Method for quickly and accurately detecting copy number variation of CYP2D6 gene

Abstract

The invention discloses a method for quickly and accurately detecting a copy number variation of a CYP2D6 gene. The method comprises the steps of 1, preparing a primer which detects CYP2D6 gene polymorphism; 2, preparing a primer and a probe of a RPP30 gene; 3, conducting fluorescent quantitation PCR reaction, wherein a method of double quantitating PCR through a single tube is adopted to detect the copy number variation of the CYP2D6 gene; 4, taking the CYP2D6 gene as a sample of a single copy so as to be a check sample, wherein four repetitive PCR reactions are conducted on a detecting object and the check sample respectively to obtain a Ct value of real-time quantification PCR amplification; 5, analyzing a fluorescent quantitation PCR result. According to a real-time quantification fluorescence PCR detecting method, no PCR aftertreatment is conducted, and thus time and manpower can be greatly saved. From DNA extraction to the end of fluorescence PCR experience and data analysis, the whole process only takes 4 hours, and entity experiment operating time is less than 2 hours.

Description

technical field [0001] The present invention relates to a method for determining the copy number change of CYP2D6 gene from a small group or individual. The method can detect the copy number change of CYP2D6 gene quickly, efficiently and accurately, and finds applications in biology and medicine. Background technique [0002] CYP2D6 is the first P450 enzyme confirmed to be controlled by a single gene. CYP2D6 is located on chromosome 22, contains 9 exons and 8 introns, and has a total length of about 5400bp. It is a complete functional gene. Modern studies have found that the content of CYP2D6 in the liver only accounts for 2% of the total P450 liver protein, but it participates in the metabolism of about 25% of clinical drugs, including antidepressants, antiarrhythmics, antipsychotics and analgesics, etc. . So far, more than 100 CYP2D6 allelic variations have been found, mainly including single nucleotide variations, loss of large fragments of genes, and copy number variat...

AI Technical Summary

Problems solved by technology

[0005] At present, domestic and foreign products for detecting CYP2D6 gene mutations, such as Luminex's xTAG liquid phase chip, Roche's AmpliChip CYP450 chip, direct sequencing method, PCR-single-strand conformation polymorphism (SSCP) detection, etc., are not very sensitive. poor repeatability

These methods require a lot of manpower, and at the same time, the detection time is relatively long. It takes more than 8 hours from nucleic acid extraction to result acquisition, which is more than 1 day of normal working time.

In addition, none of these methods can quickly and accurately determine the copy number changes of the CYP2D6 gene

At present, the more accurate methods for identifying CYP2D6 gene copy number changes include digital PCR technology (patent CN 101821619 B) and standard curve method technology based on quantitative PCR (patent CN 104263820 A), but the two methods have high reagent costs and are not suitable for promotion

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