Salmonella Indiana PCR (polymerase chain reaction) detection kit and non-diagnosis detection method thereof

A detection kit and technology for Salmonella, applied in the field of molecular biology detection, can solve the problems such as the reports on PCR detection methods and kits for Salmonella Indiana serotypes that have not yet been found, and achieve detection cost advantages, strong specificity, and detection time advantages. Effect

Active Publication Date: 2018-05-04
JIANGSU INST OF POULTRY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Find through literature retrieval to prior art, do not find yet and the repor...

Method used

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  • Salmonella Indiana PCR (polymerase chain reaction) detection kit and non-diagnosis detection method thereof
  • Salmonella Indiana PCR (polymerase chain reaction) detection kit and non-diagnosis detection method thereof
  • Salmonella Indiana PCR (polymerase chain reaction) detection kit and non-diagnosis detection method thereof

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Experimental program
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Effect test

Embodiment 1

[0023] The establishment of embodiment 1 Salmonella Indiana PCR detection method

[0024] Design of primers: According to the genomic DNA sequences of different serotypes of Salmonella known in the GenBank database, after comparative genomics analysis and comparison, the specific gene fragments of Salmonella Indiana were screened out, and on this basis, they were designed and optimized for specific The PCR primers for detection of Salmonella Indiana are listed in the table below.

[0025] Table 1 Primer sequences for PCR detection of Salmonella Indiana

[0026]

[0027] Preparation of PCR template: Salmonella Indiana ATCC51959 was cultured in Brain Heart Infusion Broth Medium, and bacterial genomic DNA was extracted using a commercial bacterial genomic DNA extraction kit as a template to be tested.

[0028] Preparation of PCR detection reagent: 10× PCR buffer (its components include 100mM KCl, 80mM (NH4) SO4, pH 9.0 of 100mM Tris-HCl, 15mM MgCl2 and 0.5% Tergitol-type NP-4...

Embodiment 2

[0032] Embodiment 2 specificity evaluation experiment

[0033] Carry out the specificity evaluation of the PCR detection method of the present invention with the method for Example 1, the test strain is enriched and cultivated in the brain-heart infusion broth medium, and the bacterial genomic DNA is extracted using a commercialized bacterial genomic DNA extraction kit, Carry out PCR detection according to the method described in embodiment 1, the result is as follows figure 2 As shown, only Salmonella Indiana (swimming lane 4) showed a specific amplification band, and other Salmonella serotypes and non-Salmonella strains had no specific amplification. The strains used and the test results are shown in the table below. In the table, "+" in the test result column means positive; "-" means negative.

[0034] Table 2 The specificity evaluation test result of the present invention

[0035]

[0036]

[0037] Since the PCR amplified sequence of the present invention contains ...

Embodiment 3

[0038] Embodiment 3 Sensitivity evaluation experiment

[0039] The method of Example 1 was used to evaluate the sensitivity of the PCR detection method of the present invention. Salmonella Indiana ATCC51959 was cultured in Brain Heart Infusion Broth Medium, and the bacterial genome was extracted using a commercially available bacterial genomic DNA extraction kit. After measuring the original concentration, the bacterial genomic DNA was diluted 10 times. Carry out PCR amplification; at the same time, the bacterial solution is subjected to 10-fold gradient dilution after bacterial counting, and bacteria of different concentrations are taken for PCR amplification (see Table 3 for the PCR detection reaction system). After PCR amplification, 5 μl of the amplification product was taken, mixed with 1 μl of 6×Loading buffer, and detected by 2% agarose gel electrophoresis. Electrophoresis results such as Figure 4 As shown, M is DL-1000 marker, and swimming lanes 1-6 are the results ...

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Abstract

The invention discloses a salmonella Indiana PCR (polymerase chain reaction) detection kit and a non-diagnosis detection method thereof. The kit comprises a 10*PRC buffer solution, 2.5U/mul of Taq DNA(deoxyribonucleic acid) polymerase, 10mM dNTPs (deoxyribonucleotide triphosphates), a specific PCR detection primer of salmonella Indiana, a positive reference substance and a negative reference substance, wherein the positive reference substance is DNA of salmonella Indiana ATCC51959 genome; the negative reference substance is sterilized double distilled water. The invention also discloses a PRCmethod using the kit to detect the salmonella Indiana. The method has the advantages that the operation is quick and simple, the specificity is strong, and the sensitivity is high; the salmonella Indiana can be detected without using salmonella diagnosis serum; compared with the traditional salmonella serological typing method, more advantages are realized in aspects of ssdetection time and detection cost, and the method is suitable of batched detection.

Description

technical field [0001] The invention belongs to the technical field of molecular biology detection, and relates to a Salmonella Indiana PCR detection kit and a non-diagnostic detection method thereof. Background technique [0002] Salmonella Indiana (Salmonella Indiana) was first isolated in 1955 from a girl suffering from vomiting, diarrhea and fever in Indiana, USA. Later, the pathogen caused multiple human and mammal infection outbreaks in North America and Europe. In my country, there were few reports of Salmonella Indiana in the early years, but in recent years, it has a high prevalence rate in different sources including humans, animals, food and the environment, and has become the second most popular serotype after Salmonella Enteritidis, surpassing other serotypes. Several common serotypes (S. Typhimurium, Salmonella Delphi, Salmonella Agona, etc.). Many studies have shown that Salmonella Indiana is closely related to vomiting, diarrhea, fever, gastroenteritis, local...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/10C12R1/42
CPCC12Q1/686C12Q1/689C12Q2565/125
Inventor 龚建森张萍张笛乐敏沈海玉庄林林许明徐敬潇盛中伟韩先干窦新红
Owner JIANGSU INST OF POULTRY SCI
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