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Method for assisting in detecting and identifying histamine-producing enterobacteria

A histamine-producing intestine and auxiliary identification technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, recombinant DNA technology, etc., can solve pollution and other problems, and achieve timely quality control, easy identification, and simple methods

Inactive Publication Date: 2014-04-09
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Food is often contaminated by intestinal bacteria, among which there are amine-producing bacteria. At present, the detection of histamine-producing bacteria mainly focuses on lactic acid bacteria, but there are few reports on intestinal bacteria

Method used

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  • Method for assisting in detecting and identifying histamine-producing enterobacteria
  • Method for assisting in detecting and identifying histamine-producing enterobacteria
  • Method for assisting in detecting and identifying histamine-producing enterobacteria

Examples

Experimental program
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Embodiment 1

[0017] Embodiment 1, design and preparation of primers

[0018] A specific primer pair (composed of primer LB1 and primer LB2) was designed according to the conserved region of the histidine decarboxylase coding gene (hdc gene) of various histamine-producing enterobacteria on NCBI. A universal primer pair (composed of primer B-for and primer B-for) for 16S rDNA of various histamine-producing bacteria was selected. Synthesize specific and universal primer pairs.

[0019] LB1 (sequence 1 of the sequence listing): 5'-AAGATACCCATTACTCCGTG-3';

[0020] LB2 (SEQ ID NO: 2 of the Sequence Listing): 5'-TCTACAGAGATCCGATCGAC-3'.

[0021] B-for (sequence 3 of the sequence listing): 5'-AGAGTTTGATCCTGGCTCAG-3';

[0022] B-rev (SEQ ID NO: 4 of the Sequence Listing): 5'-AAGGAGGTGATCCAGCCGCA-3'.

Embodiment 2

[0023] Example 2. Detection and identification of histamine-producing intestinal bacteria in fermented food

[0024] 1. Sample processing

[0025] Aseptically weigh 20 g of commercially available soy sauce into koji, cut it into pieces under aseptic conditions, place it in a triangular flask filled with 180 ml of sterilized normal saline, mix well, and shake on a homogenizer for 10 min (the dilution is 10 -1 ); then carry out 10-fold serial dilution with sterilized normal saline, as each sample dilution (dilution is 10 successively -2 、10 -3 ,...).

[0026] 2. Isolation of intestinal bacteria from samples

[0027]Preparation of crystal violet neutral red bile salt glucose agar medium (VRBGA medium): 3.0g yeast extract, 7.0g peptone, 5.0g sodium chloride, 1.5g No. 3 bile salt, 10.0g lactose, 10.0g glucose, 0.03g neutral red, 0.002g crystal violet, and 15.0g agar were adjusted to 1000mL with distilled water, dissolved by heating, and adjusted to pH 7.4±0.2.

[0028] Spread ...

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Abstract

The invention discloses a method for assisting in detecting and identifying histamine-producing enterobacteria. In the method, a specific primer pair for assisting in identifying the histamine-producing enterobacteria is provided, consists of deoxyribonucleic acids (DNAs) shown as a sequence 1 and a sequence 2 in a sequence table and can specifically amplify the genomic DNA of the histamine-producing enterobacteria. The method comprises the following steps of: culturing a sample in a solid bacterial differential medium containing bromcresol purple and histidine, and screening potential histamine-producing enterobacteria; amplifying a histidine decarboxylase (hdc) gene, and further identifying; and amplifying the 16S ribosomal DNA (rDNA) segment of the histamine-producing enterobacteria to identify the type of the histamine-producing enterobacteria. The method is simple and quick, and the histamine-producing enterobacteria are easy to identify, can be distinguished from non-histamine-producing enterobacteria on the same flat plate, and is further identified by a molecular biological method to achieve higher accuracy and reliability; and the method is particularly suitable for evaluating the risk of histamine formed in fermented food, can be used for performing quality control on daily production effectively and timely, and is a food hygiene detection technology with a wide application prospect.

Description

technical field [0001] The invention relates to a method for auxiliary detection and identification of histamine-producing intestinal bacteria. Background technique [0002] Biogenic amines are a class of low-molecular-weight nitrogen-containing compounds mainly formed by the decarboxylation of amino acids or the amination of aldehydes and ketones. Mainly putrescine, cadaverine, phenylethylamine, spermine, spermidine, histamine, tryptamine and tyramine. When the human body ingests excessive biogenic amines, it can cause damage to the human nervous system and cardiovascular system. In addition, biogenic amines can also form nitrosamines with obvious mutagenicity and potential carcinogenicity with food preservatives such as nitrite, thereby indirectly causing cancer. [0003] Histamine in food is mainly produced by microorganisms carrying amino acid decarboxylase. At present, the methods used in the detection and analysis of histamine in food mainly include microbiological ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
Inventor 韩北忠钱雨林闫寅卓陈晶瑜鲁绯
Owner CHINA AGRI UNIV
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