Multi-PCR detection kit and method for identifying poultry salmonella

A detection kit and Salmonella technology, applied in the field of PCR detection, can solve the problems of affecting accuracy, time-consuming and laborious, etc., and achieve the effects of rapid detection process, high sensitivity and strong specificity

Inactive Publication Date: 2015-07-15
JIANGSU INST OF POULTRY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Bacteriological detection is usually based on the growth characteristics of Salmonella. Selective medium is used to make Salmonella the dominant bacterial group. A series of biochemical and serological tests are carried out on the colony isolates to make identification. The whole process of traditional Salmonella detection takes at least 4-7 days, time-consuming and labor-intensive; the immunological detection method detects Salmonella according to the surface antigen characteristics of the bacteria and the specificity of the antigen-antibody reaction, and the level of individual antibodies greatly affects the accuracy of the detection
At present, there are also PCR and other detection methods established for some serotypes of Salmonella. Products on the market (Salmonella PCR Rapid Detection Kit-Beijing Fudean

Method used

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  • Multi-PCR detection kit and method for identifying poultry salmonella
  • Multi-PCR detection kit and method for identifying poultry salmonella
  • Multi-PCR detection kit and method for identifying poultry salmonella

Examples

Experimental program
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Example Embodiment

[0027] Example 1

[0028] A multiple PCR detection method for avian salmonella, comprising the following steps:

[0029] 1. The preparation method of PCR template is as follows:

[0030] (1) Salmonella ATCC14028, Salmonella enteritidis CMCC50336, Salmonella pullorum CMCC50770, Salmonella Heidelberg CMCC50111, Kentucky Salmonella CMCC50794 were cultured in sterile selenite cystine enrichment solution (SC) at 37°C for 12h.

[0031] (2) Take 1 ml of the bacterial solution cultured in the enrichment liquid culture shake flask for 12 hours, and use a commercial kit, bacterial genomic DNA extraction kit (DP302, Tiangen Biochemical Technology) to extract the bacterial genome as a template to be detected by multiple PCR;

[0032] 2. The configuration method of the multiple PCR amplification reagents for avian salmonella is as follows:

[0033] (1) Synthesize 5 pairs of specific primers bcf-F / R, Heli-F / R, rhs-F / R, sdf-F / R, fla-F / R according to the prior art, and dilute with ultrapure water to mak...

Example Embodiment

[0048] Example 2

[0049] Detection of Salmonella prevalence in cotton swabs from farms:

[0050] (1) 50 cotton swabs collected aseptically;

[0051] (2) Enrichment culture of cotton swabs:

[0052] The cotton swab was inoculated into a sterile enrichment medium (Selenite Cystine Enrichment Solution (SC)) and cultivated at 37°C for 15 hours.

[0053] (3) Preparation of PCR template:

[0054] Take the above-mentioned 1ml bacterial liquid in a sterile 1.5ml EP tube and mark. After washing with ultrapure water once, the bacterial pellet was dissolved in 500μl, boiled for 5min, cooled on ice, centrifuged at 13000rpm / 5min, and the supernatant was the multiplex PCR template.

[0055] (4) Assembly of multiplex PCR amplification reagents

[0056] Take 2.5μl of 10× PCR buffer, 0.5μl of a mixture of dGTP, dCTP, dATP and dTTP at a concentration of 25mmol / L, and a concentration of 2.5U / μl Taq DNA polymerase 1.25μl, bcf-F / R, Heli-F The final concentrations of / R, rhs-F / R, sdf-F / R, and fla-F / R are: 0....

Example Embodiment

[0063] Example 3

[0064] Detection of Salmonella prevalence in cotton swabs from farmers’ markets:

[0065] (1) 120 cotton swabs collected aseptically;

[0066] (2) Cotton swab enrichment culture

[0067] The cotton swabs were inoculated into sterile enrichment medium (Selenite Cystine Enrichment Solution (SC)) and cultured at 37°C for 18 hours.

[0068] (3) Preparation of PCR template

[0069] Take the above-mentioned 1ml bacterial liquid in a sterile 1.5ml EP tube and mark. After washing with ultrapure water once, the bacterial pellet was dissolved in 500μl, boiled for 5min, cooled on ice, centrifuged at 13000rpm / 5min, and the supernatant was the multiplex PCR template.

[0070] (4) Assembly of multiplex PCR amplification reagents

[0071] Take 2.5μl of 10× PCR buffer, 0.5μl of a mixture of dGTP, dCTP, dATP and dTTP at a concentration of 25mM, and a concentration of 2.5U / μl Taq DNA polymerase 1.25μl, bcf-F / R, Heli-F / R The final concentrations of rhs-F / R, sdf-F / R, and fla-F / R are respe...

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Abstract

The invention discloses a multi-PCR detection kit for identifying poultry salmonella. The multi-PCR detection kit comprises PCR buffer fluid, a deoxyribonucleoside triphosphate mixture, Taq DNA polymerase and primer pairs of a bcf gene, a heli gene, an rhs gene, an sdf gene, a fla gene. The invention further discloses a multi-PCR detection method for identifying the poultry salmonella through the kit. The multi-PCR detection kit can effectively determine whether the salmonella is included in a sample to be detected or not, and can further identify different epidemic salmonella serotypes in poultry production or poultry products. Through a PCR, the salmonella can be detected from the sample at the same time, the different salmonella serotypes are further identified, the detection reaction sensitivity is high, high specificity is achieved, the detection process is fast and efficient, and the kit and method are suitable for batch detection.

Description

technical field [0001] The invention belongs to the technical field of PCR detection, and in particular relates to a multiplex PCR detection kit and a method for identifying avian-derived Salmonella. Background technique [0002] Salmonella is a common zoonotic pathogen. The infection and contamination of some serotypes of Salmonella during poultry production seriously affects poultry production efficiency and endangers the poultry industry. In food poisoning incidents around the world, the poisoning cases caused by Salmonella It has risen to the first or second place, among which Salmonella infection of poultry is one of the important media transmitted to humans through the food chain. According to the US CDC and FDA yearbook reports, epidemiological investigations in poultry production in the past five years have shown that in clinical samples of poultry production, Salmonella Enteritidis is the most prevalent serotype, while Salmonella Heidelberg has the highest detection...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12R1/42
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 朱春红朱国强龚建森陶志云宋迟李慧芳宋卫涛束婧婷单艳菊徐文娟刘宏祥
Owner JIANGSU INST OF POULTRY SCI
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