PCR primers specifically targeting the flagellar hook gene flge and its application in the rapid detection of flagellar and aflagellate Salmonella
A Salmonella, non-flagellate technology, applied in the direction of microbial-based methods, biochemical equipment and methods, microbial determination/inspection, etc., to achieve rapid detection, save labor, and shorten working hours
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Embodiment 1
[0044] Example 1: Primer Design
[0045] The inventors of the present application have found through comprehensive analysis of the genomes of pullorum pullorum and Salmonella gallinarum typhi that although pullorum pullorum and Salmonella gallinarum typhi do not have flagella attachment structures on the surface, their chromosomal genomes contain flagella-related coding gene clusters. Through further sequence comparison analysis, it was found that the specific flagella-related genes in pullorum and Salmonella gallinarum typhi chromosomes had extremely high homology and similarity with the flagella-related genes of other flagellated Salmonella (such as Salmonella enteritidis).
[0046] On this basis, after comparing the flagellar-related genes of various different types of bacteria, the applicant inventor selected the flagellar hook protein coding gene flgE as the target for identifying Salmonella, and realized the simultaneous detection of flagellar and aflagellated Salmonella....
Embodiment 2
[0051] Embodiment 2: The establishment of the Salmonella PCR detection method based on flgE gene
[0052] 1. Required reagents and materials:
[0053] Reagents: rTaq DNA polymerase, dNTPs, MgCl 2 Purchased from Takara Corporation.
[0054] Strain: Salmonella Enteritidis standard strain CMCC(B)50336, donated by Professor Jiao Xin'an of Yangzhou University.
[0055] Template preparation:
[0056] Take a single colony of the sample to be tested on the agar petri dish and inoculate it in liquid LB medium. Incubate at 37°C for 18 hours, take 1.5 mL of the bacterial solution and place it in a 1.5 mL centrifuge tube, centrifuge at 12,000 rpm for 10 min, wash twice with sterile PBS, centrifuge at 12,000 rpm for 10 min, resuspend the pellet in 200 μL of sterile double-distilled water, and place in a boiling water bath for 10 min, at 12,000 rpm Centrifuge for 10 min, and take the supernatant, which can be used as a template for PCR reaction.
[0057] 2. PCR amplification method:
...
Embodiment 3
[0070] Embodiment 3: the specific detection of the PCR method of specific targeting flgE gene
[0071] 1. Strains
[0072] The bacterial strains used in the present invention are shown in Table 1.
[0073] Table 1. Test strains and related information
[0074]
[0075]
[0076] Among them, the domestic standard strain of Salmonella enteritidis CMCC (B) 50336 (hereinafter referred to as SE50336), donated by Professor Jiao Xin'an of Yangzhou University; Salmonella enteritidis T48, T64, Salmonella typhimurium W32, T14, U27, W2, A12, Salmonella typhi W33, Salmonella choleraesuis U80, U81, U82, Salmonella gallinarum typhi U20 were donated by Professor Liu Shulin of Harbin Medical University, standard strains of Salmonella pullorum CVCC523, CVCC526, CVCC535, CVCC540 were purchased from China Center for Veterinary Culture Collection (CVCC), chicken Escherichia coli CE7 was donated by Professor Lu Chengping of Nanjing Agricultural University. Staphylococcus was obtained from ...
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