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DNA methylation biomarker panels, detection methods and kits

A methylation marker and detection kit technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial assay/inspection, etc. Low protein content, etc., to achieve the effect of simple and feasible detection method, optimized reaction components, and high amplification efficiency

Active Publication Date: 2021-08-10
ANCHORDX MEDICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, cystoscopy combined with biopsy pathology is the gold standard for cystoscopy diagnosis, but this inspection method is invasive, prone to complications, and patient compliance is low
However, imaging examinations have limited diagnostic ability for small lesions, urine exfoliation cytology examination has low sensitivity, urine FISH detection is complicated, and interpretation results are subjective.
Existing tumor marker examinations are mainly based on the presence of specific proteins in urine, but due to the low content of protein in urine, the sensitivity and specificity are still limited

Method used

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  • DNA methylation biomarker panels, detection methods and kits
  • DNA methylation biomarker panels, detection methods and kits
  • DNA methylation biomarker panels, detection methods and kits

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0172] Used for bladder cancer detection, diagnosis, classification, or prediction, treatment monitoring, prognosis, or other equivalency region evaluated bladder cancer, including multiple methyl groups indicated by [CG] in the nucleic acid sequence listed in Table 1 A common methylation of the azimeticization and a plurality of methylation sites that are fully complementaryly complementary to the nucleic acid indicated by [CG] in the sequence in the sequence.

[0173] Table 1 Aminomelated composition of DNA methylation region

[0174]

[0175]

[0176]

Embodiment 2

[0178]A common methylation test kit for bladder cancer detection, diagnosis, classification, or prediction, treatment monitoring, prognosis, or other evaluation bladder cancer, including multiple methylated regions Specific primer pairs and probes, as shown in Table 2:

[0179] Table 2-1 Plastics and probe sequence combinations of 22 methylated regions

[0180]

[0181]

[0182]

[0183] Table 2-2 Plants of 22 methylated regions Aminomelated primer and probe sequence combination 2

[0184]

[0185]

[0186] Table 2-3 Plastics and probe sequence combinations of 22 methylated regions

[0187]

[0188]

[0189]

[0190] In practical applications, the corresponding primers and probes will be selected according to a combination of specific methylation regions.

[0191] Interior primers and probes:

[0192]

Embodiment 3

[0196] Multi-fluorescence quantitative PCR for among methylgenization detection of 2-3 methylated regions

[0197] 22 methylation regions (SEQ ID NO.1-22) were performed using commercially complete methylation (positive controls) and non-methylation (negative control) standards (purchased from Qiagen) to each 2-3 Aminimalized detection of a methylated region.

[0198] The specific process is as follows:

[0199] 1, DNA extraction

[0200] Extraction kits purchased from QIAGEN, follow the kit instructions.

[0201] 2, DNA bisulfite

[0202] DNA bisulfite conversion kits were purchased in Zymo, followed by the kit specification.

[0203] 3, multiple PCR amplification

[0204] The primer pair of 22 methylation regions (SEQ ID No. 1-8) is used, and multiple PCRs in 1 reaction hole (primer sequence are shown in Table 2), and the target sequence containing the target region is amplified, the product size is Around 70-130 bp.

[0205] 1) Configure a single primer concentration of 5 μm (e...

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Abstract

The present invention relates to a combination of DNA methylation markers for bladder cancer detection, which is selected from any combination of two or more of SEQ ID NO.1 to SEQ ID NO.22, or selected from SEQ ID Any combination of two or more completely complementary sequences from NO.1 to SEQ ID NO.22. The present invention also relates to a detection kit for the combination of the above methylated markers. The invention utilizes the combination of the co-methylation states of multiple specific methylated regions to discriminate and analyze the occurrence of bladder cancer. The specific methylation combination has high sensitivity for discriminating the occurrence of bladder cancer, and the detection method is simple and feasible. The primer pair combination of this kit overcomes the shortcomings of false positives caused by mismatches in the detection of single methylation sites in the design of primer sequences, and considers the interaction between multiple methylation biomarker primers and probe pair combinations. effect.

Description

Technical field [0001] The invention belongs to the biotechnology field, and more particularly to a combination of DNA methylated biomarkers and detection kits and detection methods. Background technique [0002] Bladder cancer is one of the most common malignant tumors in the urinary system. In China's bladder cancer, the number of vulnetruals in 2014 is 78,000 / year, and the incidence has increased year by year. Bladder cancer has the characteristics of high incidence and easy recurrence. His common clinical symptoms of hematuria are bladder cancer, of which about 17% of hematuria is detected bladder cancer. At present, the main diagnostic method of bladder cancer includes a blood cystoscopy, urinary decencemental cell examination, urine FISH detection, and tumor marker examination. Among them, cystoscopy combined with the pathology of biopsy tissue as the diagnosis of cystoscopy, but the inspection method has invasive, easy to produce complications, and the patient is low dep...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/112C12Q2600/118C12Q2600/154C12Q2600/16C12Q2600/166C12Q2523/125C12Q2537/143C12Q2563/107C12Q2531/113C12Q2545/101
Inventor 阮微媚蒋泽宇李霞陈志伟范建兵
Owner ANCHORDX MEDICAL CO LTD