Primer set, reagent, kit, application and detection method for detecting Lactobacillus rhamnosus lv108
A technology of Lactobacillus rhamnosus and LV108, applied in the biological field, can solve the problems of complex identification of strains, great differences in fermentation performance, intestinal tolerance, and probiotic functions, and the lack of fast and effective means for rapid confirmation.
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Embodiment 1
[0106] Embodiment 1 Primer synthesis and mother plant inspection
[0107] Whole genome sequencing of Lactobacillus rhamnosus LV108, LGG, Lc705 and hsryfm1301 as reference strains, through multi-strain gene family analysis, the unique genes of Lactobacillus rhamnosus LV108 were screened, and primers were designed in these genes: LV108 -1-F and LV108-1-R, LV108-2-F and LV108-2-R, LV108-3-F and LV108-3-R, and LV108-4-F and LV108-4-R (see table 1 shown). 4 pairs of primers were synthesized by Sangon Bioengineering Co., Ltd. (Shanghai). The mother strain of Lactobacillus rhamnosus LV108 was streaked and purified in MRS solid medium, and a single colony was picked and cultured in MRS medium at 37°C for 24h, transferred to fresh MRS medium (2% v / v), and cultured at 37°C for 24h ; Collect the bacterial cells, and use a column DNA extraction kit (Sangon Bio, Shanghai) to extract the DNA of the Lactobacillus rhamnosus LV108 mother strain. The DNA of Lactobacillus rhamnosus LV108 moth...
Embodiment 2
[0113] Embodiment 2 Non-identical lactic acid bacteria amplification detection
[0114] DNA extraction and PCR methods were the same as in Example 1. In order to verify the specificity of the four pairs of primers, the DNA of other strains of lactic acid bacteria was first amplified. The DNA of Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus delbrueckii subsp bulgaricus and Lactobacillus casei were amplified to bands ( Figure 2A ); LV108-2-F and LV108-2-R can amplify into bands from Lactobacillus helveticus, Lactobacillus delbrueckii and Lactobacillus casei DNA, but the sizes are 1000bp, 4000bp and 5000bp, respectively, which are different from rhamnose Lactobacillus LV108; LV108-4-F and LV108-4-R were able to amplify into bands from Lactobacillus fermentum, Lactobacillus helveticus and Lactobacillus casei DNA, but the sizes were 2000bp, 3000bp and 4000bp, respectively, which were different from rhamnosus Lactobacillus saccharides LV108 ( Figure 2A ). Th...
Embodiment 3
[0115] Embodiment 3 Amplification detection of different strains of Lactobacillus rhamnosus
[0116] The purpose of this example is to identify between strains, and 4 pairs of primers are used to amplify and detect other 12 strains of Lactobacillus rhamnosus isolated and preserved in the laboratory. Lactobacillus rhamnosus hsryfm1301 is also a reference strain for primer design, LV108-2-F and LV108-2-R, LV108-3-F and LV108-3-R, and LV108-4-F and LV108-4-R from The amplified bands of the DNA of Lactobacillus rhamnosus hsryfm1301 were about 2000bp, 1500bp and 4000bp ( Figure 2B ), all different from LV108, further confirming the validity of gene family analysis. In terms of other strains, in addition to LV108-1-F and LV108-1-R from strain BG, V108-2-F and LV108-2-R from strain Bm03, and LV108-3-F and LV108-3-R from strain Bm01 The bands amplified by , F and LV-1 are close to LV108, and the bands amplified from 12 strains by 4 pairs of primers are all different from LV108 ( ...
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