A multiplex PCR kit and its rapid detection method for Micrococcus luteus
A technology of Micrococcus luteus and a kit, which is applied in the directions of botanical equipment and methods, biochemical equipment and methods, and applications, etc., to achieve the effects of high sensitivity and strong specificity
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0029] Example 1: Design and effect display of PCR amplification primers
[0030] 1. PCR amplification primer design and synthesis
[0031] The CBS gene, Sig gene and Pol gene in the genome of Micrococcus luteus in GeneBank were selected as target genes. The sequence of the CBS gene is shown in SEQ ID NO:1; the sequence of the Sig gene is shown in SEQ ID NO:2; the sequence of the Pol gene is shown in SEQ ID NO:3.
[0032] According to the sequence of each described gene, select respectively a part that can best represent Micrococcus luteus in each described gene sequence, respectively correspondingly design three pairs of primers with Primer Premier 5.0 software, the concrete sequence of described each pair of primers is as shown in Table 1 Show. Among them, primer pair 1 is TCBS-1F and TCBS-1R, respectively amplifying the forward and reverse sequences of the CBS gene; primer pair 2 is Tsig-2F and Tsig-2R, respectively amplifying the forward and reverse sequences of the Sig ...
Embodiment 2
[0075] Embodiment two: Specific detection
[0076] 1. Preparation of DNA template
[0077] According to the requirements of aseptic operation, inoculate the preserved strain of Bacillus subtilis in MBS solid medium for activation culture, pick a single colony and inoculate it in MBS liquid medium for three generations at 37°C, and extract DNA from 1-5ml bacterial liquid; Inoculate the preserved strain of Enterococcus faecalis in MRS solid medium for activation culture, pick a single colony and inoculate it in MRS liquid medium for three generations at 37°C, take 1-5ml bacterial liquid to extract DNA; Micrococcus luteus, Streptococcus, Edwardsiella piscicida, Brevibacterium sanguinis, Aeromonas sobria, Vibriocholerae, Bacillus cereus, The preserved strains of Shewanella xiamenensis and Aeromonas veronii were respectively inoculated in LB solid medium for activation culture, and a single colony was picked and inoculated in LB liquid medium for three generations at 37°C, and 1 ...
Embodiment 3
[0084] Embodiment 3: Sensitivity detection
[0085] 1. Preparation of DNA template: The DNA template of Micrococcus luteus was prepared according to the method in Example 1. After measuring the concentration of the extracted Micrococcus luteus genomic DNA, the DNA samples were followed by 10 times, 20 times, 50 times, 10 times 2 times, 10 3 times, 10 4 times, 10 5 times, 10 6 times, 10 7 double dilution.
[0086] 2. PCR amplification: Take the DNA templates of various concentrations obtained in step 1, and use the primers in Table 1 to perform PCR amplification. The overall PCR system, components and reaction conditions are the same as the "triple PCR amplification" in Step 4 of Example 1.
[0087] 3. Take 5 μl of the PCR amplification product, use 1% agarose gel electrophoresis, run the electrophoresis at a constant voltage of 120V for 45 minutes, and observe the PCR amplification result under the gel imaging system. The concentration determination result of Micrococc...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More