阅读说明：本技术 一种快速检测食源性致病菌沙门氏菌用标准化质粒冻干粉 (Standardized plasmid freeze-dried powder for rapidly detecting food-borne pathogenic bacteria salmonella ) 是由 郜晓峰 崔丽丽 王深垒 马盼盼 夏丹丹 康文艺 于 2020-07-16 设计创作，主要内容包括：本发明属于食品检测技术领域,具体涉及一种快速检测食源性致病菌沙门氏菌用标准化质粒冻干粉。具体制备步骤包括：提取沙门氏菌全基因组DNA、设计引物并进行PCR扩增、与pLB质粒连接、转化、筛选、鉴定并提取质粒、制备冻干粉成品等步骤。本申请中,发明人以现有若干菌株作为标准菌株,以invA和fimI毒力基因作为目标基因,利用基因工程技术手段,以pLB质粒作为载体,构建了含有目标毒力基因的标准化质粒样品。利用该标准品,可为相关沙门氏菌的快速检测和鉴定奠定良好技术基础,无论对于沙门氏菌标准化检测体系建立、抑或对于确保食品安全,都具有十分重要的技术价值和应用意义。(The invention belongs to the technical field of food detection, and particularly relates to a standardized plasmid freeze-dried powder for rapidly detecting food-borne pathogenic bacteria salmonella. The preparation method comprises the following specific steps: extracting salmonella whole genome DNA, designing a primer, carrying out PCR amplification, connecting with pLB plasmid, transforming, screening, identifying and extracting the plasmid, preparing a freeze-dried powder finished product and the like. In the application, the inventor takes a plurality of existing strains as standard strains, takes invA and fimI virulence genes as target genes, utilizes a genetic engineering technical means, takes pLB plasmids as a carrier, and constructs a standardized plasmid sample containing the target virulence genes. The standard substance can lay a good technical foundation for the rapid detection and identification of related salmonella, and has very important technical value and application significance for establishing a salmonella standardized detection system or ensuring food safety.)

1. A standardized plasmid freeze-dried powder for rapidly detecting food-borne pathogenic bacteria salmonella is characterized by being prepared by the following steps,

extracting salmonella whole genome DNA, designing primer and carrying out PCR amplification

Extracting salmonella whole genome DNA for later use;

simultaneously, aiming at target virulence genes invA and fimI, primer sequences for PCR amplification are respectively designed as follows:

fimI-F：5’- CACTAAATCCGCCGATCAA-3’，

fimI-R：5’- CAACGGTGAGGAGGTATTATC-3’；

amplification product 262 bp;

invA-F：5’- GTGAAATTATCGCCACGTTCGGGCAA-3’，

invA-R ：5’-TCATCGCACCGTCAAAGGACCC-3’；

284bp of an amplification product;

performing sequence amplification by using the primer by using the extracted salmonella whole genome DNA as a template;

recovering and purifying PCR amplification products for later use;

(II) ligation and transformation

Connecting the amplification products obtained in the step (one) with pLB plasmids respectively by using pLB plasmids as connecting vectors, and carrying out transformation;

(III) screening, identifying and extracting plasmids

Further extracting plasmids from the transformation product in the step (II) for identification, carrying out subculture on strains with correct identification, and finally extracting plasmids for later use;

(IV) preparing the finished product of the freeze-dried powder

And (5) further preparing the plasmid prepared in the step (three) into freeze-dried powder, namely a standard plasmid standard substance for evaluating the pathogenicity of the salmonella.

2. The standardized plasmid lyophilized powder for rapidly detecting the food-borne pathogenic bacterium salmonella as claimed in claim 1, wherein in the step (one), when extracting salmonella whole genome DNA, any one of four strains of salmonella typhimurium CMCC50071, salmonella typhimurium 50115, salmonella ATCC14028 or salmonella enteritidis CICC 21482 is taken as a whole genome DNA source.

3. The application of the standardized plasmid freeze-dried powder for rapidly detecting the food-borne pathogenic bacteria salmonella in the food detection as claimed in claim 1 is characterized in that the standardized plasmid freeze-dried powder is used as a positive control template sample in PCR detection.

4. The application of the standardized plasmid freeze-dried powder for rapidly detecting the food-borne pathogenic bacteria salmonella in the food detection as claimed in claim 3, is characterized in that the standardized plasmid freeze-dried powder is used for chrysanthemum detection to judge whether chrysanthemum is polluted by pathogenic salmonella.

5. The salmonella detection method using the standardized plasmid freeze-dried powder for food-borne pathogenic bacteria salmonella as claimed in claim 1, which is characterized by comprising the following steps:

(one) treating the sample

Processing a detection sample, and extracting microbial DNA in the sample to be detected;

(II) designing primers and carrying out PCR amplification

The primer sequence is as follows:

fimI-F：5’- CACTAAATCCGCCGATCAA-3’，

fimI-R：5’- CAACGGTGAGGAGGTATTATC-3’；

invA-F：5’- GTGAAATTATCGCCACGTTCGGGCAA-3’，

invA-R ：5’-TCATCGCACCGTCAAAGGACCC-3’；

taking the microbial DNA extracted in the step (one) as a template, and respectively carrying out PCR amplification on one pair, two pairs or any pairs of the primer pairs to serve as detection samples;

meanwhile, the standardized plasmid freeze-dried powder for salmonella is used as a template, and one pair, two pairs or any pairs of the primer pairs are respectively used for PCR amplification to be used as standard samples;

(III) comparison and identification

And (5) carrying out electrophoretic analysis and comparison on the amplification products in the step (II), and if the detection sample has the same band type as the standard sample, indicating that the sample to be detected is polluted by salmonella.

Technical Field

The invention belongs to the technical field of food detection, and particularly relates to a standardized plasmid freeze-dried powder for rapidly detecting food-borne pathogenic bacteria salmonella.

Background

With the continuous improvement of living standard of people, people pay more and more attention to food safety problem. Among food safety problems, food quality problems caused by food-borne microorganisms have been the first, and food-borne diseases caused by food-borne microorganisms have become one of the most important worldwide health problems. The method is limited by technical practice and other objective conditions, and the technical level of detection is greatly limited because a qualitative detection DNA reference substance system is not incomplete when food-borne microorganisms are detected in China at the present stage and in real-time detection.

Salmonella bacteria (A), (B)Salmonella) Can cause diseases such as gastroenteritis, diarrhea and the like after being infected with water sources and foods, and seriously harms the health and safety of human bodies. As one of the main common pathogenic bacteria in food-borne microorganisms, salmonella is also an important basic research object in the fields of food hygiene and epidemiology. Genome research on salmonella shows that a salmonella invasion protein A gene (invA) encodes proteins for adsorbing and invading epithelial cell surface, and an inv gene cluster is related to invasion of intestinal epithelial cells by salmonella, wherein the protein encoded by the invA gene belongs to main virulence factors of the salmonella. The salmonella pili are encoded by fim gene determinants, and fim gene operons each comprise a gene encoding a structural component and a gene encoding an orderly assembled assembly protein of the pili, wherein the fimI gene encodes an adhesin protein. Therefore, if a standard substance for detecting virulence genes of pathogenic salmonella can be developed, the method has very important technical significance for the instant and rapid detection of related pathogenic bacteria.

Disclosure of Invention

The application aims to provide the standardized plasmid freeze-dried powder for detecting the food-borne pathogenic bacteria salmonella, and when the standardized plasmid freeze-dried powder is used as a standard substance, a certain technical basis can be laid for the establishment of a rapid and instant detection system of the salmonella and the food safety.

The technical solution adopted in the present application is detailed as follows.

A standardized plasmid freeze-dried powder for rapidly detecting food-borne pathogenic bacteria salmonella is prepared by taking four strains of salmonella typhimurium CMCC50071, CMCC50115 (Chinese medical bacterium preservation management center), salmonella typhimurium ATCC14028 (American strain preservation center) or salmonella enteritidis CICC 21482 (Chinese industrial microorganism bacterium preservation management center) as examples, taking an invA (encoding invasion protein) gene and a fimI (encoding adhesion protein) gene as target virulence genes and pLB plasmid as a carrier plasmid, and transforming and preparing a standard product containing the target virulence genes by a gene engineering technical means, thereby laying a foundation for a pathogenic bacterium standardized detection system, and the specific steps are as follows:

extracting salmonella whole genome DNA, designing primer and carrying out PCR amplification

Extracting salmonella whole genome DNA for later use by using the existing bacterial genome DNA extraction kit and referring to the instruction book;

aiming at target virulence genes invA and fimI, primer sequences for PCR amplification are respectively designed as follows:

fimI-F：5’- CACTAAATCCGCCGATCAA-3’，

fimI-R：5’- CAACGGTGAGGAGGTATTATC-3’；

invA-F：5’- GTGAAATTATCGCCACGTTCGGGCAA-3’，

invA-R ：5’-TCATCGCACCGTCAAAGGACCC-3’；

performing sequence amplification by using the primer by using the extracted salmonella whole genome DNA as a template;

recovering and purifying PCR amplification products for later use;

(II) ligation and transformation

Connecting the amplification products obtained in the step (one) with pLB plasmids respectively by using pLB plasmids as connecting vectors, and carrying out transformation;

(III) screening, identifying and extracting plasmids

Further extracting plasmids from the transformation product in the step (II) for identification, carrying out subculture on strains with correct identification, and finally extracting plasmids for later use;

(IV) preparing the finished product of the freeze-dried powder

And (3) matching with an excipient and a protective agent, and further preparing the plasmid prepared in the step (three) into freeze-dried powder, namely a standard plasmid standard substance (namely, the standard plasmid standard substance is used as a reference substance) for evaluating the pathogenicity of the salmonella.

The food-borne pathogenic bacterium salmonella standardized plasmid freeze-dried powder is applied to food detection, is specifically used for detecting chrysanthemum, and is used as a positive control for judging whether the chrysanthemum is polluted by pathogenic salmonella.

The salmonella detection method by utilizing the standardized plasmid freeze-dried powder for food-borne pathogenic bacteria salmonella comprises the following steps:

(one) treating the sample

Processing a detection sample, and extracting microbial DNA in the sample to be detected;

(II) designing primers and carrying out PCR amplification

The specific primer sequence is the same as the sequence, namely the specific primer sequence is as follows:

fimI-F：5’- CACTAAATCCGCCGATCAA-3’，

fimI-R：5’- CAACGGTGAGGAGGTATTATC-3’；

invA-F：5’- GTGAAATTATCGCCACGTTCGGGCAA-3’，

invA-R ：5’-TCATCGCACCGTCAAAGGACCC-3’；

taking the microbial DNA extracted in the step (one) as a template, and respectively carrying out PCR amplification on one pair or two pairs of the primer pairs to serve as a detection sample;

meanwhile, the standardized plasmid freeze-dried powder for salmonella is used as a template, and one pair or two pairs of primers in the primer pair are respectively used for PCR amplification to be used as a standard sample;

(III) comparison and identification

And (5) carrying out electrophoretic analysis and comparison on the amplification products in the step (II), and if the detection sample has the same band type as the standard sample, indicating that the sample to be detected is polluted by salmonella.

In the application, the inventor takes the invA and fimI virulence genes of the existing specific salmonella strains as target genes, utilizes a gene engineering technical means and takes pLB plasmids as a carrier to construct a standardized plasmid sample containing the invA and fimI virulence genes. The standard substance can lay a good technical foundation for the rapid detection and identification of the related food-borne salmonella, and has very important technical value and application significance for establishing a salmonella standardized detection system or ensuring food safety.

Drawings

FIG. 1 is an electrophoretogram of Salmonella whole genome DNA, M: marker, 1: salmonella typhi CMCC 50071; 2: salmonella CMCC 50956; 3: salmonella typhimurium ATCC 14028; 4: salmonella enteritidis CICC 21482;

FIG. 2 shows the result of PCR amplification of virulence genes in Salmonella, where (a) and (b) are the invA and fimI genes, respectively; m: d2000DNA Marker; 1: salmonella typhi CMCC 50071; 2: salmonella CMCC 50956; 3: salmonella typhimurium ATCC 14028; 4: salmonella enteritidis CICC 21482;

FIG. 3 shows the PCR identification result of transformed colonies, M: marker, 1, 2, 3, 4 are four single colonies randomly selected on the plate;

FIG. 4 shows the electrophoresis results of plasmids extracted after passage 15, M: marker, 1: an invA gene; 2: fimI gene;

FIG. 5 shows the PCR identification result of the lyophilized powder product; m is Marker; 1-3: at 25 ℃; 4-6:4 ℃; 7-9: -20 ℃ conditions;

FIG. 6 shows the results of PCR detection sensitivity for the Salmonella invA (a) and fimI (b) genes, M: d2000DNA Marker; 1-6: 10^-1、10^-2、10-³、10^-4、10^-5And 10^-6A series of concentration gradients; 7: negative control;

FIG. 7 shows the PCR detection result of invA in chrysanthemum samples, M: d2000DNA Marker; a: 1-15: a chrysanthemum sample; 16: negative control; 17: a positive control; b: 1-8: a chrysanthemum sample; 9: negative control; 10: a positive control;

FIG. 8 shows the result of PCR detection of fimI in chrysanthemum samples, M: d2000DNA Marker; a: 1-15: a chrysanthemum sample; 16: negative control; 17: a positive control; b: 1-8: a chrysanthemum sample; 9: negative control; 10: a positive control; according to the positive control, namely the glue-running result of the salmonella virulence gene invA plasmid freeze-dried powder, it can be seen that 7 and 15 chrysanthemum samples contain invA virulence genes and are possibly polluted by salmonella in different degrees.

Detailed Description

The present application is further illustrated by the following examples. Before describing the specific embodiments, a brief description will be given of some experimental background cases in the following embodiments.

Biological material:

the related primer sequence synthesis and sequencing work is completed by Beijing Liuhua Dagenetechnology Co., Ltd and Biotechnology engineering Co., Ltd;

salmonella typhimurium CMCC50071, CMCC50115 (China medical bacteria collection management center), Salmonella typhimurium ATCC14028 (American strain collection center) and Salmonella enteritidis CICC 21482 (China industrial microorganism strain collection management center) are all common microorganism strains in the research in the field;

experimental reagent:

a bacterial whole genome DNA extraction kit (centrifugal column type) (DP302), an agarose gel DNA recovery kit (centrifugal column type) (DP 209), an pLB zero background rapid cloning kit (without MCS) VT206, a plasmid miniprep kit (DP 103), a soil genome DNA extraction kit (DP 336), a 2 Xpfu PCR MasterMix (KP201), competent cells (DH-5 alpha), a Marker for electrophoresis and the like, which are all products of Tiangen Biochemical technology Co., Ltd;

6 multiplied by DNA loading Buffer, GoldView nucleic acid stain, Amp and the like, which are all products of Solarbio company;

an experimental instrument:

the nucleic acid protein detector and the PCR amplification instrument are both products of Thermo scientific company;

pulsed field gel electrophoresis apparatus, product of Junyi corporation;

dongsheng Longbing 910 gel imaging system, product of Beijing Dongsheng Innovative Biotechnology Limited.