Multiplex nested methylation specific PCR (Polymerase Chain Reaction) detection kit, using method and application thereof

Abstract

The invention discloses a multiplex nested methylation specific PCR (Polymerase Chain Reaction) detection kit which comprises the following substances: a primer, of which the nucleotide sequence is shown as SEQ ID NO.1-42, and a PCR working solution. A using method for the multiplex nested methylation specific PCR detection kit comprises the steps of obtaining serum of a patient to extract DNA; (2) obtaining a DNA solution to carry out methylation beautification; (3) carrying out PCR detection by using the kit; and (4) judging the result, that is, taking an obtained product, carrying out agarose gel electrophoresis detection, putting gel in a gel imaging system for shooting and analysis, and judging the reaction result by naked eyes. The defects of low sensitivity and low specificity of serum free DNA trace and single methylation markers are overcome to the greatest extent by the multiplex nested methylation specific PCR (MN-MSP) detection technology disclosed by the invention, the methylation states of seven target genes are efficiently detected, the multiplex nested methylation specific PCR detection kit has the advantages of strong specificity, high accuracy and the like and can be used as an effective diagnosis and detection means for early epithelial ovarian cancers.

Description

technical field [0001] The invention relates to a multiple nested methylation-specific PCR (MN-MSP) detection kit and its application method, as well as its application in the detection of ovarian cancer, especially early ovarian cancer. Background technique [0002] At present, the incidence rate of ovarian cancer ranks third among malignant tumors of the female reproductive system, and the mortality rate ranks first, accounting for 3% of all female patients who died of cancer. More than 90% of ovarian cancers are epithelial ovarian cancers, and the onset is insidious. About 80% of the patients are in the advanced stage when they are diagnosed clinically, and the prognosis is poor. The 5-year survival rate of patients with advanced ovarian cancer (FIGO stage II-IV) is only 30-45%, while the 5-year survival rate of patients with early stage ovarian cancer (FIGO stage I) is as high as 90%. Therefore, in addition to actively looking for new and effective treatment methods, it...

AI Technical Summary

Problems solved by technology

The multiplex nested methylation-specific PCR (MN-MSP) detection technology of the present invention combines multiplex PCR (Multiplex PCR), nested PCR (Nested PCR) and methylation-specific PCR (MSP) for the first time at home and abroad. In combination, use the primer design software developed by the research group to design brand-new PCR primers, and simulate the entire PCR process to avoid the formation of primer dimers and hairpin structures during the PCR process. anneal) method, to ensure the specificity of primer-template binding, to overcome the shortcomings of the trace amount of free serum DNA and the sensitivity and low specificity of a single methylation marker to the greatest extent, and can achieve a minimum of 1 / 8000ug DNA (approx. Efficient detection of the methylation status of 7 target genes in 19 genomic DNAs), and has been repeatedly verified by clinical samples, which provides a strong supplement to the existing detection technology and becomes an effective means of early ovarian cancer diagnosis and detection

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