Primer pairs and kits for detecting trimethylamine-producing genes
A trimethylamine and primer pair technology is applied in biochemical equipment and methods, microbial determination/inspection, resistance to vector-borne diseases, etc. Sensitive effect
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Embodiment 1
[0070] Preliminary verification of the differential expression of cutC in atherosclerosis model mice and normal mice by real-time fluorescent quantitative qRT-PCR
[0071] 1. Experimental materials
[0072] The first example of qRT-PCR was used to verify the difference in the expression of cutC in the bacterial genomes extracted from fecal samples of atherosclerotic mice and normal control mice.
[0073] 2. Test method
[0074] Genome extraction of fecal bacteria: MinkaGene Stool DNA kit (Catalog No. DX1050-02) was used to extract the genome of the stool sample, and a DNA sample with a final concentration of 500-2000 μg / μL was obtained.
[0075] Amplification of the target cutC gene: real-time fluorescent quantitative PCR was performed using the ChamQ SYBR qPCR Master Mix kit (product number Q331-02 / 03) of Vazyme Company.
[0076] The first set of primers was used for amplification, the upstream primer (Primer1) 5'-CCATCCATCACGACCTACCG-3', the downstream primer (Primer2) 5'-...
Embodiment 2
[0084] A ROC curve was constructed to verify the ability of the cutC gene sequence SEQ ID NO.1 for auxiliary diagnosis to distinguish atherosclerosis patients from healthy volunteers.
[0085] The receiver operating curve (ROC) method was used to verify the expression level of the marker gene cutC in the feces of patients with atherosclerosis and healthy volunteers to judge its ability to predict the risk of differentiating atherosclerotic diseases. Such as figure 2 As shown, the results show that the area under the ROC curve (AUC) for distinguishing atherosclerosis patients with basic indicators alone is only 0.53, and after adding the expression indicator of cutC, the AUC increases to 0.76, which has clinical significance for prognosis.
Embodiment 4
[0087] Used to detect the presence of the cutC gene and its ability to produce trimethylamine in bacterial samples.
[0088] The bacterial culture solution of Clostridium saccharolyticum WM1 is amplified by PCR with the first set of primers, and the amplified product is subjected to nucleic acid electrophoresis to obtain the band of cutC (such as image 3 shown) is consistent with the real sequence (approximately 2.1 kb). Then, the enzymatic activity of cutC was judged by adding increasing amounts of choline as a substrate in a specific medium (ATCC Medium 1118) and detecting the production of trimethylamine. Such as Figure 4 As shown, it was found that the ability of cutC to produce trimethylamine increased significantly with the increase of the substrate content.
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