Double gene focusing on PCR assay for the detection of Crocodylus porosus in industrial merchandise.
The demand for crocodile meat is rapidly rising due to its unique and organoleptic attraction and likewise the low content material of ldl cholesterol and lipids. Furthermore, crocodile oil and blood have been utilized in various medicines for treating bronchial asthma and several other different illnesses since historical occasions. Moreover, crocodile hides have nice demand in leather-based industries.
All of those have collectively contributed to the intensive searching, unlawful buying and selling and consequent decline of crocodiles in most elements of the world. To maintain area with the rising calls for, some crocodile species comparable to Crocodylus porosus have been raised in farms and its industrial trades have been legalised.
Nevertheless, demand for wild crocodiles in meals and medicines has continued in excessive gear. Not too long ago, a number of DNA-based strategies have been proposed for crocodile detection, however these assays are primarily based on single gene and longer-sized amplicon targets that break down throughout intensive processing.
To handle this hole, right here we developed and validated a extremely secure double gene focused multiplex PCR assay for the identification of C. porosus supplies in industrial merchandise. The assay concerned two quick websites from C. porosus atp6 (77 bp) and cytb (127 bp) genes and a common inside management (99 bp) for eukaryotes.
The PCR primers had been cross-tested in opposition to 18 species and validated underneath pure and blended matrices underneath intensive boiling, autoclaving and microwave cooking circumstances.
Lastly, it was used to determine 5 crocodile-based industrial merchandise. The decrease limits of detection for atp6 and cytb genes had been 0.001 ng and 0.01 ng DNA, respectively, in pure meat and 1% underneath blended matrices.
Some inherent options, comparable to 77-127 bp amplicon sizes, distinctive stability and superior sensitivity, prompt the assay could possibly be used for the identification of C. porosus in any forensic specimen.
A Quick and Dependable Actual-Time PCR Technique for Detection of Ten Animal Species in Meat Merchandise.
Species substitution in meat merchandise is a typical drawback reported worldwide. One of these meals fraud is, usually, an intentional act for financial achieve, utilizing sources of low-priced meats in high-value meat merchandise. Penalties embody financial, well being, and non secular issues. Extremely delicate and environment friendly methods are thus required to detect meat species.
This paper describes a way primarily based on real-time PCR to detect 10 animal species (Bos taurus, Sus scrofa, Ovis aries, Capra hircus, Gallus gallus, Meleagris gallopavo, Bubalus bubalis, Equus caballus, Felis catus, and Canis familiaris) in meat product. The strategy combines species-specific and common (used right here as inside constructive management) primers, and applies soften curve evaluation for amplicon checking. Technique accuracy was evaluated on 46 experimental meat mixtures and all species had been accurately recognized in all instances, at 1% take a look at sensitivity.
Evaluation of 14 industrial meat merchandise revealed that 6 of 14 samples had nondeclared bovine and/or rooster materials. We carried out an interlaboratory comparability utilizing the reference meat mixtures and industrial samples, attaining 100% of reproducibility. The developed take a look at proved to be efficient and dependable for routine evaluation of meat merchandise.
Description: Small Intestine tissue lysate was prepared by homogenization in modified RIPA buffer (150 mM sodium chloride, 50 mM Tris-HCl, pH 7.4, 1 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% sodium dodecylsulfate, 5 μg/ml of aprotinin, 5 μg/ml of leupeptin. Tissue and cell debris was removed by centrifugation. Protein concentration was determined with Bio-Rad protein assay. The product was boiled for 5 min in 1 x SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 50 mM DTT.
Description: Small intestine tissue lysate was prepared by homogenization in modified RIPA buffer (150 mM sodium chloride, 50 mM Tris-HCl, pH 7.4, 1 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% sodium dodecylsulfate, 5 μg/ml of aprotinin, 5 μg/ml of leupeptin. Tissue and cell debris was removed by centrifugation. Protein concentration was determined with Bio-Rad protein assay. The product was boiled for 5 min in 1 x SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 50 mM DTT.
Description: Small intestine cancer, metastatic lymph nodes and normal tissue high density tissue microarray, 69 cases/208 core, with stage and grade data
Small intestine disease spectrum (small intestine cancer progression) tissue array
Description: Small Intestine tumor tissue lysate was prepared by homogenization in lysis buffer (10 mM HEPES pH7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM ethylenediaminetetraacetic acid, 10% glycerol, 1% NP-40, and a cocktail of protease inhibitors). Tissue and cell debris was removed by centrifugation. The product was boiled for 5 min in 1 x SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 50 mM DTT.
Description: Human small intestine tissue membrane protein lysate was prepared by isolating the membrane protein from whole tissue homogenates using a proprietary technique. The human small intestine tissue was frozen in liquid nitrogen immediately after excision and then stored at -70°C. The membrane protein is provided in a buffer including HEPES (pH 7.9), MgCl2, KCl, EDTA, Sucrose, Glycerol, sodium deoxycholate, NP-40, and a cocktail of protease inhibitors. For quality control purposes, the isolated small intestine tissue membrane protein pattern on SDS-PAGE gel is shown to be consistent for each lot by visualization with coomassie blue staining. The isolated small intestine tissue membrane protein is then Western analyzed by either GAPDH or β-actin antibody to confirm there is no signal or very weak signal.
Description: Human small intestine tissue cytoplasmic protein lysate was prepared by isolating the cytoplasmic protein from whole tissue homogenates using a proprietary technique. The human small intestine tissue was frozen in liquid nitrogen immediately after excision and then stored at -70°C. The cytoplasmic protein is provided in a buffer including HEPES (pH 7.9), MgCl2, KCl, EDTA, Sucrose, glycerol, and a cocktail of protease inhibitors. For quality control purposes, the isolated small intestine tissue cytoplasmic protein pattern on SDS-PAGE gel is shown to be consistent for each lot by visualization with coomassie blue staining. The isolated small intestine tissue cytoplasmic protein is then Western analyzed by GAPDH antibody, and the expression level is consistent with each lot.
Description: Human small intestine tissue membrane protein lysate was prepared by isolating the membrane protein from whole tissue homogenates using a proprietary technique. The human small intestine tissue was frozen in liquid nitrogen immediately after excision and then stored at -70°C. The membrane protein is provided in a buffer including HEPES (pH 7.9), MgCl2, KCl, EDTA, Sucrose, Glycerol, sodium deoxycholate, NP-40, and a cocktail of protease inhibitors. For quality control purposes, the isolated small intestine tissue membrane protein pattern on SDS-PAGE gel is shown to be consistent for each lot by visualization with coomassie blue staining. The isolated small intestine tissue membrane protein is then Western analyzed by either GAPDH or β-actin antibody to confirm there is no signal or very weak signal.
Description: Human small intestine tissue lysate was prepared by homogenization using a proprietary technique. The tissue was frozen in liquid nitrogen immediately after excision and then stored at -70°C. The human small intestine tissue total protein is provided in a buffer including HEPES (pH7.9), MgCl2, KCl, EDTA, Sucrose, Glycerol, Sodium deoxycholate, NP-40, and a cocktail of protease inhibitors. For quality control purposes, the small intestine tissue pattern on SDS-PAGE gel is shown to be consistent for each lot by visualization with coomassie blue staining. The small intestine tissue is then Western analyzed by either GAPDH or β-actin antibody, and the expression level is consistent with each lot.
Description: Small intestine carcinoma test tissue array, with normal small intestine tissues, including pathology grade, TNM and clinical stage, 6 cases/24 cores, replacing T421a
Description: The Matched Pair Paraffin Tissue (MPPT) slides are designed for identifying tumor-specific/metastasis genes or proteins. Slices from normal and malignant tissues are mounted on each MPPT slide which can then be treated as a single histological slide for H&E staining, immunohistochemistry, or in situ hybridization. This format allows a rapid analysis of protein expression and localization across normal and cancerous tissue.
Total Protein - Mouse Normal Tissue: Small Intestine
Description: The Multiple Species Tissue Array (MSTA) slides were designed to study protein expression patterns in different cells and tissues from multiple species. Tissue slices from three different species are mounted on each MSTA slide which can then be treated as a single histological slide for H&E staining, immunohistochemistry, or in situ hybridization. This format allows a rapid analysis of protein expression and localization across different species. MSTA slides can also be used to quickly determine the species reactivity of a given antibody.
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UNASSIGNED
This paper describes a quick and dependable methodology for species detection in meat merchandise primarily based on real-time PCR. It may be utilized for evaluation of in natura or processed meat. The strategy proposed right here can play an vital position in controlling the origin of meat merchandise, guaranteeing their high quality and security for the complete meals industry-producers to shoppers.
A common mini-vector and an annealing of PCR merchandise (APP)-based cloning technique for handy molecular organic manipulations.
Presently, essentially the most extensively used methods for molecular cloning are sticky-end ligation-based cloning, TA cloning, blunt-end ligation-based cloning and ligase-independent cloning. On this examine we have now developed a novel mini-vector pANY1 which may concurrently meet the necessities of all these cloning methods.
As well as, the choice of applicable restriction digestion websites is tough in some instances due to the presence of inside websites. On this examine, an annealing of PCR merchandise (APP)-based sticky-end cloning technique was launched to keep away from this challenge. Moreover, false positives happen throughout molecular cloning, which will increase the workload of isolating constructive clones.
The plasmid pANY1 incorporates a ccdB cassette between a number of cloning websites, which effectively avoids these false positives. Subsequently, this mini-vector ought to function a great tool with large functions in biosciences, agriculture, meals applied sciences.
Description: Small Intestine tissue lysate was prepared by homogenization in modified RIPA buffer (150 mM sodium chloride, 50 mM Tris-HCl, pH 7.4, 1 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% sodium dodecylsulfate, 5 μg/ml of aprotinin, 5 μg/ml of leupeptin. Tissue and cell debris was removed by centrifugation. Protein concentration was determined with Bio-Rad protein assay. The product was boiled for 5 min in 1 x SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 50 mM DTT.
Description: Small intestine tissue lysate was prepared by homogenization in modified RIPA buffer (150 mM sodium chloride, 50 mM Tris-HCl, pH 7.4, 1 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% sodium dodecylsulfate, 5 μg/ml of aprotinin, 5 μg/ml of leupeptin. Tissue and cell debris was removed by centrifugation. Protein concentration was determined with Bio-Rad protein assay. The product was boiled for 5 min in 1 x SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 50 mM DTT.
Description: Small intestine cancer, metastatic lymph nodes and normal tissue high density tissue microarray, 69 cases/208 core, with stage and grade data
Description: Small Intestine tumor tissue lysate was prepared by homogenization in lysis buffer (10 mM HEPES pH7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM ethylenediaminetetraacetic acid, 10% glycerol, 1% NP-40, and a cocktail of protease inhibitors). Tissue and cell debris was removed by centrifugation. The product was boiled for 5 min in 1 x SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 50 mM DTT.
Description: Human small intestine tissue membrane protein lysate was prepared by isolating the membrane protein from whole tissue homogenates using a proprietary technique. The human small intestine tissue was frozen in liquid nitrogen immediately after excision and then stored at -70°C. The membrane protein is provided in a buffer including HEPES (pH 7.9), MgCl2, KCl, EDTA, Sucrose, Glycerol, sodium deoxycholate, NP-40, and a cocktail of protease inhibitors. For quality control purposes, the isolated small intestine tissue membrane protein pattern on SDS-PAGE gel is shown to be consistent for each lot by visualization with coomassie blue staining. The isolated small intestine tissue membrane protein is then Western analyzed by either GAPDH or β-actin antibody to confirm there is no signal or very weak signal.
Description: Human small intestine tissue cytoplasmic protein lysate was prepared by isolating the cytoplasmic protein from whole tissue homogenates using a proprietary technique. The human small intestine tissue was frozen in liquid nitrogen immediately after excision and then stored at -70°C. The cytoplasmic protein is provided in a buffer including HEPES (pH 7.9), MgCl2, KCl, EDTA, Sucrose, glycerol, and a cocktail of protease inhibitors. For quality control purposes, the isolated small intestine tissue cytoplasmic protein pattern on SDS-PAGE gel is shown to be consistent for each lot by visualization with coomassie blue staining. The isolated small intestine tissue cytoplasmic protein is then Western analyzed by GAPDH antibody, and the expression level is consistent with each lot.
Description: Human small intestine tissue membrane protein lysate was prepared by isolating the membrane protein from whole tissue homogenates using a proprietary technique. The human small intestine tissue was frozen in liquid nitrogen immediately after excision and then stored at -70°C. The membrane protein is provided in a buffer including HEPES (pH 7.9), MgCl2, KCl, EDTA, Sucrose, Glycerol, sodium deoxycholate, NP-40, and a cocktail of protease inhibitors. For quality control purposes, the isolated small intestine tissue membrane protein pattern on SDS-PAGE gel is shown to be consistent for each lot by visualization with coomassie blue staining. The isolated small intestine tissue membrane protein is then Western analyzed by either GAPDH or β-actin antibody to confirm there is no signal or very weak signal.
Membrane Protein - Human Tumor Tissue: Small Intestine
Description: Human small intestine tissue lysate was prepared by homogenization using a proprietary technique. The tissue was frozen in liquid nitrogen immediately after excision and then stored at -70°C. The human small intestine tissue total protein is provided in a buffer including HEPES (pH7.9), MgCl2, KCl, EDTA, Sucrose, Glycerol, Sodium deoxycholate, NP-40, and a cocktail of protease inhibitors. For quality control purposes, the small intestine tissue pattern on SDS-PAGE gel is shown to be consistent for each lot by visualization with coomassie blue staining. The small intestine tissue is then Western analyzed by either GAPDH or β-actin antibody, and the expression level is consistent with each lot.
Total RNA - Human Adult Normal Tissue: Small Intestine
Description: Small intestine carcinoma test tissue array, with normal small intestine tissues, including pathology grade, TNM and clinical stage, 6 cases/24 cores, replacing T421a
Membrane Protein - Human Adult Normal Tissue: Small Intestine
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