Method for diagnosing colorectal cancer from human feces sample by quantitive pcr, primers and kit

A technology for colorectal cancer and stool samples, which is applied in the direction of biochemical equipment and methods, and microbial measurement/testing, and can solve the problems of industrial differences in microbial groups and high costs

Inactive Publication Date: 2017-04-19
DR JOSEP TRUTTA GIRONA BIOMEDICAL RES INST FOUNDATION +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the main disadvantages associated with this technique are: higher cost (because of the need for trained personnel), risk of anesthesia-related death and risk of bowel perforation (Garborg K. et al., Ann Oncol., 2013;
[0019] However, it is well known in the art that there are differences in microbiota between mucosal biopsies and stool samples

Method used

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  • Method for diagnosing colorectal cancer from human feces sample by quantitive pcr, primers and kit
  • Method for diagnosing colorectal cancer from human feces sample by quantitive pcr, primers and kit
  • Method for diagnosing colorectal cancer from human feces sample by quantitive pcr, primers and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0181] Example 1.- Materials and methods

[0182] 1. Biological samples

[0183] Stool samples were obtained from 27 patients with recently diagnosed colorectal cancer (CRC) (stages 0-I), 24 Lynch syndrome carriers (L) and 19 healthy individuals (C). All patients underwent colonoscopy at least 15 days before sample collection.

[0184] All personnel signed the corresponding informed consent. Exclusion criteria included antibiotic treatment within 1 month before the study and age <18 years.

[0185] 2.16S rDNA bacterial sequence:

[0186] B3, B10, B41, B46, B48 and B50 sequences correspond to denaturing gradient gel electrophoresis (DGGE) gel bands previously isolated from uncultured bacterial isolates.

[0187] -16S rDNA bacterial sequence SEQ ID NO: 1 (B3), published as GQ411111.1 in the EMBL-EBI European Nucleotide Archive (ENA) database;

[0188] -16S rDNA bacterial sequence SEQ ID NO: 4 (B10), published as GQ411118.1 in the EMBL-EBI European Nucleotide Archive (ENA)...

Embodiment 2

[0216] Example 2: Primer Design and Verification

[0217] Primers for the quantification of bacterial markers B3, B10, B46, B48, B41 and B50 in biopsies were designed based on comparative analysis with sequences previously obtained from phylogenetic groups using the following bioinformatic tools: Bioinformatics from Europe ClustalX (European Bioinformatics Institute (www.ebi.ac.uk)), Netprimer ((Premier Biosoft) and PrimerExpress (Life Tecnologies-Thermo Fischer).

[0218] The detection system chosen is Primer sets with less than 3 different positions relative to adjacent sets were discarded, as were primer sets with Tm values ​​in dissociation curves different from the expected value. Primer validation was performed in biopsy samples and stool samples. Dissociation curves were measured to analyze primer performance.

[0219] exist Figures 8a to 13b In , the amplification curves and dissociation curves of the primers designed to amplify B3, B10, B46, B48, B41 and B50 a...

Embodiment 3

[0222] Example 3: Analysis of 16S rDNA biomarkers in colorectal cancer patients VS healthy subjects

[0223] A total of 16 stool samples from 7 controls and 9 patients with colorectal cancer were analyzed. The quantitative analysis of each bacterial marker as described in Example 1 in the analysis of stool samples was expressed as Ct value. Ct (cycle threshold) was defined as the number of q-PCR cycles required for the fluorescence signal to exceed the threshold. The Ct level is inversely proportional to the logarithm of the target nucleic acid concentration in the sample (ie, the lower the CT level, the higher the amount of target nucleic acid in the sample). Real-time analysis was performed over 40 amplification cycles. The results obtained are shown in Tables 1 and 2 below. Table 1 is the absolute value of Ct, and Table 2 is the ratio of Ct. A two-sample t-test was used.

[0224] Table 1: Absolute value of Ct

[0225]

[0226]

[0227] *Determination of accuracy...

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Abstract

The present invention relates to the field of detection of colorectal cancer (CRC), Specifically it relates to methods for the early detection, risk screening and monitoring of CRC and / or adenomatous polyps in a human subject based on the quantification of one or more 18S rDNA bacterial sequences in feces. It further relates to the use of said bacterial sequences as biomarkers of colorectal cancer and / or adenomatous polyps; to a kit comprising a reagent and instructions for the quantification of said bacterial sequences; and to nucleic acids for the quantification of said 16S rDNA bacterial sequences.

Description

technical field [0001] The present invention relates to the field of colorectal cancer (CRC) detection. In particular, it relates to methods for early detection, risk screening and monitoring of CRC and / or adenomatous polyps in human subjects based on quantified levels of one or more 16S rDNA bacterial sequences in stool. It also relates to the use of said bacterial sequence as a biomarker for CRC and / or adenomatous polyps; to a kit comprising reagents and instructions for quantifying said bacterial sequence; and to a method for quantifying said 16S rDNA Nucleic acid of bacterial sequences. Background technique [0002] Colorectal cancer (CRC) is the second leading cause of cancer death in Europe and the United States, and is the most frequently diagnosed cancer in Europe, with more than 400,000 new cases and 200,000 deaths in 2008. These data point to the need for precautionary measures. [0003] The most effective and cost-effective measures to reduce CRC morbidity and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/689C12Q2600/118C12Q2600/158C12Q1/6886C12Q1/6806
Inventor 马里奥娜·萨拉·佩吉斯杰西·加西亚-吉尔特里萨·马斯·德·萨萨尔斯赛维尔·奥尔迪格尔
Owner DR JOSEP TRUTTA GIRONA BIOMEDICAL RES INST FOUNDATION
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