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PCR detection kit for rapidly detecting salmonellae and identifying salmonella pullorum/salmonella gallinarum and application thereof

A technology of Salmonella typhimurium and detection kit, which is applied in microorganism-based methods, microbial determination/inspection, microorganisms and other directions, can solve the problems of pathogen contamination, high false positives, interference, etc., and achieves good repeatability and high sensitivity , good specificity

Pending Publication Date: 2020-10-02
YANGZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Traditional detection methods, namely non-selective and selective enrichment, biochemical characterization and serological identification are laborious and time-consuming, taking 4-7 days to complete, while other methods such as antibody detection, although fast, have a high false positive rate making it unsuitable for routine testing
In addition, factors such as low levels of pathogenic bacteria contamination, "injury" of Salmonella after food processing and interference from other food ingredients have limited the detection of Salmonella

Method used

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  • PCR detection kit for rapidly detecting salmonellae and identifying salmonella pullorum/salmonella gallinarum and application thereof
  • PCR detection kit for rapidly detecting salmonellae and identifying salmonella pullorum/salmonella gallinarum and application thereof
  • PCR detection kit for rapidly detecting salmonellae and identifying salmonella pullorum/salmonella gallinarum and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Bioinformatics method to identify the distribution of cigR gene

[0042] In NCBI, use the Blastn online comparison software (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to search the cigR gene in the whole genome database. The search results show that the cigR gene only exists in Salmonella, while chicken The cigR gene of pullorum and Salmonella gallinarum lacks an intermediate nucleotide fragment (such as figure 1 ), that is, there is no pullorum and Salmonella gallinarum typhi. According to the distribution characteristics of the cigR gene, pullorum pullorum and Salmonella gallinarum typhi can be distinguished from other strains.

Embodiment 2

[0043] The preparation of embodiment 2 kit

[0044] Primer design and synthesis: using the cigR gene of Salmonella typhimurium as a template, primers were designed based on the common fragments of all Salmonella to distinguish whether it is Salmonella, and designed for more fragments of Salmonella typhimurium than pullorum and Salmonella gallinarum The primers were selected to further identify Pullorum pullorum and Salmonella gallinarum typhi, and the specificity of the primers was compared online through Primer-BLAST on the NCBI website, and the best pair of detection primers was manually selected. Among them, cigR-F and cigR-R1 primers amplify the partial sequence of non-pulmonary pullorum and Salmonella gallinarum typhi (463bp) or partial sequence of cigR of pullorum and Salmonella gallinarum typhi (421bp), and cigR-F and cigR-R2 primers amplify The partial fragment (65bp) of non-pulmonary pullorum and Salmonella gallinarum typhi, its nucleotide sequence is shown in Table 1...

Embodiment 3

[0065] Example 3 Specific identification of kits for detection of Salmonella and pullorum / Salmonella gallinarum typhi

[0066] Two sets of primer pairs in the kit described in Example 2 were used, and the genomes of different serotypes of Salmonella and other bacteria were respectively used as templates, and the multiplex PCR method was used to detect the specificity of Salmonella and pullorum / Salmonella gallinarum.

[0067] The PCR reaction system is (25μL): 2x Taq Master mix 12.5μL, cigR-F 80nM, cigR-R140nM, cigR-R2 40nM, template 1μL, ddH 2 O to 25 μL.

[0068] The PCR program was 95°C for 3min; 95°C for 15s, 50°C for 15s, 72°C for 30s, 30 cycles; 72°C for 10min.

[0069] The PCR product was subjected to 1% agarose gel electrophoresis, and the PCR electrophoresis results showed that two bands could be amplified in the swimming lanes using the non-pulmonary pullorum and Salmonella typhi genome as templates, which were 463bp and 65bp respectively; Only one band was amplifie...

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Abstract

The invention provides a PCR detection kit for rapidly detecting salmonella and identifying salmonella pullorum / salmonella gallinarum and an application thereof. According to the applicant, a primer is designed for a cigR gene, a PCR detection technology is adopted for detecting the cigR gene, and whether a detected object belongs to salmonellae or not or whether the detected object is the salmonella pullorum / salmonella gallinarum or not can be analyzed and judged according to amplification and detection conditions. In a specific scheme, the detection kit at least comprises a forward primer asshown in SEQ ID NO.1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.2. The kit is simple, rapid, good in specificity, high in sensitivity and good in repeatability.

Description

technical field [0001] The invention relates to a biological detection kit, in particular to a PCR detection kit for rapidly detecting Salmonella and identifying pullorum / Salmonella gallinarum typhi, and its preparation and application. Background technique [0002] Salmonellosis is one of the important zoonoses in public health. Its pathogen Salmonella belongs to Enterobacteriaceae. Livestock, poultry and egg and meat products are the main media. It can not only cause a variety of livestock and poultry diseases , cause systemic sepsis and enteritis, and can also be used as the pathogen of foodborne diseases, causing human gastroenteritis and food poisoning. Pullorum pullorum and typhoid fever are caused by Salmonella Pullorum and Salmonella Gallinarum respectively. Salmonella pullorum, also known as Salmonella pullorum, can cause pullorum in poultry, and it mostly affects young chicks less than 3 weeks old, with high morbidity and mortality; adult chickens can also be infe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/10C12R1/42
CPCC12Q1/689C12Q1/686C12Q2600/16C12Q2537/143
Inventor 焦新安潘志明周莹莹康喜龙孟闯顾丹殷俊磊李求春
Owner YANGZHOU UNIV
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