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Quintuple PCR detection method and detection kit for avian escherichia coli, chicken typhoid salmonella, salmonella pullorum and the like

A technology for Salmonella typhi and Salmonella pullorum is applied in the biological field to achieve the effects of good repeatability, sensitive detection and high sensitivity

Active Publication Date: 2017-09-26
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, a variety of detection methods have been established for avian Escherichia coli and different serotypes of Salmonella, however, there are no rapid detection and identification methods for avian Escherichia coli, Salmonella gallinarum typhi, Salmonella pullorum, Salmonella enteritidis, and Salmonella typhimurium Diagnostic PCR method

Method used

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  • Quintuple PCR detection method and detection kit for avian escherichia coli, chicken typhoid salmonella, salmonella pullorum and the like
  • Quintuple PCR detection method and detection kit for avian escherichia coli, chicken typhoid salmonella, salmonella pullorum and the like
  • Quintuple PCR detection method and detection kit for avian escherichia coli, chicken typhoid salmonella, salmonella pullorum and the like

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 5

[0038] Example 1 Five-fold PCR detection method

[0039] In this embodiment, the primer sets used for multiplex PCR to detect avian Escherichia coli, Salmonella typhi, Salmonella pullorum, Salmonella enteritidis, and Salmonella typhimurium are used to detect avian Escherichia coli, Salmonella typhi, Salmonella pullorum, and enteritis. The detection of Salmonella and Salmonella typhimurium includes the following steps:

[0040] Step 1. Prepare a PCR template based on the bacteria to be detected

[0041] Inoculate the bacterial strains to be tested into LB liquid medium and cultivate to OD at a temperature of 37℃ 600 =1.0, the bacterial solution that can be used as a PCR template is obtained.

[0042] Step 2. Prepare specific primer set

[0043] Preparation of primer sets for multiplex PCR detection of avian Escherichia coli, Salmonella gallisepticum, Salmonella pullorum, Salmonella enteritidis, and Salmonella typhimurium: the concentration of each primer set is 10pM, and the primers of ...

Embodiment 2 5

[0071] Example 2 Five-fold PCR detection kit for detection

[0072] This embodiment is consistent with the steps of embodiment 1, except that this embodiment directly uses a five-fold PCR detection kit including the primer set mentioned in embodiment 1 to establish the above PCR reaction system.

[0073] The composition of the five-fold PCR detection kit at this time includes 2×PCR PreMix and a primer set. The volume of 2×PCRPreMix is ​​12.5μL. The volume of the primer pair ECphoA-F and ECphoA-R in the primer set is 0.5μL each. The volume of SGglgC-F and SGglgC-R is 0.2μL, the volume of primer pair SGPspeC-F and SGPspeC-R is 0.3μL, the volume of primer pair SEsdfⅠ-F and SEsdfⅠ-R is 0.4μL, primer pair STstm4495 The volume of -F and STstm4495-R are each 0.5μL.

[0074] Evaluation example 1 specific evaluation

[0075] In this example, the five-fold PCR detection kit of Example 2 is used as an example to specifically detect the primer sets in Example 1 and Example 2:

[0076] Select Riem...

Embodiment 1

[0105] The function and effect of Example 1 and Example 2

[0106] From Evaluation Example 1 to Evaluation Example 3, it can be seen that the primer set used to amplify the positive strains stored in the laboratory can amplify specific bands, while other bacterial strains and other control samples are not amplified To the band, the specificity of the primer set is strong, and the minimum detection sensitivity of using this primer set to detect positive samples of different dilutions can reach 1000CFU bacterial solution and 500pg bacterial genomic DNA, indicating the primer set’s High sensitivity, at the same time, the primer set has good repeatability, indicating that its stability is strong. Therefore, Example 1 and Example 2 provided by the five-fold PCR detection method using the above-mentioned primer set or the five-fold PCR detection kit containing the above-mentioned primer set can detect avian Escherichia coli, Salmonella typhi, Salmonella pullorum, and Salmonella enterit...

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Abstract

The invention provides a multiple PCR primer group for avian escherichia coli, chicken typhoid salmonella, salmonella pullorum, salmonella enteritidis and bacillus breslaviensis, a quintuple PCR detection kit comprising the primer group and a quintuple PCR detection method. The primer group comprises primer pairs ECphoA-F and ECphoA-R for specific amplification of avian escherichia coli phoA genes, primer pairs SGg1gC-F and SGg1gC-R for specific amplification of chicken typhoid salmonella glgC genes, primer pairs SGPspeC-F and SGPspeC-R for specific amplification of chicken typhoid salmonella and salmonella pullorum genes, primer pairs SEsdf I-F and SEsdf I-R for specific amplification of salmonella enteritidis sdf-1 genes and primer pairs STstm4495-F and STstm4495-R for specific amplification of bacillus breslaviensis stm4495 genes.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a multiple PCR primer set for detecting avian Escherichia coli, Salmonella typhi, Salmonella pullorum, Salmonella enteritidis, and Salmonella typhimurium, and a five-fold PCR including the primer set A detection kit and a five-fold PCR detection method using the primer set. Background technique [0002] With the rapid development of the large-scale poultry industry, the occurrence and prevalence of diseases have increased correspondingly, resulting in a decline in the economic benefits of the poultry industry. Important bacterial infectious diseases such as poultry colibacillosis, pullorum, typhoid fever and other important bacterial diseases seriously harm the poultry industry. [0003] At present, the country mainly relies on antibiotics to prevent and control bacterial diseases, but due to the long-term and unreasonable use of antibiotics: on the one hand, there is widespread...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10C12N15/11C12R1/19C12R1/42
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2531/113
Inventor 于圣青王少辉梁华吴晓君丁铲
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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