Method based on the polymerase chain reaction for the detection of hepatitis A virus in eggs and shellfish.

Outbreaks of gastroenteritis suspected to be caused by the virus are on the rise. Therefore, there is a need for regulatory agencies responsible for food safety to develop adequate techniques for the detection of viruses in food. We have established a general procedure for the detection of hepatitis A virus (HAV) in shellfish that, with minor modifications, can also be used for fresh produce such as coriander. Total RNA was isolated from mussels or coriander, followed by isolation of poly (A) -containing RNA. Since HAV genomic RNA contains poly (A) tails, isolation of RNA -containing poly (A) also enriches HAV genomic RNA. Reverse transcription is used to convert RNA to cDNA, and then amplification is performed by polymerase chain reaction (PCR). Boosters with internal primers are used to improve the quality and quantity of amplified DNA, enabling post-PCR analysis such as the identification of viral strain sequences. With this procedure, a trained person can test multiple samples in four business days. The nominal sensitivity of the detection method was 0.15 TCID50 (50% of the tissue culture infective dose) per 0.62 g of tissue with the test virus. The direct RNA isolation protocol avoids the pitfalls associated with the entire virus removal procedure by replacing viral precipitation steps involving polyethylene glycol and Procipitate with phenol extraction. The method is simple and reliable. We successfully used this procedure to detect naturally occurring HAV in shellfish involved in gastroenteritis outbreaks, as well as in coriander artificially contaminated with the test virus.


Biotechnology support group launched: DNA enrichment from protein samples with ProCipitate™

In order to perform sequencing, polymerase chain reaction (PCR), microarrays, and similar techniques for molecular biology studies, researchers routinely implement DNA extraction or enrichment techniques to separate DNA from samples. Traditionally, DNA from biological samples such as blood, humans, animals, plant tissues, bacteria, and cell cultures is separated from DNA by a mixture of proteins and organic solvents, such as phenol. The phenol extraction method for nucleic acid solution decomposition is toxic, requires re-distillation before use, and phenolic oxidation products (quinone, di-acid, etc.) accumulate during storage, which are purified by hemodialysis. ProCipitate™ is non-hazardous and can replace phenol/chloroform with the added benefits of solid phase suspensions: adaptability to filtration and automation. It is commonly used for plasmids, conjugates, BACs, genomic DNA, as well as RNA. ProCipitate™ can also be used to remove proteinase K and other enzymes. ProCipitate™ provides high-quality DNA suitable for automated sequencing, southern blotting and limited digestion. ProCipitate™ binds to proteins and the DNA/RNA remains unreacted.

Preparation of high-quality, quantitative genomic DNA requires harvesting cells containing DNA, releasing DNA from harvested cells, separating DNA from proteins and cellular contaminants, and isolating concentrated DNA for use in genomic and genomic analysis techniques, such as genetic mapping, TILLING (targeting ) local lesions induced by the genome) and next generation sequencing. ProCipitate™ is a high-throughput, high-throughput, cost-effective enrichment of genomic DNA for small or large samples. It is compatible with high-throughput genomic DNA isolation and produces an improved yield of high-quality DNA compared to alternative ligation and elution systems. ProCipitate™ is available as a suspended reagent and in ProPrep™ kits for specific applications and high performance 96-well filter formats.


P0050-100 Biotech Support Group 100 mL 1217 EUR


P0050-30 Biotech Support Group 30 mL 511 EUR

Localization of membrane cations in the anterior limbs of human neutrophils during chemotaxis.

Potassium pyroantimonate is used to localize cation binding sites in human neutrophils under conditions of random migration, stimulated random migration (chemokinesis), and directed migration (chemotaxis). Cells were placed in a standard chamber where a 0.45 micron micropore filter separated the cells from the stimulus (buffer, serum-activated Escherichia coli endotoxin, or synthetic chemotactic peptide N-formyl-Met-Leu-Phe). The small pore filter allows the formation of pseudopods but prevents cell migration through the filter. The cells examined under all conditions had a dense precipitate of antimonate salts in various granules. However, antimonate deposits have been localized to nuclear condensed chromatin during random migration and are largely associated with uncondensed nuclear chromatin during chemokinesis and chemotaxis. Under chemokinesis deposition conditions, antimonate precipitates appear in the cytoplasm of the neutrophil plasma membrane, whereas under chemotaxic cation deposition conditions below the cell membrane, they are located in pseudopods directed to chemoattractants. In addition to endotoxin-activated serum, N-formyl-Met-Leu-Phe concentrations that induce neutrophil chemotaxis (10 (8) M) also cause cation deposition beneath the cell membrane at the primary end of the cell. , regardless of whether it is albumin. found in incubation media. However, with higher concentrations of synthetic peptides (10 (5) M) that produce granular and non -chemotactic releases, submembrane cation deposition is not visible. EDTA (10 mM) and EGTA (10 mM) removed nuclear, granular and submembranous cation deposits from neutrophils examined under chemotaxic conditions. X-ray microprobial analysis of antimonate deposits revealed the possible presence of calcium but did not detect sodium or magnesium. The data indicate that chemotactic factors induce the deposition of submembrane cations, most likely Ca ++, that localize at the major margins of cells exposed to the chemoattractant gradient.

Removal of foreign matter from biological fluids containing nucleic acids and recovery of nucleic acids.

A method for removing unwanted protein and aggregated DNA from biological media containing nucleic acids by exposing the starting material to a water-insoluble compound consisting of ProCipitate™ and protein intercalated with ferric oxide particles under magnetic force.

The present invention relates to a means to remove unwanted aggregated proteins and DNA from biological media containing the desired nucleic acids by subjecting the starting material to a water-insoluble compound consisting of ProCipitate™ protein and phenoxide-infiltrated aggregated DNA to a magnetic process. duty. or by incorporating heavy metal oxides such as bismuth oxide chloride into the accumulator ProCιpιtate™ and allowing the resulting aggregate to settle under unit gravity. The described method involves many steps and is expensive. First, the magnetic particles must be chemically broken apart to allow DNA binding. The present invention utilizes non-decomposing magnetic particles and is not limited to solvent composition and ionic strength.

Nucleic acids are polyminic acids. In addition to having a large number of nucleotides and nbose residues, it possesses a set of negatively charged phosphate groups. Because of its strong negative charge, it must bond strongly to the surface of a positively charged fuming metallic oxide. (Kummert R, and Strum W, International Journal of Colloid and Interface Science, 75 (2) 373, 1980) showed that organic molecules with molecular masses less than 200 Daltons and with carboxylic functional groups, phenol-OH or amino group can form covalent bonds With structural metal, bonded to a fumigated aluminum oxide surface. The compounds used in these studies are phthalic acid, benzoic acid, salicylic acid, and catechol. Since the main focus and objective is to bind polyminic acids to the oxide. On the surface, very little can be learned from studies using monomeπc particles. In general, binding of a polyelectrolyte (eg DNA) to a surface containing multiple permanent charges of an opposite tag is more convenient than binding of an isolated monomeric unit (eg deoxy πbonucleoside tπphosphate) to the same surface. The simultaneous existence of multiple interactions when multiple electrolytes and surface bonding can lead to their cooperation, and together they can be much stronger than would be expected from the sum of the individual binding forces.


Leave a Comment