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Kit for rapidly detecting SLCO1B1 gene mutation and detection method

A detection method and kit technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of poor specificity and low detection accuracy, and achieve the effect of easy operation, simple equipment requirements, and low cost

Inactive Publication Date: 2017-08-18
安徽安龙基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve the defects of low detection accuracy and poor specificity in the prior art, and provide a kit and detection method for rapidly detecting SLCO1B1 gene mutation to solve the above problems

Method used

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  • Kit for rapidly detecting SLCO1B1 gene mutation and detection method
  • Kit for rapidly detecting SLCO1B1 gene mutation and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] (1), extract blood DNA, and dilute to 100ng / ul;

[0042] (2) Take 20ul ARMS-PCR reaction system, specifically including: 10×PCR Buffer 2μl, FI 1μl, RI 1μl, FO 5μl, RO 5μl dNTP mixture 1.6μl, Taq DNA polymerase 0.4μl, Mg2+1.6μl, DNA Template 1μl, add ddH2O to 20μl;

[0043] (3) Put the mixed reaction solution into the PCR instrument, set the program as follows: pre-denaturation at 95°C for 10 minutes, then enter 1 cycle, denaturation at 95°C for 15s, annealing at 45°C for 45s, extension at 72°C for 45s, a total of 32 cycles Cycle, and then continue to extend at 72° C. for 8 min, 1 cycle, and then analyze the PCR reaction product by gel electrophoresis using 3% agarose gel for gel electrophoresis analysis.

[0044] The present invention initially designed 4 sets of primers for primer screening, the specific sequences are as follows:

[0045] FI-1: 5'-TCTTATCTACATAGGTTGTTTAAAGGATTC-3'

[0046] RI-1:5'-ACCATATATCCACATGTATGACCGAC-3'

[0047] FO-1: 5'-CAGTCTCAGGTATGTATTTA...

Embodiment 2

[0063] (1), extract blood DNA, and dilute to 100ng / ul;

[0064] (2) Take 20ul ARMS-PCR reaction system, specifically including: 10×PCR Buffer 2μl, FI 1μl, RI 1μl, FO 5μl, RO 5μl dNTP mixture 1.6μl, Taq DNA polymerase 0.4μl, Mg2+1.6μl, DNA Template 1μl, add ddH2O to 20μl;

[0065] (3) Put the mixed reaction solution into the PCR instrument, set the program as follows: pre-denaturation at 95°C for 10 minutes, then enter 1 cycle, denaturation at 95°C for 15s, annealing at 45°C for 45s, extension at 72°C for 45s, a total of 32 cycles Cycle, and then continue to extend at 72° C. for 8 min, 1 cycle, and then analyze the PCR reaction product by gel electrophoresis using 3% agarose gel for gel electrophoresis analysis.

[0066] PCR reaction products were analyzed by gel electrophoresis using 3% agarose gel.

[0067] The ratio of endogenous primer and exogenous primer in embodiment 2 is 1:1.

Embodiment 3

[0069] (1), extract blood DNA, and dilute to 100ng / ul;

[0070] (2) The optimal reaction system for ARMS-PCR is 20ul, specifically including: 10×PCR Buffer 2μl, FI 1μl, RI 1μl, FO 10μl, RO 10μl dNTP mixture 1.6μl, Taq DNA polymerase 0.4μl, Mg2+1.6μl, DNA template 1μl, add ddH2O to 20μl;

[0071] (3), put the mixed reaction solution into the PCR instrument, the setting procedure is as follows:

[0072]

[0073] PCR reaction products were analyzed by gel electrophoresis using 3% agarose gel.

[0074] The ratio of endogenous primer and exogenous primer in embodiment 2 and 3 is respectively 1:1 and 1:10, all do not obtain ideal result, so applicable endogenous primer and exogenous primer ratio in the present invention are 1:5 .

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Abstract

The invention discloses a kit for rapidly detecting SLCO1B1 gene mutation and a detection method of the kit. The kit comprises Taq DNA polymerase, 10*PCR reaction buffer, MgCl2, dNTPs, an endogenous upstream primer FI, an endogenous downstream primer RI, an exogenous upstream primer FO and an exogenous downstream primer RO. The invention further discloses the detection method of the kit for rapidly detecting SLCO1B1 gene mutation. The detection method comprises the following specific steps: blood DNA is extracted and diluted to 100 ng / mu l; a 20 mu l ARMS-PCR reaction system is taken; a mixed reaction liquid is placed into a PCR instrument, and a setting procedure is performed as follows: initial denaturation is performed for 10 min at 95 DEG C, then one cycle is performed, denaturation is performed for 15 s at 95 DEG C, annealing is performed for 45 s at 45 DEG C, extension is performed for 45 s at 72 DEG C, 32 cycles are totally performed, then extension is performed for 8 min at 72 DEG C, one cycle is performed, and then a PCR reaction product is subjected to gel electrophoresis analysis. The kit and the detection method have the advantages of high detection accuracy, high specificity and the like.

Description

technical field [0001] The invention relates to the field of biological technology, in particular to a kit and a detection method for rapidly detecting SLCO1B1 gene mutation. Background technique [0002] Hyperbilirubinemia is the most common clinical condition in newborns, 8%-11% of newborns will have severe bilirubinemia, but the total serum bilirubin (TSB) rises to a very high level When it occurs, it will lead to long-term diseases, including heme encephalopathy and kernicterus. Therefore, it is very important to be aware of the severity of neonatal hyperbilirubinemia. There are many factors that cause hyperbilirubinemia. Includes ABO, Rh incompatibility, deficiency of glucose-6-phosphate dehydrogenase (G6PD) and pyruvate dehydrogenase, hereditary spherocytosis, defective hemoglobin synthesis, hypothyroidism, breast milk jaundice, intracranial hematoma Wait. [0003] UDP glycosyltransferase family 1, polypeptide A1 (UGT1A1), organic anion transporting polypeptide 2 (OA...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6883C12Q1/6858C12Q2600/156C12Q2535/137
Inventor 韦玉军李航李静苏军吴远航
Owner 安徽安龙基因科技有限公司
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