157 results about "Cronobacter sakazakii" patented technology

The invention relates to the field of microbial detection and discloses a method for rapid detection of enterobacter sakazakii. The method comprises the steps of: adding immunized superparamagnetism nanometer magnetic beads into a sample, adding a stabilizer, and incubating for 45-60min at 30-40 DEG C, wherein the magnetic beads are specifically bonded and enriched on microbial food-borne pathogenic bacteria, namely the enterobacter sakazakii, in the sample; and measuring spinning-spinning relaxation time and calculating a value deltaT2 by taking a bacteria-free sample as a blank control. The method disclosed by the invention has high specificity and sensitivity to the detection of the enterobacter sakazakii, is rapid, and is particularly suitable to detection of low-concentration enterobacter sakazakii, especially the enterobacter sakazakii in dairy products; in addition, the method is convenient and simple to operate and has good reliability and low requirement on assorted equipment.

The invention discloses a fourteen-food-borne pathogenic bacterium multiplex PCR detection primer set and a kit. The detection primer set is composed of primer pairs for detecting salmonella, Shigella, Vibrio parahemolyticus, campylobacter jejuni, campylobacter coli, staphylococcus aureus, bacillus cereus, Listeria monocytogenes, yersinia enterocolitica, enterobacter sakazakii, Escherichia coli, vibrio cholerae, Escherichia coli O157, aeromonas hydrophila and internal positive control. A multiplex PCR detection method based on an ordinary PCR platform is built, multiplex PCR reactions are carried out on genomic DNA, extracted from a sample to be tested, of bacteria in the same reaction system through the primer pairs acquired through analysis and design, and whether the food-borne pathogenic bacteria are contained in the sample or not is judged through the electrophoretic analysis of reaction products.

The invention discloses a loop-mediated isothermal amplification detection primer group, a detection method and a detection kit for enterobacter sakazakii. Aiming at ompA genes of the enterobacter sakazakii, in the invention, a specific detection primer group, a detection kit containing the detection primer group, and a detection method utilizing the detection kit are designed and selected, so that detection is carried out on samples to be detected, especially milk powder, thus determining whether the specific gene segments of the enterobacter sakazakii exist, and further determining whether the samples to be detected contains the enterobacter sakazakii. According to the invention, the detection kit and the detection method have the advantages of high sensitivity, strong specificity, short detection time, without a PCR (polymerase chain reaction) instrument and an electrophoresis apparatus, are simple for operation process, are especially suitable for a basic detection mechanism and food enterprises for self check, and have important meaning for fast detecting milk powder and ensuring the safety of the milk powder.

The invention discloses a kit used for rapid detection of enterobacter sakazakii in milk, and applications thereof, and belongs to the field of food safety technology. The kit comprises a loop-mediated isothermal amplification (LAMP) reaction system, Bst DNA polymerase, a probe and a colloidal gold test strip. The kit is capable of solving a problem that result interpretation of existing LAMP detection methods is not capable of realizing on-site detection; result interpretation method of the test strip of the kit is simple, no instrument is needed, and interpretation can be realized by the naked eye.

The present invention provides a gene chip for detecting common pathogen in dairy, which comprises a solid-phase vector and an oligonucleotide probe fixed on the solid-phase vector, wherein the oligonucleotide probe includes DNA fragment or its complementary DNA or RNA sequence selected from E. sakazakii, streptococcus pyogenes, staphylococcus aureus, Klebsiella pneumoniae, Klebsiella oxytoca, Listeria monocytogenes, salmonella and 16S-23S rDNA intergenic spacer region of Bacillus cereus, as well as ipaH gene of Shigella or gyrB gene of citrobacter freundii. The invention further provides a reagent box which uses the gene chip to detect pathogen in dairy. The gene chip and the reagent box of the invention for detecting pathogen in dairy are easy to operate, highly precise and greatly repeatable.

The invention discloses a method for detecting cronobacter sakazakii as well as a kit and a primer thereof. The method comprises the following steps of (1) extracting a genome DNA (deoxyribonucleic acid) of a sample to be detected; (2) taking the genome DNA obtained in the step (1) as a template, carrying out PCR (polymerase chain reaction) on the primers shown in sequences SEQ ID NO.1 and SEQ ID NO.2; and (3) detecting existence of a 498bp single amplification product in the reaction product obtained in the step (2), wherein the primers comprise the primers shown in the sequences SEQ ID NO.1 and SEQ ID NO.2. The kit comprises the primers. The method disclosed by the invention is short in detection time, low in cost, reliable in detection result, and simple to judge the result, and has single specificity; the method for simply, rapidly and sensitively detecting the cronobacter sakazakii is provided for the technical field of food safety inspection; and the method has significance on food safety in China.

Particular aspects provide novel compositions and methods useful for the growth, isolation and detection of microorganisms that have α-glucosidase activity (e.g., the bacterium E. sakazakii). Certain embodiments provide a novel growth and/or plating media, comprising a fluorogenic α-glucosidase substrate, which is both selective for and differential to E. sakazakii. In particular embodiments, the α-glucosidase substrate comprises 4-methylumbelliferyl-α-D-glucoside. Additional embodiments relate to a selection media. Further embodiments relate to a selective medium that is based on Tryptone Bile agar. Still further embodiments relate to OK media as defined herein. Other embodiments of the invention relate to methods for growing bacterial cultures on media that is selective for and differential to microorganisms that have α-glucosidase activity (e.g., the bacterium E. sakazakii).

The invention discloses a method for rapidly detecting and screening Enterobacter sakazakii, which comprises the following steps: adding immunized super-paramagnetic nanoparticles in a sample to be detected to enable the immunized super-paramagnetic nanoparticles to be specifically combined with Enterobacter sakazakii; collecting the immunized super-paramagnetic nanoparticles, and after washing the immunized super-paramagnetic nanoparticles, adding immunized luminescent quantum dots to enable the immunized luminescent quantum dots to be specifically combined with Enterobacter sakazakii carried by the immunized super-paramagnetic nanoparticles; and using an applied magnetic field to adsorb and collect Enterobacter sakazakii and immunized luminescent quantum dot combinations carried by the immunized super-paramagnetic nanoparticles, and after washing the combinations, conducting qualitative or quantitative fluorescence detection. By adopting the method, the target bacteria can be rapidly separated from all kinds of samples and can be efficiently enriched, the operation is convenient and simple, the reliability is high and the requirement on the supporting equipment is low.

The invention relates to a rapid detection reagent kit for Enterobacter sakazaii by using a loop-mediated isothermal amplification technology and a detection method thereof. A loop-mediated isothermal amplification reaction liquid, Bst DNA polymerase and a chromogenic reagent are placed in the reagent kit; and the reaction liquid contains a reaction buffer liquid, dNTP, magnesium sulfate, an upstream inner primer 5- TATGCGGGATCGAACCGCAGA-GGCTATAGCTCAGCTGGGA-3, a downstream inner primer 5- GCTCCACCATCACTTCGGAGTG-TTCAGCTTGTTCCGGATTGT-3, an upstream outer primer 5- TCCGCAGGAGTTGAAGAGG-3, a downstream outer primer 5-CAGCAGCGTGTCTGTTTCA-3 and lycine. The method for detecting the Enterobacter sakazaii comprises the extraction of bacterial DNA, the loop-mediated isothermal amplification of the Enterobacter sakazaii, and chromogenic detection. The rapid detection reagent kit and the detection method have the advantages of quickness, strong specificity, high sensitivity and low cost.

The invention discloses a detecting method (ELISA) of enzyme link immune suction of banqi-enterobacteria and antibody and alpha-glucosidase and coded nucleic acid and carrier of the antibody, which is characterized by the following: using the amino acid sequence of alpha-glucosidase as Seq ID No:2; using ELISA of antibody to make agent box of the antibody; detecting the banqi-enterobacteria rapidly and effectively; providing high sensitivity, strong specificity and reliable result.

The invention provides a fluorogenic culture medium of Enterobacter sakazakii, a test kit and a test method thereof, and relates to a method for testing microbe and a composition used by the method. The fluorogenic culture medium comprises the compositions as follows: 8 to 20 grams of peptone, 4 to 7 grams of beef extract powder, 4 to 7 grams of sodium chloride, 1.0 to 2.0 grams of cholate, 12 to 20 grams of agar, 0.05 to 0.5 grams of X-a-glucopyranoside, 0.01 to 0.04 grams of Na2CO3, 0.3 to 2.0 grams of ferric ammonium citrate, and 0.3 to 2.0 grams of sodium thiosulfate. The test kit consists of the fluorogenic culture medium of Enterobacter sakazakii, an enriched liquid A ,namely buffer peptone water culture medium, and an enriched liquid B, namely modified lauryl sulfate pancreas peptone broth. The test kit is configured simply, and the test method has high sensitivity in detection, so the method is suitable for treating large flux samples.

The invention provides a PCR (Polymerase Chain Reaction) method and kit for quickly detecting Enterobacter sakazakii in a baby formula. In the method, Enterobacter sakazakii is efficiently extracted from an artificially polluted baby formula, and is evaluated by comparison. The method comprises the following specific steps: amplifying an Enterobacter sakazakii gene used as the target gene, thus constructing a target recombinant plasmid as a standard substance for quantification; constructing an internal standard recombinant plasmid, adding into a reaction system, and amplifying together with the target gene; monitoring the reaction system, and eliminating the false negative; optimizing the fluorescent quantitative reaction system; determining the fluorescent quantitative method and the artificial pollution detection limit; and carrying out the actual detection. The invention has the advantages of high detection speed, good specificity, simple use steps and high repeatability, can further implement dual or multiple real-time PCR, and can complete the simultaneous detection of multiple food-borne pathogens.

The invention relates to a double-antibody sandwich method for detecting enterobacter sakazakii in food. The method is characterized in that the enterobacter sakazakii (ATCC29544) and lipopolysaccharide (LPS) extracted by coupling the enterobacter sakazakii (ATCC29544) with BSA (bovine serum albumin) are respectively used as immunogen to simultaneously immunize BALB/c mice, and 11 enterobacter sakazakii monoclonal antibodies are obtained through immunization, fusion and screening to respectively label HRP (horse radish peroxidase) and are paired through the enterobacter sakazakii. A No.7 monoclonal antibody CGMCC No. 7207 is used as a coating antibody, a No.8 monoclonal antibody CGMCCNO.7208 is used as an enzyme labeled antibody, a sandwich ELISA method of the enterobacter sakazakii is established by adopting the ATCC29544 as a standard product, and LOD is 53000CFU/Ml. By adopting the monoclonal antibody with high physicochemical property, good uniformity, good specificity and capability of being in mass preparation, the established sandwich method is high in sensitivity and low in cost, no cross reaction with salmonella enteritidis, E.coli, E.coliO157: H7, salmonella typhimurium, staphylococcus aureus and listeria monocytogenes exists, and thus a rapid and high efficient analysis means is provided for detecting the enterobacter sakazakii in the food.

The invention relates to a method for detecting enterobacter sakazakii based on a nucleic acid chromatography biosensing technique. According to a toxicity gene ompA of the enterobacter sakazakii, a loop-mediated isothermal amplification primer (LAMP) (shown in SEQ ID NO:1-4) is designed, and an enterobacter sakazakii detection method based on an LAMP nano-enzyme sensor is established by combining with a nano-enzyme nucleic acid test strip. The method provided by the invention can be successfully used for distinguishing viable bacterial cells and dead bacterial cells, and lower detection limit on the enterobacter sakazakii can reach 10CFU/mL.

The invention discloses a double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) method for detecting listeria in food on the basis of monoclonal antibodies, belonging to the technical field of immunoassay. The method comprises the following steps: immunizing 8-week BALB mouse with prokaryotically expressed listeria monocytogenes P60 protein, and obtaining 15 strains of listeria specific monoclonal antibodies by immunization, fusion and multiple screening; respectively marking horse radish peroxidase HRP, and carrying out pairwise coupling by taking the listeria monocytogenes CMCC54003 as targets. The sandwich ELISA method which is established by screening different paired cross reactions and taking 2G7 as a coating antibody and 2H8-HRP as an enzyme labelled antibody has cross reactions to listeria including the listeria monocytogenes, and has no cross reactions to tested staphylococcus aureus, escherichia coli, escherichia coli O157, salmonella, enterobacter sakazakii, campylobacter jejuni and campylobacter coli. According to the method disclosed by the invention, the monoclonal antibodies having listeria specificity are prepared and the double-antibody sandwich ELISA method is established, and the method provides a technical means for specific rapid detection of the listeria in food.

The invention belongs to the technical field of food safety rapid detection, and more specifically relates to a preparation method and a detection method of a nano immunosensor used for rapid detection of enterobacter sakazakii. According to the preparation method, a glassy carbon electrode is taken as a base electrode; a polyamide dendritic macromolecule material combined reductive graphene nano composite material is taken as an electroactivity modification membrane layer, and is immobilized onto the surface of the glassy carbon electrode; physical absorption or bovine serum albumin-glutaraldehyde crosslinking is adopted to immobilize horse radish peroxidase-labelled enterobacter sakazakii antibody so as to obtain the nano immunosensor. According to the detection method, the nano immunosensor is applied to enterobacter sakazakii detection in food, and obtained results accord with results obtained via a national standard method; the detection method is high in sensitivity, specificity, and accuracy, is short in detection time, is simple, convenient, and rapid, and possesses a promising application prospect.

The invention discloses an Enterobacter sakazakii enrichment reagent and preparation method thereof, the reagent comprises immunomagnetic beads coated with polyclonal antibody of Enterobacter sakazakii. The reagent concentrates the object micro-organism by directly enriching the object micro-organism in sample or bacterial enrichment culture medium and the key problem of the sample pre-processing is solved and the detection sensitivity and detection rate can be improved.

The invention relates to a Cronobacter sakazakii O antigen specific nucleotides and use thereof. The nucleotides comprises: 1) at least one of nucleotides represented by sequences from SEQ ID No.1 to SEQ ID No.14; and 2) at least of nucleotides complementary to the nucleotides represented by the sequences from SEQ ID No.1 to SEQ ID No.14. The nucleotides can be used for preparing polymerase chain reaction (PCR) kits and gene chips for detecting Cronobacter sakazakii. The Cronobacter sakazakii O antigen specific nucleotides, and the PCR kits and the gene chips containing the nucleotides have high practicality; the preparation method of the PCR kits are simple and convenient, the detection period is short, the detection speed is high, the operability of the kits is high, the industrial production of the kits is easy, and the detection cost is relatively low; the accuracy is high; and the sensitivity is high.

The invention discloses a method, a primer pair, a target probe, an internal standard probe and a kit for detecting Cronobacter sakazakii. The method comprises the following steps: (1) adding the deoxyribose nucleic acid (DNA) of a sample to be detected, the primer pair, the target probe, the internal standard probe and an internal standard template into a polymerase chain reaction (PCR) system, and performing fluorescent quantitative PCR amplification, wherein the sequences of the primer pair are shown as SEQ ID NO.1 and SEQ ID NO.2, the sequence of the target probe is shown as SEQ ID NO.3, the sequence of the internal standard probe is shown as SEQ ID NO.4, and the sequence of the internal standard template is shown as SEQ ID NO.5; (2) acquiring fluorescent signals in a PCR process. The method is short in detection time, low in cost and high in practicality; a detection result is specific and is easy to determine; a false negative result can be well avoided.

The invention discloses a Cronobacter sakazakii CRISPR typing method. The method is as follows: performing DNA sequencing on CRISPR1, CRISPR2, CRISPR3 and CRISPR6 locus sequences of Cronobacter sakazakii, extracting spacer sequences of the four locus sequences, and determining CRISPR type of the Cronobacter sakazakii based on the combination of the four spacer sequences. The method is simple, rapid and low-cost, the resolution of the method is higher than the resolution of MLST typing method, and the Cronobacter sakazakii CRISPR typing method has no environmental pollution, and has low requirements on laboratory equipment and software, CRISPR molecules preserve a lot of information such as historically infected phage and plasmid, by joint with a database, national and even global standardization molecule traceability can be realized, and the method can be extended to the fields of food, inspection and quarantine.

The invention discloses a detection kit of 16 food born pathogenic bacteria. The detection kit comprises a PCR amplification reagent and reference reagents; the PCR amplification reagent is composed of PCR buffer solution A, enzyme system B, and a primer probe mixed solution of the 16 food-borne pathogenic bacteria; the reference reagents are mainly composed of a negative control and a positive control; the PCR buffer solution A contains 5*Buffer, 25mM dN(U)TPs; the enzyme system B is mainly composed of HotStart Hi Taq DNA polymerase and UNG anti-pollution enzyme; the primer probe mixed solution of the 16 food-borne pathogenic bacteria contains a mixed solution of detection primer probes of salmonella, Shigella, enteropathogenic Escherichia coli, enterotoxigenic E. coli, enteroinvasive escherichia coli, adherent escherichia coli, Escherichia coli O157, Staphylococcus aureus, listeria monocytogenes, Bacillus Cereus, campylobacter jejuni, vibrio parahaemolyticus, Vibrio cholerae group O1, Vibrio cholerae group O139, Yersinia enterocolitica, and enterobacter sakazakii. The detection kit is high in detection sensitivity, is simple, and is convenient.