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Primer pair and kit for detecting butyric acid synthetic gene

A technique for synthesizing genes and primer pairs, which is applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc. It can solve problems such as limited sensitivity and specificity, high incidence of CRC, and hidden early symptoms , to achieve the effect of improving prediction rate, high sensitivity and good stability

Active Publication Date: 2019-01-18
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

While the incidence of CRC is high, the prognosis is also worrying. One of the main reasons is that its early symptoms are hidden and non-specific, and early diagnosis is difficult.
Nowadays, the early specific markers of CRC are mostly plasma and serum biomarkers, and most of them have limited sensitivity and specificity. It is urgent to find more ideal CRC-specific biomarkers to improve the current CRC diagnostic strategy

Method used

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  • Primer pair and kit for detecting butyric acid synthetic gene
  • Primer pair and kit for detecting butyric acid synthetic gene
  • Primer pair and kit for detecting butyric acid synthetic gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Real-time fluorescence quantitative qRT-PCR preliminarily verified the abundance difference of Alistipes in CRC model mice and normal control mice.

[0070] 1. Experimental materials

[0071] The bacterial genomes extracted from feces samples of CRC model mice and normal control mice were verified for the first time by qRT-PCR on the difference in the abundance of Alistipes.

[0072] 2. Test method

[0073] 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.

[0074] Amplification of the target gene but: real-time fluorescent quantitative PCR was performed using the ChamQ SYBR qPCR Master Mix kit (product number Q331-02 / 03) of Vazyme Company.

[0075] The first set of primers was used for amplification, the upstream primer (Primer1) 5'-CAGCGTCTACATTCAGGGCA-3', the downstream primer (Primer2) 5'-CCGGAGTAGAGCGTG...

Embodiment 2

[0082] A ROC curve was constructed to verify the ability of the but gene sequence SEQ ID NO.1 for auxiliary diagnosis to distinguish CRC patients from healthy volunteers.

[0083] The receiver operating curve (ROC) method was used for verification, and the expression level of the signature gene but of characteristic bacteria in the feces of CRC patients and healthy volunteers was used to judge its ability to predict the risk of CRC disease. The result is as figure 2 As shown, it shows that the area under the ROC curve (AUC) for distinguishing CRC patients by basic indicators alone is only 0.51, and after adding the expression indicator of but, the AUC increases to 0.72, which has clinical significance for prognosis.

Embodiment 3

[0085] Used to explore the effect of Alistipes putredinis bacteria on the proliferation of CRC cells.

[0086] Use standard medium (ATCC medium 260) to cultivate Alistipes putredinis bacterial culture value bacterial liquid density OD 600 = 0.5 to confirm that the bacteria have reached log phase. The medium was collected by centrifugation (1000 xg, 15 minutes) and filtered through a 0.22 μm pore size filter to adjust the pH to 7.2-7.4. Then the bacterial culture solution is mixed with the culture medium to a final concentration of 5%, 10%, 20%, 40% of the total culture medium. Different concentrations of bacterial culture solution and colon cancer Caco-2 cells were placed in a 96-well plate (the cell density was 5×10 3 / well) were incubated together for 24h, 48h, and 72h to observe the proliferation of Caco-2 cells. Finally, 10 μL of CCK-8 solution was added to each well, and the plate was incubated at 37° C. for 2 h, and the cell viability was determined by absorbance read...

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Abstract

The invention discloses a primer pair and a kit for detecting butyric acid synthetic gene (butyryl-CoA: acetate CoA transferase). Based on the selected butyric acid synthesis gene from the intestinalbacteria Alistipes putredinis, it is highly expressed in humans (named the but gene), and its gene sequence is SEQ ID NO. As shown in Fig. 1, a combination of primer pairs having specificity and quantitative effects is found, and a detection kit was designed. The invention effectively overcomes the deficiency of the prior art for detecting risk genes of colorectal cancer (CRC) in intestinal bacteria, and can improve the prediction rate of CRC diseases. The kit of the invention is suitable for all types of fluorescence quantitative gene amplification instruments currently on the market, has high sensitivity, rapid and accurate quantitative, good stability, has good application prospect, and has high industrial utilization value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a primer pair and a kit for detecting butyryl-CoA: acetate CoA transferase (butyryl-CoA: acetate CoA transferase) synthesis gene. Background technique [0002] In recent years, the morbidity and mortality of sporadic colorectal cancer (CRC) have been increasing globally, and it is necessary to find new targets for diagnosis and treatment. While the incidence of CRC is high, the prognosis is also worrying. One of the main reasons is that its early symptoms are hidden and non-specific, and early diagnosis is difficult. Nowadays, the early specific markers of CRC are mostly plasma and serum biomarkers, and most of them have limited sensitivity and specificity. It is urgent to find more ideal CRC-specific biomarkers to improve the current CRC diagnostic strategy. [0003] A large number of studies at home and abroad have shown that the changes in intestinal flora in CRC pati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/6886C12Q2600/158C12Q2531/113C12Q2563/107
Inventor 李菁王雪王马浩
Owner CHINA PHARM UNIV
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