Primer group, agent and kit for detecting lactobacillus rhamnosus LV108, application and detecting method
A technology of Lactobacillus rhamnosus and LV108, which is applied in the biological field, can solve the problems of complex identification of strains, great differences in fermentation performance, intestinal tolerance and probiotic functions, lack of fast and effective means for rapid confirmation, etc., and reach clinical The effect of wide application range, high sensitivity and short detection cycle
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Embodiment 1
[0106] Example 1 Primer Synthesis and Mother Plant Inspection
[0107] The entire genome of Lactobacillus rhamnosus LV108 was sequenced, and LGG, Lc705 and hsryfm1301 were used as reference strains, and the unique genes of Lactobacillus rhamnosus LV108 were screened out through multi-strain gene family analysis, 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). 4 pairs of primers were synthesized by Sangon Bioengineering Co., Ltd. (Shanghai). The Lactobacillus rhamnosus LV108 mother strain was streaked and purified in MRS solid medium, and a single colony was picked and cultured in MRS medium at 37°C for 24h, then transferred to fresh MRS medium (2% v / v), and cultured at 37°C for 24h ; Collect the thalli, and extract the DNA of the mother strain of Lactobacillus rhamnosus LV108 using a column DNA extraction kit (Sangon Biotechnology, Shanghai). Use rTaq DNA polymer...
Embodiment 2
[0113] Embodiment 2 Non-homogeneous lactic acid bacteria amplification detection
[0114] DNA extraction and PCR methods are the same as in Example 1. In order to verify the specificity of the 4 pairs of primers, the DNA of other strains of lactic acid bacteria was first amplified. The DNA of Lactobacillus, 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 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 can amplify bands from DNA of Lactobacillus fermentum, Lactobacillus helveticus and Lactobacillus casei, but the sizes are 2000bp, 3000bp and 4000bp respectively, which are different from Rhamnos Lactobacillus sugar LV108 ( Figure 2A ). The results show...
Embodiment 3
[0115] Embodiment 3 Amplification detection of different strains of Lactobacillus rhamnosus
[0116] The purpose of this example is to carry out inter-strain identification, 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 the 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 DNA amplified bands of Lactobacillus rhamnosus hsryfm1301 were about 2000bp, 1500bp and 4000bp respectively ( Figure 2B ), are different from LV108, which further confirmed the validity of gene family analysis. Other strains, except 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 were close to LV108, and the bands amplified by 4 pairs of primers from 12 strains were different from LV108 ...
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