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Aminoglycosides related 12s rRNA gene mutation site detection kit

A technology of aminoglycosides and detection kits, applied in DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problem of lack of rapid and effective detection of mitochondrial 12SrRNA gene, etc., to reduce the risk of repeated freezing and thawing , short time-consuming, highly specific effect

Inactive Publication Date: 2018-09-28
宁波美丽人生医药生物科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently a lack of rapid and effective methods for the detection of mitochondrial 12S rRNA gene

Method used

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  • Aminoglycosides related 12s rRNA gene mutation site detection kit
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  • Aminoglycosides related 12s rRNA gene mutation site detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] An aminoglycoside drug-related 12s rRNA gene polymorphism detection primer composition, including polymorphism detection primers and internal control gene detection primers, and its sequence composition is shown in Table 1 and Table 2 below.

[0067] Table 1 12s RRNA gene polymorphism detection primers

[0068] Primer name

Base sequence (5'→3' direction)

P1-FW1 (i.e. polymorphism primer one)

5′-TACGCATTTA TATAGAGGATA-3′

P1-FW2 (i.e. polymorphic primer 2)

5′-TACGCATTTA TATAGAGGACG-3′

P1-R (i.e. polymorphic primer three)

5′-GTGCACTTGGACGAACCAGA-3′

P2-FW1 (i.e. polymorphic primer four)

5′-GCGTACACAC CGCCCGTCAAC-3′

P2-FW2 (i.e. polymorphic primer five)

5′-GCGTACACACCGCCCGTCAGT-3′

P2-R (i.e. polymorphic primer six)

5′-TAAATGCGTAGGGGTTTTAG-3′

[0069] Table 2 Internal control gene detection primers

[0070] Primer name

Embodiment 2

[0072] Such as figure 1 As shown, an aminoglycoside drug-related gene 12srRNA gene mutation detection kit equipped with the primer composition described in Example 1 includes: a box body 1 with a box cover, a baffle 3, two sponges 2, four One test tube with distilled water 4, four test tubes with PCR reaction master mix 5, one test tube with positive quality control 12s rRNA gene A1555G mutant plasmid 6, one test tube with positive quality control 12s rRNA gene A1555G wild plasmid 7, One test tube 8 containing positive quality control 12s rRNA gene C1494T mutant plasmid, one test tube 9 containing positive quality control 12s rRNA gene C1494T wild plasmid, two test tubes 10 containing internal control gene detection primers, three containing 12s rRNA Test tube 11 of gene A1555G detection primer, three test tubes 12 containing 12s rRNA gene C1494T detection primer. Wherein, the baffle 3 is placed in the box body 1, and the box body 1 is divided into two independent cavity part...

Embodiment 3

[0075] The operation method of the kit described in the above-mentioned embodiment 2 is:

[0076] (1) Sample processing and nucleic acid extraction

[0077] Use a commercial DNA extraction kit to process the sample. For specific operations, refer to the kit instruction manual. The concentration and purity of the extracted DNA need to be measured with a UV spectrophotometer. The DNA OD 260 / OD 280 The value should be between 1.8 and 2.0, and the concentration should be between 5 and 50 ng / μL. If the quality of the sample DNA is unqualified, it should not be used for detection. It is recommended that the nucleic acid extraction should be performed again if the quality of the sample DNA is lower than 5ng / μL, and if it is higher than 50ng / μL, it should be properly extracted. Dilute to the specified concentration range, and it is recommended to test the extracted DNA immediately, otherwise, please store it below -20°C, and the storage time should not exceed 6 months. The kit qual...

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Abstract

The invention relates to an aminoglycosides related 12s rRNA gene mutation site detection kit, in particular to a detection primer composition, a kit and a method for an aminoglycosides related 2s rRNA gene polymorphism mutation site. The primer composition comprises polymorphism detection primers and internal control gene detection primers, wherein sequences of the polymorphism detection primersare as follows: a first polymorphism primer: 5'-TACGCATTTA TATAGAGGATA-3'; a second polymorphism primer: 5'-TACGCATTTA TATAGAGGACG-3'; a third polymorphism primer: 5'-GTGCACTTGG ACGAACCAGA-3'; a fourth polymorphism primer: 5'-GCGTACACAC CGCCCGTCAAC-3'; a fifth polymorphism primer: 5'-GCGTACACAC CGCCCGTCAGT-3'; a sixth polymorphism primer: 5'-TAAATGCGTA GGGGTTTTAG-3'; sequences of the internal control gene detection primers are as follows: a first internal control primer: 5'-AGCAAGCAGG AGTATGACG-3'; a second internal control primer: 5'-GAAAGGGTGT AACGCAACT-3'. Compared to the prior art, the aminoglycosides related 12s rRNA gene mutation site detection kit has the advantages of high sensitivity, high specificity, short time consumption and the like in the process of detecting 12s rRNA gene mutation.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a primer composition, a kit and a method for detecting mutation sites of 12s rRNA gene polymorphisms related to aminoglycoside drugs. Background technique [0002] Aminoglycoside antibiotics are commonly used antibiotics in clinical practice. They have some common characteristics in molecular structure, metabolism in vivo, antibacterial spectrum and toxic and side effects. Its main side effect is aminoglycoside antibiotics can cause deafness (AAID). Aminoglycoside antibiotics damage the cells of the 8th cranial nerve, vestibule and cochlea causing deafness. AAID patients can be divided into two categories, one is deaf due to receiving toxic doses of aminoglycoside antibiotics, most of these patients have no genetic background. Another type of deafness has been caused by regular doses or single doses of aminoglycoside antibiotics, and these patients have a genetic family history. [...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/106C12Q2600/156
Inventor 田晓丽
Owner 宁波美丽人生医药生物科技发展有限公司
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