Qualitative real-time PCR [25 (link)] was performed on specimens before 2006, where 5 μl of extracted DNA was used for amplification and detection. Specimens received year 2007 onwards, quantitative assay was performed using artus® HSV-1/2 LC PCR Kit (Qiagen Inc., Valencia, CA, USA). DNA from 0.1 to 1 mL of CSF was extracted using Nuclisens® extraction kit (bioMérieux, Boxtel, The Netherlands). The amplification was undertaken using a LightCycler® instrument (Roche Diagnostics, Germany). HSV-1 and -2 DNA PCR product was differentiated by melting curve analysis in the LightCycler® PCR instrument. The standard curve was generated from four quantitation standards of HSV (10, 100, 1000 and 10,000 copies/μl) supplied by the manufacturer. Preparation of PCR assay, PCR profile and data analysis was conducted according to the manufacturer’s protocol. The internal control supplied by the manufacturer was included in all reactions to check for possible PCR inhibition. The analytical detection limit of the PCR Kit was 1 copy/μl (p = 0.05) (Qiagen Inc., CA, USA). For qualitative assay, all steps were the same as quantitative assay, except that the standard curve was not used for calculation, and only one standard positive control was performed along with the assayed sample.
Nuclisens extraction kit
Manufactured by bioMérieux
Sourced in France
The NucliSens extraction kit is a laboratory equipment product designed for the extraction and purification of nucleic acids, such as DNA and RNA, from various biological samples. The core function of this kit is to isolate and concentrate the target nucleic acids for further analysis or testing, without any interpretations or extrapolations on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Lightcycler, by Roche (1 mentions)
Artus hsv 1 2 lc pcr kit, by Qiagen (1 mentions)
Bead beater, by Biospec (1 mentions)
Zr bashingbead lysis tube, by Zymo Research (1 mentions)
Kingfisher 96 flex system, by Thermo Fisher Scientific (1 mentions)
Rna minelute clean up, by Qiagen (1 mentions)
Elution buffer, by bioMérieux (1 mentions)
Qiaamp tissue mini kit, by Qiagen (1 mentions)
Nuclease free water, by Qiagen (1 mentions)
Mx3000p qpcr system, by Agilent Technologies (1 mentions)
Proteinase k solution, by Merck Group (1 mentions)
8 protocols using nuclisens extraction kit
1
HSV-1/2 Detection by Real-Time PCR
2
HAV Detection in Bivalve Molluscs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 746 samples, including four bivalve mollusc species (Mytilus galloprovincialis, Solen vagina, Venus gallina, and Donax trunculus), were taken over 2015–2018 from 27 production areas of the coast and from biomonitoring points. In detail, 398 samples were taken during the 2015 HAV outbreak involving the Campania Region (February to August), 87 from September to December 2015, and the remaining 261 samples were collected periodically between 2016 and 2018. Analysis was performed according to ISO 15216-1, revision 2013 or 2017 based on processing year [22 ,23 ]. Briefly, depending on species’ size, 10 to 60 individuals were randomly selected and digestive tissue was dissected and finely chopped. Two-gram aliquots were spiked with 10 μL of Mengovirus MC0 process control (1.6 × 105 TCID50/mL), digested with 2 mL of proteinase K (0.1 mg/mL; 37 °C for 60 min with shaking), and then placed at 60 °C for 15 min. Finally, the sample supernatant was collected after centrifugation at 3000× g for 5 min, and its volume was recorded. Nucleic acid extraction and purification were performed on 500 μL of supernatant using the NucliSens extraction kit (bioMerieux, France) according to the manufacturer’s instructions, and RNA was eluted in 100 μL and stored at −80 °C until molecular analysis.
3
Wastewater Virus Concentration for ddPCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We prepared influent samples for ddPCR following the method described in Steele et al. (63 (link)). Briefly, we first added bovine coronavirus (BoCoV) vaccine (Bovilis; Merck & Co, Kenilworth, NJ) to 20 ml of wastewater as a sample processing control to assess viral RNA recovery. We then added MgCl2 to a final concentration of 25 mM and adjusted the pH to <3.5 with 20% HCl on a mixed cellulose ester membrane (type HA; Millipore, Bedford, MA) in replicates of six. We then transferred the HA filters to preloaded 2-ml ZR BashingBead lysis tubes (Zymo, Irvine, CA) and bead beat the samples with a BioSpec beadbeater (BioSpec Products, Bartlesville, OK) for 1 min. We then extracted total nucleic acids with a bioMérieux NucliSENS extraction kit with magnetic bead capture (bioMérieux, Durham, NC) following the manufacturer’s protocol.
4
Viral Nucleic Acid Extraction using NucliSens Kit
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The viral nucleic acids were extracted from the PEG pellets and the ultrafiltered concentrate using the NucliSens extraction kit (BioMerieux, France) on a Kingfisher 96 Flex system (Thermo Scientific, USA) as described previously [32 (link), 34 (link)]. The final volume of the eluent was 100 μl. To assess extraction performance and cross-contamination, extraction positive (deionised water spiked with phi6) and negative controls (distilled water) were used.
5
Virus Concentration and Purification from Wastewater
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Raw wastewater samples were homogenized and 11 mL were ultracentrifugated as described previously (Wurtzer et al., 2020 (link)). Viral pellets were resuspended in 200 μL of PBS 1×. The membranes of zetapor and nylon were immersed directly in 8 ml of lysis solution to which 4 ml of 1× PBS were added. For virus titration, RNA was extracted from 10 μl of virus stock solution and from 1 ml of exposure mixture. All nucleic acids were extracted using a NucliSENS extraction kit (bioMérieux, Lyon France) as previously described (Vincent-Hubert et al., 2021 (link)), eluted with 100 μl of nuclease-free water and kept frozen at −20 °C until purification. Nucleic acids extracted from membranes exposed in the laboratory were then purified using a Qiagen kit (RNA MinElute Clean up, Qiagen, France) to eliminate potential PCR inhibitors, eluted with 100 μl of nuclease-free water (Qiagen, France) and kept frozen at −20 °C. As partial inhibition of qRT-PCR was found with wastewater samples from field study, we used a One Step PCR Inhibitor removal kit (Zymo Research Kit, USA) for all these samples. Inhibited samples represented 25% and 37% of membranes exposed respectively in seawater and wastewater.
6
Nucleic Acid Extraction from Membranes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Membranes were rinsed in sterile water and nucleic acids were directly extracted from the membrane, the entire membrane was used for extraction. For the first field study, a NucliSENS extraction kit (bioMérieux, Lyon France) was used for NoV, Vibrio spp., and Bacteroidales markers, and a QiAamp tissue mini kit® (QIAgen, France) was used for OsHV-1 as published previously (Vincent-Hubert et al., 2017 (link)). For the second field study, a single extraction procedure was used for all microorganisms using a NucliSENS extraction kit. Nucleic acids were eluted from the paramagnetic silica into 100 μl of elution buffer (bioMérieux, France), further purified using a Qiagen kit (RNA MinElute, Qiagen, France) to eliminate potential PCR inhibitors, and eluted with 120 μl of nuclease-free water (Qiagen, France).
7
Detection and Quantification of Human Noroviruses in Shellfish
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For detection of HuNoVs, shellfish were shucked, and the digestive tissues (DT) were recovered and frozen under aliquots of 2 g. For analysis, Mengovirus (MgV) (2.106 RNA copies) was added to each sample and incubated with 2 ml of proteinase K solution (30 U/mg, Sigma-Aldrich, France) at 37°C under shaking for 15 min and then at 60°C for 15 min. After centrifugation at 3,000 g for 5 min, the supernatant was recovered. Nucleic acids (NAs) were extracted using the NucliSens extraction kit (BioMérieux) with increasing the lysis buffer volume (Le Mennec et al., 2017 (link)). After checking the extraction efficiency by amplification of MgV, HuNoVs GI and GII were detected as previously described using 5 μl of undiluted and 10-fold dilutions of the NA extract on a Mx3000P qPCR System (Agilent Technologies, France). Only sample with a MgV extraction efficiency >1% were considered for quantification. The number of RNA copies present in each positive evaluable sample was estimated by comparing the sample Cq value to standard curves. Calculated concentrations for GI and GII were added to express the final result as HuNoV RNAc/g of DT.
8
Norovirus Detection in Shellfish
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Upon reception, shellfishes were shucked, weighed, and their digestive tissues (DTs) dissected as previously described [25 (link)]. As an extraction process control, Mengovirus (MgV) (2.106 RNA copies) was added to each DT sample (2 g) before incubation with 2 mL of proteinase K solution, following the ISO norm for NoV detection in foods (ISO 15216–1:2017). The NucliSens extraction kit (BioMérieux, Marcy-l’Étoile, France) was used to recover nucleic acids (NA) from 0,5 mL of DT supernatant or MgV control, following the manufacturer’s recommendations, with a final elution step of 5 min at 60 °C. The eluted nucleic acids were directly used for NoV GII and MgV genome detection and further stored at −20 °C.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!