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Kit and method for detecting aminoglycoside drug-induced deafness-sensitive gene

A technology of drug-induced deafness and aminoglycosides, which is applied in the fields of biochemical equipment and methods, and microbial measurement/inspection, and can solve the problems of long operating time of microarray chip products, inability to perform quality control, and the need for special instruments, etc. Achieve the effect of high cost, fast detection and low cost

Active Publication Date: 2013-02-13
亚能生物技术(深圳)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Disadvantages common to common PCR products: the genotype needs to be judged by the size of the band when interpreting the results, which lacks intuition
[0015] To sum up: the existing common methods, such as fluorescent quantitative PCR method for gene detection, only detect a single mutation site on the mitochondria, and most fluorescent quantitative PCR methods have no internal standard and cannot be used for quality control; the operating time of microarray state chip products Long, high cost, low accuracy and requires special equipment
Large-scale screening of mitochondrial gene mutations requires fast, low-cost, and wide detection range, which cannot be met by the above products at the same time

Method used

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  • Kit and method for detecting aminoglycoside drug-induced deafness-sensitive gene
  • Kit and method for detecting aminoglycoside drug-induced deafness-sensitive gene
  • Kit and method for detecting aminoglycoside drug-induced deafness-sensitive gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] The preparation and use of embodiment 1 kit of the present invention

[0063] The kit of the invention comprises PCR reaction solution and quality control products. The PCR reaction solution includes Taq enzyme, DNA double-stranded chimeric dye, amplification primer, amplification buffer, dN(U)TP, etc., and dN(U)TP is the dNTP mixture. The PCR reaction program is run on a fluorescent PCR instrument and the detection results are read.

[0064] 1. Design of primers for the detection of susceptibility genes for aminoglycoside-induced deafness

[0065] According to the principle of site-specific primer design, the mutation site was set as the 3' end of the primer, and point mutations were introduced at the 2nd, 3rd, and 4th positions of the 3' end to improve specificity. Finally, 5 usable site-specific primers were selected from 20 primers, including two for 1555 site and three for 1494 site. The position of the mutation site is crucial to the specificity of the primer. ...

Embodiment 2

[0092] The use effect of embodiment 2 kit of the present invention

[0093] 1, the comparison of kit of the present invention and existing similar technology

[0094] Compared with the prior art, the present invention establishes a screening method for sites of aminoglycoside antibiotic drug-induced deafness, which is suitable for rapid detection and mass screening. Only one tube reaction can detect two mitochondrial mutation sites popular in the Chinese population, avoiding the problems of cumbersome operations, cross-contamination, and low accuracy.

[0095] 2. Detection of C1494T and A1555G mutations in clinical samples using the kit of the present invention

[0096] This kit was used to detect 200 samples from the general population, and to detect the C1494T and A1555G mutations in clinical samples. All samples were sequenced and verified by the gold standard sequencing method. The detection results and sequencing results of the kit of the present invention and their comp...

Embodiment 3

[0120] Embodiment 3 A specific embodiment of the kit of the present invention

[0121] 1. The main components of the kit of the present invention are as shown in Table 6

[0122]

[0123] Table 6

[0124] 2. Applicable instruments

[0125] Including ABI Prism 7300 / 7500 / 7700 / 5700 / 7000 / 7900, Roche LightCycler 480 and other fluorescent PCR amplification detectors.

[0126] 3. Storage conditions and validity period

[0127] Storage conditions: The kit should be stored at -20°C protected from light.

[0128] Validity period: 6 months.

[0129] 4. Sample requirements

[0130] (1) Sample type

[0131] Dried blood film, anticoagulated peripheral blood, oral swab.

[0132] (1)Sample storage

[0133] Store at -20°C for no more than one year.

[0134] (2) Specimen transportation

[0135] Dried blood spots and oral swabs can be transported at room temperature, and blood samples are transported sealed with ice.

[0136] (3) Sample size

[0137] The upper limit of detection i...

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Abstract

The invention relates to the field of gene detection, and particularly relates to a kit and method for detecting an aminoglycoside drug-induced deafness-sensitive gene through a fluorescent PCR (polymerase chain reaction)-melting curve method. The technical scheme of the invention provides a kit for detecting an aminoglycoside drug-induced deafness-sensitive gene. The kit comprises a PCR solution, a negative quality control sample and a positive quality control sample, wherein the PCR solution comprises primers, a PCR Buffer, a dNTP (deoxyribonucleotide triphosphate) mixed solution, saturated fluorescent dyes, a Taq enzyme and a UNG enzyme. In the invention, the specific PCR primers are designed according to SNP (single nucleotide polymorphism) sites of the aminoglycoside drug-induced deafness-sensitive gene, two aminoglycoside drug-induced deafness-sensitive sites can be detected in one PCR, the operation can be finished in one tube, and the detection is quick, accurate and wide in range.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a kit and a method for detecting aminoglycoside drug-induced deafness susceptibility genes by fluorescent PCR-melting curve method. Background technique [0002] According to my country's second national sampling survey of disabled people in 2006, there were 20.04 million people with hearing disabilities, accounting for 24.16% of the country's 82.96 million disabled people. Among them, drug-induced deafness accounted for 30%-40%, and the number reached 8 million. Drug-induced deafness refers to the deafness caused by the use of certain drugs to treat diseases or the exposure of the human body to certain chemical agents. More than 100 ototoxic drugs have been found so far, and ototoxic antibiotics are the most common. Aminoglycoside antibiotics are the most common ototoxic antibiotics, including streptomycin and gentamicin commonly used in clinical practice. In the past ten years, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 王小薇刘晶晶危林耿任维
Owner 亚能生物技术(深圳)有限公司
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