Polymerase chain reaction (PCR) detection kit for rapidly identifying salmonella pullorum or salmonella gallinarum
A technology of Salmonella typhi and detection kit, which is applied in the field of biotechnology detection, can solve the problems of cumbersome process, time-consuming and laborious, and achieve good repeatability
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Embodiment 1
[0037] Embodiment 1 Bioinformatics method identifies the distribution of SPUL_2694 gene
[0038] In NCBI, use the Blastn online comparison software (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to search for the SPUL_2694 gene in the genome-wide database, and the search results show that all pullorum / Salmonella gallinarum contain SPUL_2694 gene, and the nucleotide sequence of the gene length is 2619bp, in which Salmonella Chester was identified by PCR in this paper, and the results showed that the target band could not be amplified; the other two serotypes were Salmonella paratyphi A , Java Salmonella, both of which are not of avian origin, so they have no effect on the identification of pullorum / salmonella typhi. The results of bioinformatics identification showed that SPUL_2694 had good specificity and could be used to establish a PCR method for identifying pullorum / Salmonella gallinarum ( figure 1 ).
[0039] The nucleotide sequence of pullorum / salmonella gallinarum typhi SPU...
Embodiment 2
[0041] The preparation of embodiment 2 kit
[0042] Primer design and synthesis: Using the SPUL_2694 gene as a template, design and analyze primers, and select the best detection primer pair according to the genomic DNA sequence. The nucleotide sequences are shown in Table 1 below:
[0043] Table 1
[0044]
[0045] The above-mentioned primer pairs can be packaged individually, or can be made into a PCR detection solution. In the PCR detection solution, the amount of the above-mentioned primer pairs can be the conventional amount known to those skilled in the art.
[0046] That is to say, the kit of the present invention may contain the aforementioned independently packaged primer pair, or may contain a configured PCR detection solution containing the primer pair.
[0047] Further, the kit can also contain double distilled water (ddH 2 O), 2×Taq Master Mix, sample genomic DNA extraction reagents, etc.
Embodiment 3
[0048] Embodiment 3 Kit detects the specific identification of pullorum / salmonella gallinarum typhi
[0049] Using the primers in the kit described in Example 2, using the genomes of different serotypes of Salmonella and other bacteria as templates, the distribution characteristics of the SPUL_2694 gene in different bacteria were identified by PCR.
[0050] The PCR reaction system is (25 μL): 9.5 μL double distilled water (ddH 2O), 12.5 μL 2×Taq Master Mix, SPUL_2694-F 1 μL, SPUL_2694-R 1 μL, template 1 μL.
[0051] The PCR program is (a) 94°C for 5min; (b) 94°C for 45s; (c) 58.8°C for 30s; (d) 72°C for 2min 37s, steps (b) to (d) cycled 25 times; (e) 72°C for 10min .
[0052] The PCR product is carried out 1% agarose gel electrophoresis, and PCR electrophoresis result shows that all have 2619bp objective band ( figure 2 , image 3 ), while other serotypes of Salmonella did not amplify the bands ( figure 2 , image 3 , Figure 4 , Figure 5 ), show that with the speci...
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