Method for evaluating stable state of flora in excrement sample and application of method in colorectal cancer screening

An evaluation method, a technology of colorectal cancer, applied to the indicator bacteria combination and its calculation indicating the relationship between flora balance in individual stool samples, non-invasive diagnosis and screening of colorectal cancer, can solve the problem of out-of-control flora balance, etc. Achieve the effect of low cost, good intestinal health screening and promotion value of clinical auxiliary diagnosis

Pending Publication Date: 2018-10-23
SUN YAT SEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But when the internal environment of the body...

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  • Method for evaluating stable state of flora in excrement sample and application of method in colorectal cancer screening
  • Method for evaluating stable state of flora in excrement sample and application of method in colorectal cancer screening
  • Method for evaluating stable state of flora in excrement sample and application of method in colorectal cancer screening

Examples

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Embodiment example 1

[0022] Implementation Case 1: Total DNA Extraction from Stool Samples

[0023] 1. Sample collection: The stool samples collected in this study included 215 cases of CRC, 100 cases of NGC, and 156 cases of HS. 178 cases of BCD. Among the 215 CRC patients, 97 were early stage patients: 38 cases of stage I CRC and 59 cases of stage II CRC. Before collecting the specimens and background information of each patient, an informed consent was signed with the patient to obtain permission to use their information as research.

[0024] The average age of 215 CRC patients was 61.2 years old, ranging from 22 to 93 years old, including 118 males and 97 females; the average age of 178 BCD patients was 54.2 years old, ranging from 31 to 81 years old, including 86 males and 92 females ; The average age of 100 NGC patients was 58.4 years old, ranging from 34 to 73 years old, including 48 males and 52 females; the average age of 156 HS patients was 48.6 years old, ranging from 20 to 78 years o...

Embodiment example 2

[0028] Implementation Case 2: Quantitative Analysis of Microbial Indicator Bacteria

[0029] 1. Production of standard products: The bacterial DNA of Fn standard strain ATCC25586, Fp standard strain ATCC27766, and Bb standard strain BB-12 was used as standard products for conventional PCR reactions. PCR reaction system 25 μL: 10×PCR Buffer 2.5 μL, dNTP Mixture 1 μL, 5 μmol / L upstream and downstream primers 2 μL each, DNA template 2 μL, DNA Taq enzyme (5U / μL) 0.2 L, H 2 O15.3 μL. Amplification program: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 1 min, annealing at 56°C for 1 min, extension at 72°C for 1 min, cycled 35 times.

[0030] The PCR amplification products of the above three kinds of bacteria were purified and recovered as the target DNA fragments respectively, and connected to the pGEM-T vector by the conventional cloning method, and the recombinant plasmid was transformed into competent bacteria DH5a, and the positive clones were obtained by blue-wh...

Embodiment example 3

[0034] Example 3: Evaluation of Fecal Microbial Markers

[0035] 1. The amount of Fn in the feces of the CRC group was significantly higher than that of the three control groups: HS, NGC, and BCD groups (pfigure 1 a.

[0036] 2. Compared with the HS group and NGC group, the amount of Bb and Fp in the feces of the CRC group was significantly reduced (HS group; pfigure 1 b, 1c).

[0037] 3. The ratio Fn / Fp of bacteria in feces in CRC and BCD groups was significantly higher than that in HS and NGC groups (pfigure 1 d.

[0038] 4. Compared with HS, BCD and NGC groups, the ratio Fn / Bb of the number of bacteria in feces in CRC group was significantly higher (pfigure 1 e.

[0039] 5. The Fn / Fp and Fn / Bb in feces of stage I CRC were significantly higher than those of HS group (all pfigure 1 d, 1e.

[0040] 6. These results indicate that the Fn / Bb ratio in feces has the ability to evaluate CRC; the Fn / Fp ratio has the ability to evaluate CRC and BCD

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Abstract

The invention relates to a method for calculating a flora balanced relation index in an individual excrement sample, and an application of the method in screening, diagnosis or auxiliary diagnosis incolorectal cancer (CRC). By extracting bacteria in excrement during DNA sequencing, the types and quantity characteristics of the bacteria can be obtained, and a CRC diagnosis by taking quantity ratiocharacteristic of a plurality of bacteria as a base is carried out. Compared with the methods used in clinical diagnosis or noninvasive screening of CRC with an applied patent, the method is completely noninvasive, and can realize accurate diagnosis of CRC. The analysis result displays that a ratio of fusobacterium nucleatum Fn to bifidobacteria Bb quantity (Fn/Bb) has high susceptibility and specificity on CRC screening, which can respectively reach 84.6% and 92.3% (AUC=0.911). the ratio of fusobacterium nucleatum Fn to clostridium leptum Fp (Fn/Fp) quantity is combined to increase the diagnosis value on CRC, and the Area Under Curve (AUC) of a subject work characteristic curve can reach 0.943. In addition, combination of Fn/Bb and Fn/Fp quantity ratio for screening I-stage CRC has 60% of specificity and 90% of sensitivity.

Description

technical field [0001] The present invention relates to the field of colorectal cancer (Colorectal cancer, CRC) in vitro diagnosis, and the present invention provides a combination of index bacteria indicating the balance of flora in individual stool samples and a calculation method thereof, which are used for non-invasive diagnosis and screening of colorectal cancer cancer Background technique [0002] Colorectal cancer is one of the most common malignant tumors in the world. According to the WHO report, colorectal cancer is the third most common malignant tumor in men and the second most common in women. In recent years, the morbidity and mortality rate in my country have been rising continuously, with more than 170,000 new cases and nearly 100,000 deaths each year. The incidence rate ranks third among malignant tumors in my country. [0003] Early colorectal cancer is not easy to find, and the vast majority of patients come to the hospital three to six months after sympt...

Claims

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Application Information

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IPC IPC(8): C12Q1/04C12R1/01
CPCC12Q1/04G01N2800/7028
Inventor 张革郭松鹤陆永帆刘万里
Owner SUN YAT SEN UNIV
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