogen). The reactions were incubated in a 96-well optical plate at 45°C for 15 min, then 95°C for 3 min, followed by 50 cycles of 95°C for 10 sec, 50°C for 10 sec and 72°C for 20 sec and a cooling step at 40°C for 30 sec.
4
Quantitative PUUV RNA Detection
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A volume of 100 μL of plasma or urine plus 40 μL of DNase/RNase-free water was first spiked with 10 μL of a known amount of a Sigmavirus from Drosophila (used as an internal control of RNA extraction), and then RNA was extracted from the 150-μL mixture using the NucleoSpin Dx virus kit (Macherey-Nagel, Germany). Extracted RNA quality was finally assessed by the partial amplification of the Sigmavirus genome using a real-time reverse transcription-PCR (RT-PCR) method (21 (link)). RNA extraction was validated if the cycle threshold (CT) value was in the expected range. Otherwise, the extraction was repeated once and then considered validated or not.
PUUV RNA detection was carried out in duplicate according to a real-time RT-PCR performed according to Kramski et al.’s method (22 (link)). Positive results were qualitatively accepted even if the RNA extraction was not validated, while negative results were considered undetermined when the extraction was not validated. Absolute quantification was achieved using a standard curve established with the European Virus Archive Global project, reference PUUV RNA (Ref-SKU 007N-EVA370). A total of 101 RNA copies/assay mixture, corresponding to 2 × 103 equivalent RNA copies/mL of sample was the last point detected in 100% of the assays.
Both assays were conducted under ISO 15189 accreditation.
PUUV RNA detection was carried out in duplicate according to a real-time RT-PCR performed according to Kramski et al.’s method (22 (link)). Positive results were qualitatively accepted even if the RNA extraction was not validated, while negative results were considered undetermined when the extraction was not validated. Absolute quantification was achieved using a standard curve established with the European Virus Archive Global project, reference PUUV RNA (Ref-SKU 007N-EVA370). A total of 101 RNA copies/assay mixture, corresponding to 2 × 103 equivalent RNA copies/mL of sample was the last point detected in 100% of the assays.
Both assays were conducted under ISO 15189 accreditation.
5
Isolation and Detection of Hepatitis B Virus DNA
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Serum samples from HBV positive patients were obtained from Dr. C. Nagaraj, Kuppam hospital, India. All the samples were handled with adequate precautions in BSL-II facility of Cancyte Technologies Pvt. Ltd, Bangalore, India. IVD certified NucleospinDx virus kit (Macherey-Nagel, GmbH) was used to isolate HBV DNA from clinical samples as per manufacturer’s instructions.
PCR for HBV detection in clinical samples was carried out in 25 μl reaction volume. The PCR mixture comprised of 1 X ammonium sulphate buffer, pH 8.3, 2.5 mM magnesium chloride, 0.2 mM dNTP’s, 5% DMSO, primers 0.4 μM primers and suitable amount for S-Taq fusion protein. PCR was carried out for 40 cycles which included initial denaturation of 95 °C, 10 min, followed by 40 cycles of denaturation step of 95 °C, 30 s, 60 °C, 30 s and 72 °C, 45 s and a final extension of 10 min at 72 °C.
PCR for HBV detection in clinical samples was carried out in 25 μl reaction volume. The PCR mixture comprised of 1 X ammonium sulphate buffer, pH 8.3, 2.5 mM magnesium chloride, 0.2 mM dNTP’s, 5% DMSO, primers 0.4 μM primers and suitable amount for S-Taq fusion protein. PCR was carried out for 40 cycles which included initial denaturation of 95 °C, 10 min, followed by 40 cycles of denaturation step of 95 °C, 30 s, 60 °C, 30 s and 72 °C, 45 s and a final extension of 10 min at 72 °C.
6
SARS-CoV-2 RT-qPCR Detection in Saliva
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RNA extraction from saliva was performed as described earlier23 . Briefly, samples were treated in the presence of DTT (10 mM) for 30 min at room temperature. Then, RNA extraction was performed using Nucleospin Dx Virus Kit (Macherey Nagel). 2 μL of purified RNA were added to Invitrogen superscript III Platinium One step reaction mix (#11,732,020) containing 1X reaction mix, MgSO4 (0.8 mM) a DNA‐primer mix aiming two RdRp targets (IP2 and IP4). Primers and probes (nCoV_IP2 and nCoV_IP4) were designed to target the RdRp gene spanning nt 12,621–12,727 and 14,010–14,116 (positions according SARS-CoV, NC_004718)44 (link). Real-time detection and thermal cycling conditions were identical to previously described23 . Saliva samples were analyzed in triplicate and RT-qPCR Ct values were the mean of these results. An absence of amplification on replicates was expressed as Ct = 40 which is the maximum value obtained by our method.
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