Abstract

Porphyromonas gingivalis (P. gingivalis) is a periodontal pathogen that can accumulate with other organisms in subgingival plaque biofilms and is associated with periodontal disease. P. gingivalis fimbriae (FimA) is a filamentous structure on the surface of bacteria that is closely associated with bacterial adhesion and colonization of host tissues and plays an essential role in biofilm formation. The present study aimed to construct prokaryotic P. gingivalis FimA expression plasmids, purify a FimA fusion protein, and explore the effect of a recombinant FimA protein on the inflammatory response in human peripheral blood mononuclear cells (PBMC).

Porphyromonas gingivalis Recombinants FimA prokaryotic expression plasmids were constructed using gene cloning and recombination technology. SDS-PAGE was used to evaluate purified recombinant FimA protein. Cell proliferation rate and inflammatory cytokine expression of PBMC treated with FimA fusion protein with or without toll-like receptor 4 (TLR4) small interfering (si) RNA transfection were detected by CCK-8 assays and ELISA, respectively. Expression levels of TLR4, nuclear factor kappa light chain enhancer of activated B cells (NF-κB), and primary myeloid differentiation response 88 (MyD88) in PBMC were detected by Western blot analysis and quantitative polymerase chain reaction with reverse transcription.

A high purity FimA fusion protein was obtained. FimA fusion protein treatment significantly increased PBMC proliferation and promoted the release of tumour necrosis factor-α (TNF-α), interleukin (IL)-6, matrix metalloproteinase (MMP)-8, and MMP-9 in PBMC. TLR4 interference reversed the effects of FimA fusion protein on PBMC proliferation and inflammatory cytokine release. The expression levels of TLR4, NF-κB and MyD88 in PBMCs were significantly increased after FimA fusion protein treatment, whereas the expression levels of these genes at mRNA and protein levels were significantly decreased in PBMCs after treatment. FimA fusion protein treatment and TLR4 interference.

Materials and methods

• Subject Recruitment, Blood Sampling, and PBMC Culture

A total of 12 healthy volunteers in good periodontal health (37.5 ± 6.3 years) with no known periodontal pocket formation or attachment loss, no alveolar bone resorption, and no systemic disease were recruited from the Second Affiliated Hospital of the Medical University of Jinzhou from September 2017 to December 2017. Additional exclusion criteria were the presence of gingivitis, periodontal disease, orthodontic treatment, or a history of long-term use of antibiotics or other medications. All subjects who participated in the experiment underwent a periodontal examination by the same dentist. Subjects clearly understood the purpose of the experiment, agreed to participate and gave written informed consent.

Specimen collection was approved by the Ethics Committee of the Second Affiliated Hospital of Jinzhou Medical University (Jinzhou, China). A total of 10 ml of venous blood was collected from each volunteer and stored in tubes with EDTA anticoagulant (BD Biosciences, San José, CA, USA). PBMCs were isolated by the Ficoll-Paque density gradient centrifugation method (650 × g; 18 °C for 20 min) and cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc., Waltham, MA, EE). bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) All cells were grown in an incubator containing 5% CO2 at 37°C with 100% humidity.

• P. gingivalis culture and genome extraction

A frozen liquid culture (-80°C) of P. gingivalis 33277 strain [American Type Culture Collection (ATCC), Manassas, VA, USA] was thawed at room temperature and inoculated into a Petri dish containing brain heart infusion (BHI) agar with 1 mg/l vitamin K, 1 g/l yeast extract and 5 mg/l hemins (China Medical University, Shenyang, China). Cells were grown under anaerobic conditions at 37°C for 4 days. Surface colonies were scraped with sterile loops onto a sterile ultraclean bench and inoculated into BHI liquid culture medium.

Cells were cultured for ~24 h until bacterial growth reached the log phase. Then, the supernatant was discarded after centrifugation at 4000 x g for 15 min at 4 °C to obtain the bacteria, and P. gingivalis genomic DNA was extracted. Genomic DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega Corporation, Madison, WI, USA), following the manufacturer’s instructions.

• Semi-quantitative polymerase chain reaction (PCR) amplification of target genes

Based on the FimA sequence of P. gingivalis ATCC 33277 available in the National Center for Biotechnology Information GenBank database, primers (forward, 5′- ATT AGG ATC CAT GGT GGT ATT GAA GAC CAG C-3′; reverse, 5′-ATA TCT CGA GCC AAG TAG CAT TCT GAC CAA CGA G-3′) were designed using Premier 5.0 software (Premier Biosoft International, Palo Alto, CA, USA) to remove the stop codon from the original sequence and add a start codon. Primers were synthesized by Nanjing GenScript Biotechnology Corporation (Nanjing, China).

The FimA gene was amplified by the LA Taq enzyme (Takara Biotechnology Co., Ltd., Dalian, China). Cycling conditions included 94 °C for 5 min, followed by 25 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 2 min, with a final extension at 72 °C for 10 min. The PCR products were identified by 1% agarose gel electrophoresis in tris acetate buffer (40 mM tris acetate, 1 mM EDTA, pH 8.0). Products were visualized with ethidium bromide by UV transillumination.

• RNA interference assay

Shanghai GenePharma Co., Ltd., (Shanghai, China) synthesized small interfering RNAs (si) targeting toll-like receptor 4 (si-TLR4) and negative siRNA with a random sequence. The target sequences for TLR4 were: Sense, 5′-GGG CUU AGA ACA ACU AGA ATT-3′; and antisense, 5′-UUC UAG UUG UUC UAA GCC CTT-3′, and the sequences for the negative siRNA were: Sense, 5′-UUC UCC GAA CGU GUC ACG UTT-3′; and antisense, 5′-ACG UGA CAC GUU CGG AGA ATT-3′. The concentration used was 20 nM. All plasmids and oligonucleotides were transfected using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions. After 48 h, cells were harvested and subjected to further experimentation.

• Cell proliferation assay

PBMC (1 × 103 cells/well) were seeded in 96-well plates and treated with FimA fusion protein (2, 4, and 6 μg/ml) with or without siTLR4 transfection at 37 °C and 5% CO2 for 12, 24 and 48 hours. Cell proliferation was detected using a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). An aliquot of 10 μl of CCK-8 solution was added to the treated PBMCs and incubated at 37 °C for an additional 4 h. Cells were washed twice with PBS and then incubated at 37°C for 1-4 h. The absorbance value at 450 nm of each well was measured with a 96-well plate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

• ELISE

PBMCs were seeded uniformly in 6 wells at a density of 1 × 106 cells/mL and treated with FimA fusion protein (2, 4, and 6 μg/mL) with or without transfection with siTLR4 at 37 °C and 5 %. CO2 for 12, 24 and 48 h. Cell culture media from all groups were collected and centrifuged at 8,000 × g for 20 min at 4 °C to remove debris.

The supernatant (400 μl) was collected from PBMCs to measure concentrations of TNF-α, IL-6, MMP-8, and MMP-9 with an ELISA-based capture assay using commercial TNF-α (cat. no. PT518) and IL-6 ELISA Kits (Cat. No. PI330) (Beyotime Institute of Biotechnology, Haimen, China), and MMP-8 (Cat. No. DMP800B) and MMP-9 ELISA Kits ( Cat. No. DMP900) (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer’s instructions. The absorption was measured at 450 nm. The concentration of inflammatory cytokines was determined by comparing the relative absorbance of the samples with the standards.

• Quantitative reverse transcription PCR (RT-qPCR)

Total RNA was extracted from PBMC treated with FimA fusion protein with or without siTLR4 transfection using TRIzol® reagent (Thermo Fisher Scientific, Inc.). The extracted RNA was then transcribed into double-stranded cDNA using a commercial reverse transcription kit (Invitrogen; Thermo Fisher Scientific, Inc.).

The cDNA was amplified using the following primers: TLR4 forward, 5′-CCG CTT TCA CTT CCT CTC AC-3′; reverse TLR4, 5′-CAT CCT GGC ATC ATC CTC AC-3′; nuclear factor of activated B cells (NF-κB) kappa light chain enhancer forward, 5′-CTT GCT TAG TTG GTC CTC-3′; reverse NF-κB, 5′-ACC CGA AGA GAA ACG A-3′; myeloid differentiation primary response 88 (MyD88) forward, 5′-AGA TGG ACC TCG GGA G-3′; reverse MyD88, 5′-ATC AAT CAC GCA CGA TTT-3′; β-direct actin, 5′-TCC CTG TAT GCC TCT G-3′; β-reverse actin, 5′-ATG TCA CGC ACG ATT-3′. The reaction system contained cDNA template (1 μl), primers (1 μl), 2X SYBR Green Mix (10 μl; Shanghai GeneCore BioTechnologies Co., Ltd., Shanghai, China), and RNase-free water (8 μl), with a total volume of 20 μl.

PCR was performed using the ABI 7500 system platform (Applied Biosystems; Thermo Fischer Scientific, Inc.). The 2−ΔΔCq method was used to calculate the relative gene expression of TLR4, NF-κB and MyD88 normalized to β-actin (20). PCR amplification was repeated in triplicate for each gene.

• Western blot analysis

Total protein was extracted from PBMC treated with FimA fusion protein with or without siTLR4 transfection by radioimmunoprecipitation assay (Beyotime Institute of Biotechnology). Protein concentration was evaluated by the BCA protein assay kit (Beyotime Institute of Biotechnology). Total lysates (50 mg) were resolved by 10% SDS-PAGE (Beyotime Institute of Biotechnology), followed by blocking for 1 h at room temperature in blocking buffer (cat. no. P0023B; Beyotime Institute of Biotechnology ).

Membranes were incubated with primary antibodies (rabbit anti-TLR4, 1:500 dilution, cat. no. ab13556; rabbit anti-NF-κB, 1:500 dilution, cat. no. ab207297; anti-rabbit Rabbit MyD88, 1:500 dilution, Catalog No. ab2064; rabbit anti-β-actin, 1:1000 dilution, Catalog No. ab8227; Abcam, Cambridge, UK). After rinsing with TBST, the secondary goat anti-rabbit horseradish peroxidase-conjugated antibody (1:5000 dilution; cat. no. ab6721; Abcam, Cambridge, UK) was added and the membranes were incubated at room temperature. environment for an additional 2 h. Image J software version 1.48 (National Institutes of Health, Bethesda, MD, USA) was used to analyze the relative density of the protein band.

• Statistic analysis

The statistical software SPSS 17.0 (SPPS Software, Inc., Chicago, IL, USA) was used for data analysis. Data are expressed as mean ± standard deviation. All experiments were repeated at least three times. Comparisons between two groups were analyzed using an independent samples t-test, while comparisons between multiple groups were analyzed using a one-way analysis of variance followed by Tukey’s honestly significant difference test. P<0.05 was considered to indicate a statistically significant difference.

Product name: Neosartorya fumigata Recombinants

Purity: > 90% determined by SDS-PAGE.

Endotoxin: < 1.0 EU per μg of protein as determined by the LAL method.

Activity: Test in progress

Protein Construction:

A DNA sequence encoding neosartorya fumigata recombinants AspF9 (XP_752985.1) (Met1-Gly370) was expressed with a polyhistidine tag at the C-terminus.

Accession number: XP_752985.1

Expressed Host: HEK293 cells

Species: Neosartorya fumigata

Terminal N planned: Gln 20

Molecular mass

Recombinant AspF9 from neosartorya fumigata consists of 362 amino acids and predicts a molecular mass of 37.5 kDa.

Formulation

• Lyophilized from sterile PBS, pH 7.4.
• Please contact us with any concerns or special requirements.
• Typically 5%-8% trehalose, mannitol and 0.01% Tween80 are added as protectants prior to lyophilization.

Shipping

Recombinant proteins are generally provided as a lyophilized powder that is shipped at room temperature. Recombinant protein bulk packs are provided as a frozen liquid. They are shipped with blue ice unless customers require otherwise.

Stability and Storage

Samples are stable for up to twelve months from the date of receipt at -20℃ to -80℃. Store it under sterile conditions between -20℃ and -80℃. It is recommended to divide the protein into aliquots for optimal storage. Avoid repeated cycles of freezing and thawing.

Reconstitution

A printed copy of the COA with instructions for reconstitution is shipped with the products. Please refer to it for detailed information.

Bottom

The fungal pathogen Candida albicans is commonly seen in immunosuppressed patients, and resistance to one of the most widely used antifungals, fluconazole (FLC), can evolve rapidly. In recent years it has become clear that the plasticity of the Candida albicans genome contributes to drug resistance through loss of heterozygosity (LOH) in resistance genes and macroscopic chromosomal rearrangements that amplify gene copy number. associated with resistance. This study addresses the role of the homologous recombination factors Rad54 and Rdh54 in cell growth, DNA damage and resistance to FLC in Candida albicans.

Materials And Methods

• Strains and growth conditions.

Candida albicans recombinants wild-type strain SC5314 was used to construct all mutants created for this study. Deletion and replacement of Candida albicans RAD54 and Candida albicans RDH54 was performed using the nourseothricin resistance marker SAT1 (generously provided by Dr Joachim Morchauser) to create homozygous null mutants Candida albicans rdh54Δ/rdh54Δ, Candida albicans rad54Δ/rad54Δ and the reconstructed strain Candida albicans rad54Δ/RAD54 (+).

The rad54Δ/RAD54 (+) reconstructed strain was prepared from one of the rad54Δ/rad54Δ strains. For routine growth, strains were maintained at 30°C in YPD (10 g Difco yeast extract, 20 g Bacto peptone and 20 g dextrose per litre) with or without 200 µg/ml nourseothricin. Spider medium was used for agar invasion tests, with a final pH of 7.2 (10 g nutrient broth, 10 g mannitol, 2 g K2PO4 and 25 g agar per litre).

• Plasmid Construction

To create null mutants of Candida albicans RAD54 and Candida albicans RDH54, approximately 500 base pairs (bp) of sequence upstream and downstream of these ORFs were cloned on either side of the SAT1-FLP cassette into the pFS2A vector. Fragments were designed such that the entire coding sequence from ATG to stop codon was replaced by the SAT1 cassette. For both genes, the upstream fragment was cloned using the restriction enzymes ApaI and XhoI and the downstream fragment was cloned using NotI and SacII. To create the Candida albicans RAD54 reconstruction vector, the entire coding region, including the promoter and terminator sequence, was cloned into the ApaI-XhoI site in the Candida albicans RAD54 deletion vector.

• Yeast transformation and screening

SC5314 was transformed with linearized Candida albicans RAD54 or Candida albicans RDH54 deletion vectors (linearized with ApaI and SacII) using the standard lithium acetate method with the following modifications. Heat shock was performed at 42°C overnight, and cells were resuspended in YPD and allowed to grow for 4 hours at 30°C before seeding on YPD containing 200 µg/mL cloNAT (Werner BioAgents, Jenna, Germany). Recycling of the SAT1 marker was performed by culturing cells overnight in non-selective media (YPD) and plating on YPD containing 25 µg/ml nourseothricin.

Small colonies that had the marker removed were screened by PCR and used in a successive round of transformation. These transformants were then selected by PCR for the homozygous deletion of Candida albicans RAD54 and Candida albicans RDH54. To create the Candida albicans reconstruction strain RAD54, recycling of the SAT1 marker was performed again and the reconstruction plasmid was introduced into the native locus by another round of transformation.

• Determination of growth rate

Overnight YPD cultures of three independent colonies were used to inoculate 3 ml of YPD at an OD600 of 0.05. Cultures were grown at 30°C with shaking. OD measurements were taken every hour for 9 hours to generate growth curves. Doubling times for each strain were calculated using time points within the log phase of growth. This test was repeated three times, the mean and standard deviations for each strain are shown.

• Colony morphology and microscopic analysis

For evaluation of colony morphology, cells were grown in YPD for 2 days at 30°C and single colonies were photographed. For agar colony invasion, strains were plated on Spider agar plates (1% nutrient broth, 1% mannitol, 0.2% K2HPO4, and 20 g Bacto agar per litre) and incubated at 37°C. C for seven days and images were taken. For cell morphology, cells were grown in YPD to early log phase from overnight YPD cultures. Samples were taken, washed and resuspended in PBS buffer and sonicated for 5 seconds at 30% amplitude in a Fisher Scientific 150T Series sonic dismemberer (Fisher Scientific, USA).

Light microscopy was used to quantify the number of individual non-budding cells, budding cells, and cells with abnormal or pseudohyphal-like morphology. To assess nuclear integrity, cells were grown to the early log phase and stained with DAPI according to a previously published protocol. Overnight cultures were diluted to an OD600 of 0.05 in 5 mL YPD and grown for 4 hours at 30°C. Samples were centrifuged, washed in 1 ml 1X PBS and fixed overnight at 4°C in 1 ml 70% ethanol. Fixed cells were washed and treated for 2 hours in 55 mM HCl with 5 mg/ml pepsin at 37 °C, then washed and resuspended in 1 ml 1X PBS containing 2.5 μg/ml DAPI (Sigma- Aldrich, St. Louis, MO, USA).

• DNA damage and sensitivities to antifungal drugs

To test the sensitivity of the strains in this study to various agents, the agar spot dilution method was used. Overnight YPD cultures were diluted to an OD 600 of 1.0 and ten-fold serial dilutions were made to 10-6. Volumes of 2 μL of each dilution were plated onto YPD plates and YPD plates containing FLC, MMS, or menadione (Sigma-Aldrich, St. Louis, MO, USA) at the indicated concentrations. Plates were incubated for 48 hours at 30°C and images were taken.

An E-test analysis for common antifungals was performed, using overnight cultures diluted to an OD600 of 0.05 to spread a lawn on CAS plates (9.0 g casitone, 5.0 g yeast extract, 0. 54 g of KH2PO4, 3.34 g of K2HPO4, 20.0 g of dextrose and 20.0 g of agar per litre). Test strips E were plated on plates, which were incubated for 48 hours at 30°C. Two independent nulls of the RAD54 gene were tested. The MIC was read as the point where the zone of inhibition intersected test strip E.

Results

The data presented here support a role for homologous recombination in cell growth and sensitivity to DNA damage, as Candida albicans rad54Δ/rad54Δ mutants were hypersensitive to MMS and menadione, and had aberrant cellular and nuclear morphology. The Candida albicans rad54Δ/rad54Δ mutant was defective in the invasion of Spider agar, presumably due to altered cell morphology. In contrast, mutation of the related gene RDH54 did not contribute significantly to DNA damage resistance or cell growth, and deletion of Candida albicans RAD54 or Candida albicans RDH54 did not alter susceptibility to FLC.

Conclusions

Together, these results support a role for homologous recombination in genome stability under non-damaging conditions. Nuclear morphology defects in rad54Δ/rad54Δ mutants show that Rad54 plays an essential role during mitotic growth and that, in its absence, cells arrest in G2. The viability of the rad54Δ/rad54Δ single mutant and the inability to construct the rad54Δ/rad54Δ rdh54Δ/rdh54Δ double mutant suggests that Rdh54 may partially compensate for Rad54 during mitotic growth.

Why test for COVID-19 at home?

• To determine if you need to self-isolate.
• To protect others from infection by COVID-19.
• To support decisions about your care.

When to use it?

• If you want to diagnose a current COVID-19 infection.
• If you are concerned that you have been infected with COVID-19.

Description

Home test STANDARD Q COVID-19 Ag

Nose swab

• Insert the swab about 1.5 cm into the nostril.
• Less invasive and technically less complex than a sampling of other upper airway sites.

Reliable test performance

82.5% sensitivity and 99.9% specificity when tested by patients in Germany*

Fast test time

Getting a quick result in 15 minutes.

Simple test method

The STANDARD Q COVID-19 Ag Home test does not require a qualified professional or equipment to interpret the test results.

What is the rapid antigen test?

Rapid antigen tests could help quickly identify those who may have been exposed to the virus and isolate them from the community. STANDARD Q COVID-19 Ag Home Test is a rapid antigen test to detect infection in people suspected of having COVID-19.

How can this diagnose COVID-19 in just 15 minutes?

This test detects the presence of viral proteins called the COVID-19 virus antigen. If an antigen is present in the respiratory sample, the test device will capture it and display it to you through a coloured band. With technology from global diagnostic company SD Biosensor, you can get highly accurate results within 15 minutes.

This is a safe test for the detection of COVID-19 infection. This test is ideally designed to protect you and your family from the spread of the virus. During the test, you will place a swab soaked in the sample into the “solution tube.” This solution works to perform tests, while at the same time inactivating the virus. This avoids the risk of infection by inactivating the virus in 2 minutes without affecting the test results.

Process any sample at any scale

Our wide range of genomic DNA extraction kits are grouped here by sample type, so you can easily find the best DNA extraction kit for your needs. Regardless of sample type, you can expect high yields and high-quality DNA to use in your downstream applications.

Our products cover a variety of performance options and processing methods, giving you flexible options. If you want to run fewer than 24 samples at a time, choose from our manual single-prep solutions. If you’re looking for an automated solution, our cartridge-based kits for use with Maxwell® Instruments can process up to 48 samples in the same run. We also offer kits designed for fully automated plate-based processing methods.

DNA Purification Kits

Classic DNA Purification Kits use a silica resin to tightly bind DNA under high-salt conditions, allowing proteins, small RNAs, and other molecules to be removed by washing steps with a salt/ethanol solution. After washing, the purified DNA is eluted in water or TE buffer. A broad portfolio of GenElute™ DNA purification kits tailored to specific sample types is available, enabling purification of genomic DNA (gDNA) from cell culture, mammalian tissue, blood, bacteria, viruses, plant tissue, soil, water, urine and faeces.

GenElute™ Cell-Free DNA Kits provide rapid and efficient purification of circulating free DNA (cfDNA) to recover cell-free DNA fragments in the 100 bp to 500 bp range, suitable for a wide range of downstream applications, including next-generation sequencing, qPCR and bisulfite sequencing.

Characteristics:

• Silica-based DNA purification method
• High-purity, high-yield DNA at the lowest cost per preparation
• Most common technique used for DNA purification in laboratory workflows
• ~90 min protocol (varies based on lysis time)
• The complete portfolio of kits tailored to specific sample types
• Vacuum and spin formats
• Multi-analyte (RNA/DNA dual co-purification kits) available

Evaluation of DNA extraction

• Spectrophotometer and Qubit Measurements.

The purity of the DNA extracted with each method was evaluated using an Eppendorf photometer. The absorbance ratio at 260 nm and 280 nm was used to assess protein contamination, while the absorbance ratio at 260 nm and 230 nm was calculated to assess guanidine contamination. Both spectrophotometric measurements constituted criteria for the evaluation of DNA quality with higher values ​​associated with better DNA purity. The amount of DNA extracted by the different methods was evaluated using the Qubit 2.0 fluorometer (Invitrogen, Life technologies).

The Qubit fluorometer calculates the concentration based on the fluorescence of a dye that binds to double-stranded DNA (dsDNA). The Qubit fluorometer captures this fluorescence signal and converts it to a measure of DNA concentration using DNA standards of known concentration. The Qubit dsDNA BR assay kit was used for DNA quantification. Based on the DNA concentration derived from the Qubit measurements and the volume of the DNA extract, the total DNA yield was calculated with a simple multiplication.

• Gel electrophoresis.

The integrity of the DNA extracted by each method was assessed by gel electrophoresis. Specifically, 1 μl of each DNA extract was analyzed on a 1.5% agarose gel containing 0.5% ethidium bromide and visualized by U.V. illumination.

• Real-time PCR.

Real-time PCR targeting the ovine prion protein (PRNP) gene was used to assess the presence of amplifiable DNA in blood sample extracts. The set of primers (which amplify a 168 bp PRNP genomic region), amplification reaction setup and thermocycling conditions described in a previous study were also applied here. Ct values ​​were used to assess the amount of amplifiable DNA obtained. In this regard, smaller Ct values ​​are desirable.

A second real-time PCR protocol was applied to assess the ability of different genomic DNA extraction protocols to remove PCR inhibitors from blood samples. The presence of PCR inhibitors in genomic DNA extracts was tested by adding 1000 bacterial genomic copies of DNA (Campylobacter coli C. coli, strain ATCC 43478) to 100 ng and 1000 ng sheep DNA extracts, respectively, followed by real-time PCR amplification. of the hydroxymethyltransferase (GLA) gene. Real-time PCR amplifications were performed with a Biorad CFX96 real-time system.

All samples were tested in triplicate, while three controls containing only the C. coli DNA tip (no sheep DNA) were included in each PCR test. The Ct values ​​obtained in the process were used to evaluate the presence of PCR inhibitors. Specifically, the resulting inhibition of amplification was evaluated in comparison to the unenriched control.

Casein peptones are manufactured by enzymatic hydrolysis of selected milk proteins. Traditionally used enzyme preparations have long been based on natural active ingredients such as pancreatin. Biotechnological enzymes obtained by fermentation are increasingly used today. To avoid any BSE contamination, Organotechnie exclusively uses milk proteins produced in non-affected countries such as New Zealand. In addition, the casein we use comes from milk intended for human consumption and meets the requirements of regulatory agencies.

Definition

Casein Peptone Plus is a multipurpose pancreatic casein hydrolyzate enriched with growth factors

Description

• The fine cream powder is easily soluble in water.
• Contains a blend of peptides, free amino acids and growth factors.
• Casein Peptone Plus is made only from New Zealand milk material.

Use

Source of organic nitrogen and growth factors recommended in media for:

– Analytical Microbiology
– Industrial fermentation (pharmaceutical, food, cosmetic)
– Life sciences

Documentation

A certificate of analysis and a health certificate is delivered with each delivery.

Packaging and storage

• 25 kg net corrugated cardboard box with inner polyethene bags.
• On request: 5 kg plastic drum.
• Store in its original closed packaging when not in use.
• at room temperature in a dry place.
• Hygroscopic product.
• Shelf life: 5 years.

What’s new in this update?

• The reference list of available assays has been updated.
• Delta and Omicron variant assays have been included.
• The Rapid Antigen Detection Tests chapter has been updated to include information related to the Health Security Committee’s list of mutually recognized tests in EU/EEA countries.

Key messages

• Several SARS-CoV-2 VOCs have emerged in recent months and monitoring their circulation in all countries is of vital importance to prevent and control the spread of VOCs.
• Complete sequencing of the SARS-CoV-2 genome, or at least complete or partial sequencing of the S gene, should be used to confirm infection with a specific variant and to characterize the variant.
• For early detection and estimation of VOC prevalence (or where sequencing capacity is limited), alternative methods such as NAAT-based diagnostic tests should be used. To contain or delay the introduction of a VOC, ideally, positive samples should be tested using a NAAT-based assay that can offer the advantage of rapid results.
• When NAAT-based methods are employed, sequencing should be used to characterize at least a subset of the variants.
• In case of the low prevalence of a VOC in the population and when the goal is to delay the introduction and spread of the VOC, ideally, all NAAT-based results indicative of the VOC should be confirmed by sequencing.
• The selection of samples and methods is key and will depend on the objectives (eg, evaluating the circulation of the different variants of SARS-CoV-2 using representative samples of the community, or genomic characterization to monitor the evolution of the virus and inform decisions about vaccine composition or outbreak analyses).
• Assay validation should be performed to ensure that the laboratory testing system is working properly for circulating viruses.
• Laboratories must remain vigilant to ensure that they detect reduced sensitivity or failure to detect/identify circulating variants by different PCR or antigen-based assays.
• If diagnostic capacity is insufficient, priority should be given to severe cases, fatal cases, and cases with suspected high contagiousness of the pathogen that caused the outbreak, especially among those vaccinated or with a history of COVID-19.
• SARS-CoV-2 consensus sequences should be submitted to GISAID or other public databases. Raw sequences should also be submitted to the European Nucleotide Archive (ENA).
• Detection of new VOCs or outbreaks of currently circulating VOCs must be reported immediately through the Early Warning and Response System (EWRS), while variant detections must be reported to Tessy on a weekly basis.

SARS-CoV-2 variants with CDC classification

1. Lineage (VOC).

Originally from the UK, The B.1.1.7 variant contains many genetic mutations. the variant was first identified in September 2020. Since then, the variant it can been found in numerous countries around the world. The tension was found in the United States in late December 2020. It has become the most common strain in the US and is associated with higher transmissibility (i.e., more efficient with rapid transmission) and increased mortality.

2. Lineage B.1.351 (VOC).

In South Africa, the variant B.1.351 arose independently of B.1.1.7, although the variant shares some genetic mutations with the strain. B.1.351 was the first identified in South Africa in samples dating from early October 2020. Since then, cases have been detected in several countries, including the US in 2021.

3. Lineage P.1 (VOC).

The P.1 variant was first identified in Brazil Travellers. The variant has 17 unique genetic mutations, including three in the receptor-binding domain (RBD) region of the spike protein. Surveillance detected the variant in the US in late January 2021. Variant P.1 represents a branch of the B.1.1.28 lineage. Evidence suggests that some of the mutations in the P.1 variant may affect transmissibility and antigenic profile. The appearance of the variant and the association with higher viral density raised concerns about possible increased transmission or a propensity for reinfection.

4. B.1.427 and B.1.429 (VOC).

Both strains of coronavirus California are now officially characterized. Deformations can be 20% more transmissible than the common strains found in California, Nevada, Arizona, Wisconsin and Virginia.

5. B.1.526 (VOI).

First detected in New York in November 2020, the B.1.526 is a variant of interest. For February 2021 mutations appeared in about 25% of the entire COVID genomes.

Thermo Scientific Pierce Poly-HRP Dilution Buffer is optimized to ensure optimal performance with Poly-HRP Streptavidin.

Characteristics of Poly-HRP Dilution Buffer:

• Formulation: 1% biotin-free casein in a PBS buffer solution.
• Application: Dilution of Poly-HRP Streptavidin (Product # N200): Gently invert bottle and shake to mix before use.

do not shake Use it at full strength. See the Streptavidin Poly-HRP Certificate of Analysis for recommended dilution ranges. Dilute Poly-HRP Streptavidin immediately before use.

Related products: Streptavidin poly-HRP

Specifications

Reagent type: Diluent

Format: Liquid

Content and storage

Shipped in cold packs. Store at 2°C to 8°C immediately upon receipt.

Diluent for long-term storage of HRP conjugates

Improved formulation to improve stability and activity

• HRP-Protector™ is a long-term stabilizer for horseradish peroxidase (HRP) coupled to antibodies or neutravidin/streptavidin. HRP-Protector™ gives peroxidase conjugates crucial long-term stability for several years, even when stored at low concentration dilutions. HRP-Protector™ can be used directly as assay buffer to incubate HRP conjugates.
• Typical concentrations for detection are between 40 and 500 ng/mL.
• If you have any background or interference, eg from cross-reactivities, we recommend that you use LowCross® HRP-Stab as a long-term stabilizer for the conjugate.
• The old formulation is still available.
• Order number 220 050 (50 mL), order number 220 125 (125 mL), and order number 220 500 (500 mL).