Primer for detecting salmonella enteritidis and detecting reagent kit containing primer

A technology of Salmonella enteritidis and detection kit, which is applied in the direction of microbial determination/inspection, resistance to vector-borne diseases, biochemical equipment and methods, etc., can solve the problems of no specificity and inability to directly distinguish serotypes, etc., and achieve Strong specificity, easy to promote and use, and easy to grasp the effect

Inactive Publication Date: 2012-08-22
CHINA AGRI UNIV
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  • Abstract
  • Description
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Problems solved by technology

However, the current general PCR technology can only identify Salmonella, and cannot directly distinguish the serotypes, and usually requires a combination of biochemical identification and serological experiments for typing
In recent years, with the completion of the sequencing of some Salmonella serotypes, foreign studies have begun to conduct PCR detection based on the unique gene sequence of Salmonella Enteritidis, in order to achieve the purpose of specific detection of Salmonella Enteritidis, and there are few domestic related studies
Most of the specific PCR detection of Salmonella Enteritidis is based on the sef operon gene, for example, the detection of Salmonella Enteritidis is based on the sef14 and sefA genes, but it has been found that these genes also exist in other individual serotypes of Salmonella, mainly and Salmonella enteritidis belongs to the serotypes of group D, such as S.Dublin, S.Pullorum, so the relevant detection methods are no longer completely specific

Method used

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  • Primer for detecting salmonella enteritidis and detecting reagent kit containing primer
  • Primer for detecting salmonella enteritidis and detecting reagent kit containing primer
  • Primer for detecting salmonella enteritidis and detecting reagent kit containing primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1 adopts specific primer of the present invention to carry out PCR detection

[0020] 1.1 Primers

[0021] Prot6E-F: 5'-ACAGGGGCACAATAACCGTA-3' and

[0022] Prot6E-R: 5'-TGCATCCCTGTCACAACATT-3'

[0023] 1.2 Bacteria enrichment

[0024] After sampling, add buffered peptone water (BPW) at a ratio of 1:9 and shake for 18 hours, draw 1 ml of the culture solution and add it to 9 ml of selenite cystine selective enrichment solution (SC), and shake for 18 hours.

[0025] 1.3 DNA extraction

[0026] Take 2 mL of the selective enrichment solution and place it in an Ep tube, centrifuge at 12000 r / min for 5 min, wash with ultrapure water twice, suspend with 0.2 mL of ultrapure water, boil in water for 15 min, insert into ice to cool for 2 min, and centrifuge at 12000 r / min for 15 min , and the supernatant was used as a DNA template.

[0027] 1.4PCR amplification

[0028] In the PCR reaction, the standard strain DNA of Salmonella enteritidis was used as a positive ...

Embodiment 2

[0030] Embodiment 2 adopts specific primer of the present invention to carry out the sensitivity analysis of PCR detection

[0031] The standard strain DNA of Salmonella enteritidis was extracted according to the method of 1.3 in Example 1, and the DNA concentration was determined to be 138 ng / μl by a spectrophotometer, and it was diluted in proportion and the accurate concentration after dilution was determined. Take 138ng / μl, 55.5ng / μl, 20.5ng / μl, 13ng / μl, 4.2ng / μl, 1.3ng / μl, 0.65ng / μl, 0.2ng / μl, 0.05ng / μl DNA at 9 concentrations Each 1 μl was used as a template for PCR amplification, and the amplification conditions were the same as 1.4 in Example 1. The result is as figure 2 As shown, the results show that the PCR method can detect template DNA with a lower limit of 0.2ng, so as long as the template DNA content in the sample is not less than 0.2ng, this method can be used for effective amplification detection.

Embodiment 3

[0032] Embodiment 3 adopts specific primer of the present invention to carry out PCR detection and adopts traditional separation method to detect the comparison of Salmonella enteritidis

[0033] Get 62 4-week-old white legion laying hens, and inoculate 10 by eating feed 4 After CFU of Salmonella Enteritidis, on the 7th and 14th days, the detection method of Example 1 and the traditional isolation and culture method were used to detect the contents of the cecum, wherein the traditional isolation and culture methods included pre-enrichment, selective enrichment, and isolation and culture , biochemical identification, serological typing a total of 5 steps.

[0034] The contents of the cecum contained many bacteria, mixed strains, and many uncertain interfering factors, which could test the sensitivity and accuracy of the detection method. The results show that the detection rate of the PCR method is obviously higher than that of the traditional culture separation method, and th...

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PUM

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Abstract

The invention provides a specificity primer for detecting salmonella enteritidis and a detecting reagent kit containing the primer according to special Prot6Es of the salmonella enteritidis. The primer comprises Prot6E and Prot6E-R (shown as the sequence identification number (Seq ID No).1 and 2). When the specificity primer provided by the invention is adopted for detecting salmonella enteritidis, the operation is simple and convenient, the mastering is easy, the popularization and the use in basic laboratories are convenient, and the detection can be completed only through two days. Meanwhile, the specificity primer and the detecting reagent kit have the advantages of high antijamming capability, high sensitivity and high specificity, the requirement of fast and specificity detection of the salmonella enteritidis in poultry and poultry products can be completely met, and very important effects on epidemic situation diagnosis and control are realized, so the economic loss and the harm to the public health are avoided to the greatest degree.

Description

technical field [0001] The invention belongs to the field of bacteria inspection, and in particular relates to a primer for detecting Salmonella enteritidis and a kit containing the primer. Background technique [0002] PCR technology is a technique developed in the past two decades to amplify specific DNA fragments in vitro. It has the advantages of simplicity, rapidity, high sensitivity and strong specificity. Therefore, since the 1990s, attempts to use this technology to detect Salmonella in food and in clinical samples and the environment. However, the current general PCR technology can only identify Salmonella, and cannot directly distinguish the serotypes. Usually, a combination of biochemical identification and serological experiments is required for typing. In recent years, with the completion of the sequencing of some Salmonella serotypes, studies abroad have begun to conduct PCR detection based on the unique gene sequences of Salmonella Enteritidis to achieve the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10C12N15/11
CPCY02A50/30
Inventor 徐桂云樊世杰杨宁郑江霞
Owner CHINA AGRI UNIV
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