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Primer group, kit and method for identifying salmonella pullorum and salmonella gallinarum

A technology for Salmonella and pullorum, applied in the field of PCR detection, can solve the problems of indistinguishability, time-consuming, poor repeatability, etc., and achieve the effects of rapid detection process, high detection sensitivity and strong specificity

Inactive Publication Date: 2015-11-25
JIANGSU INST OF POULTRY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, some great-grandfather and ancestral breeding chicken farms in my country have unsatisfactory detection results for pullorum antibody. Some chickens in ancestral breeding chicken farms are still positive for pullorum antibody. The establishment of a new rapid and accurate detection method is expected to speed up this process.
[0004] Salmonella pullorum and Salmonella gallinarum have the same bacterial antigen, and common serological methods cannot distinguish between the two
The distinction between the two is mainly based on the differences in biochemical characteristics. They have different fermentation modes for maltose, dulcitol, ornithine, etc., but the above methods have many shortcomings, such as time-consuming and poor repeatability.

Method used

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  • Primer group, kit and method for identifying salmonella pullorum and salmonella gallinarum
  • Primer group, kit and method for identifying salmonella pullorum and salmonella gallinarum
  • Primer group, kit and method for identifying salmonella pullorum and salmonella gallinarum

Examples

Experimental program
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Effect test

Embodiment 1

[0023] A kind of double PCR detection method of differentiating Salmonella pullorum and Salmonella gallinarum of the present invention, the method comprises the following steps:

[0024] 1. The preparation method of PCR template is as follows:

[0025] (1) Salmonella pullorum CMCC50771 and Salmonella gallinarum typhi CMCC50770 were respectively cultured in a selenite cystine enrichment solution at 36°C for 10 hours;

[0026] (2) Take 1ml of the bacterial solution cultured in a shake flask with the enrichment solution for 10 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 double PCR.

[0027] 2. The configuration method of dual PCR amplification reagents for Salmonella pullorum and Salmonella gallinarum is as follows:

[0028] (1) As shown in Table 1, synthesize two pairs of specific primer sequences according to the prior art: primer 1 and primer 2, pr...

Embodiment 2

[0043] A method for detecting farm suspected Salmonella pullorum and Salmonella gallinarum typhi live poultry cotton swab samples, the method comprises the following steps:

[0044] 1. Collect 20 copies of cloacal cotton swabs;

[0045] 2. Cotton swab enrichment culture:

[0046] Inoculate the cotton swab in a sterile enrichment medium (selenite cystine enrichment solution) and incubate at 37°C for 12h.

[0047] 3. Preparation of PCR template

[0048]Take the above 1ml of the enrichment culture solution to be tested, centrifuge at 13000rpm for 5min, wash once with ultrapure water, resuspend the precipitate to be tested in 100μl ultrapure water, boil for 5min, and ice-bath for 5min, centrifuge at 13000rpm to get the supernatant, and get the PCR to be tested template.

[0049] 4. Assembly of double PCR amplification reagents

[0050] Take 2.5 μl of 10× PCR buffer, 0.5 μl of dGTP, dCTP, dATP and dTTP mixture with a concentration of 25 mM each, 1.25 μl of Taq DNA polymerase wi...

Embodiment 3

[0057] Detecting the prevalence of Salmonella in cotton swabs in live poultry trading markets:

[0058] 1. Sterile collection of 550 cotton swabs;

[0059] 2. Cotton swab enrichment culture

[0060] The cotton swabs were inoculated in a sterile enrichment medium (selenite cystine enrichment solution (SC)) and incubated at 37°C for 18h.

[0061] 3. Preparation of PCR template

[0062] Take the above 1ml of the enrichment culture solution to be tested, centrifuge at 13000rpm for 5min, wash once with ultrapure water, resuspend the precipitate to be tested in 100μl ultrapure water, boil for 10min, and ice-bath for 8min, then centrifuge at 13000rpm to get the supernatant, and get the PCR to be tested template.

[0063] 4. Assembly of double PCR amplification reagents

[0064] Take 2.5 μl of 10× PCR buffer, 0.5 μl of dGTP, dCTP, dATP and dTTP mixture with a concentration of 30 mM each, 1.5 μl of Taq DNA polymerase with a concentration of 3.0 U / μl, and 1 μl of primer mixture in a...

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Abstract

The invention discloses a primer group, a kit and a method for identifying salmonella pullorum and salmonella gallinarum. The primer group comprises two pairs of specific primers, wherein the two pairs of specific primers are respectively primer 1 and primer 2 as well as primer 3 and primer 4, wherein the primer 1 is as shown in SEQ ID NO: 1, the primer 2 is as shown in SEQ ID NO: 2, the primer 3 is as shown in SEQ ID NO: 3 and the primer 4 is as shown in SEQ ID NO: 4; the kit contains the primer group; and the primer group is used for detecting to-be-detected bacteria by virtue of double PCR amplification. The primer group for identifying salmonella pullorum and salmonella gallinarum disclosed by the invention is strong in detection reaction specificity; through a PCR reaction, the salmonella pullorum and the salmonella gallinarum can be simultaneously detected from a sample, with high detection reaction sensitivity; and a detection process is rapid and efficient, and suitable for mass detection.

Description

technical field [0001] The invention belongs to the technical field of PCR detection, and in particular relates to a primer set, a kit and a method for distinguishing Salmonella pullorum and Salmonella gallinarum typhi. Background technique [0002] Salmonella is a common zoonotic pathogen. The infection and contamination of some serotypes of Salmonella in the poultry production process seriously affects the poultry production efficiency and endangers the poultry industry. Two different biotypes of Salmonella enterica serovar Gallinarum—Salmonella enterica serovar Gallina rumbiovars Pullorum and Salmonella enterica serovar Gallina rumbiovars Gallinarum cause pullorum disease and fowl typhoid respectively, both of which are important pathogenic bacteria of chickens. Under normal circumstances, Salmonella pullorum mostly affects young chicks within 20 days of age, causing white diarrhea, and the mortality rate is extremely high. There are only sporadic deaths in adult chickens...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10C12N15/11C12R1/42
CPCC12Q1/689C12Q1/686C12Q2600/16C12Q2537/143
Inventor 朱春红徐文娟束婧婷龚建森宋卫涛陶志云单艳菊刘年华姬改革
Owner JIANGSU INST OF POULTRY SCI
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