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Extraction-free reagent kit for detecting gene polymorphism of human APOE and SLCO1B1

A gene polymorphism and kit technology, applied in the field of molecular biology, can solve the problems of increasing clinical testing steps, man-hours, costs, operational errors, safety, etc., to control pollution and monitor the entire experimental process, prevent pollution, The effect of simple detection process

Pending Publication Date: 2020-04-03
苏州天隆生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fluorescent PCR molecular diagnostic products currently on the market need to separate and purify the nucleic acids in whole blood and test samples before performing nucleic acid detection on whole blood samples and test samples, which not only increases clinical testing steps, man-hours, and costs, but also In the process of extraction and purification, there are still operational errors and safety problems

Method used

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  • Extraction-free reagent kit for detecting gene polymorphism of human APOE and SLCO1B1
  • Extraction-free reagent kit for detecting gene polymorphism of human APOE and SLCO1B1
  • Extraction-free reagent kit for detecting gene polymorphism of human APOE and SLCO1B1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1. kit preparation

[0063] The main components of the main reaction solution are DNA polymerase, four deoxyribonucleotides, Tris buffer, Mg 2+ , Ultra-pure BSA.

[0064] 1. Main reaction formula:

[0065] 1.1: The raw material of DNA polymerase is Taq enzyme, the concentration is 2U-5U.

[0066] The raw material of DNA polymerase is purchased Taq enzyme, and the purchased suppliers include TOYOBO (product name TTX) and Xiamen Tongrenxin (product name HS-D-Taq). These two enzymes are highly resistant to interference and high amplification efficiency, and can be used for fluorescent PCR amplification.

[0067] Enzyme usage test optimization in the platform system.

[0068] Test method: the total system is 20μl, the enzyme usage is 0.5U-5U, and the optimal enzyme usage is 2U-5U. For usage see figure 1 and figure 2 .

[0069] figure 1 It is the test result of optimizing the amount of HS-D-Taq enzyme. The amount of enzyme used is 0.5U-5U, which can be a...

Embodiment 2

[0160] Example 2. Kit whole blood sample test

[0161] Selected 9 cases of known APOE gene SNP sites rs429358 (c.388T>C) and rs7412 (c.526C>T), SLCO1B1 gene SNP sites (c.388A>G) and (c.521T>C) types Different whole blood samples were detected according to the operation steps in the detection method of the present invention, and the detection results can be found in Figure 7-Figure 24

[0162] The known genotypes of the samples and the genotypes detected by the kit of the present invention are shown in Table 15 below.

[0163] Table 15 Test result of kit of the present invention and known result contrast

[0164]

[0165] Conclusion: By analyzing 9 cases of known APOE gene SNP sites rs429358 (c.388T>C) and rs7412 (c.526C>T), SLCO1B1 gene SNP sites (c.388A>G) and (c.521T>C ) type of whole blood samples for extraction-free detection, and through the comparative analysis of the interpretation results in Table 15, the detection results of the kit of the present invention are...

Embodiment 3

[0166] Example 3. Comparison of detection results after nucleic acid extraction and extraction-free detection results

[0167] Experimental process: Select 4 known APOE gene SNP sites rs429358 (c.388T>C) and rs7412 (c.526C>T), SLCO1B1 gene SNP sites (c.388A>G) and (c.521T>C ) type whole blood samples were divided into 2 groups, one group of nucleic acid extraction and purification kit was used for nucleic acid extraction and purification, and the other group was directly detected according to the operation steps in the detection method of the present invention without nucleic acid extraction. For the detection results, see Figure 25-Figure 40 , see Table 16 for the specific interpretation results.

[0168] Table 16 Comparison of nucleic acid extraction and extraction-free detection results

[0169]

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Abstract

The invention discloses an extraction-free reagent kit for detecting gene polymorphism of human APOE and SLCO1B1. The reagent kit comprises main reaction liquor, ApoE primer and probe mixed liquor 1,ApoE primer and probe mixed liquor 2, SLCO1B1 primer and probe mixed liquor 1, SLCO1B1 primer and probe mixed liquor 2, positive quality control and feminine quality control, wherein the main reactionliquor comprises DNA polymerase, four kinds of deoxyribonucleotide, Mg2+, Tris buffer liquor and ultrapure BSA, and the DNA polymerase includes TTX enzymes and / or HS-D-Taq enzymes. The reagent kit can directly complete test on whole blood samples and swab samples through a unique amplification system, and a primer and probe sequence and system taking account of specificity and sensitivity, the detection is completed in four reaction system closed pipes, so that pollution can be avoided; and in addition, the feminine quality control and the positive quality control in the reagent kit can effectively monitor false positivity and validity of reagents and operations, and can effectively control pollution and monitor the whole experiment process.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to an extraction-free kit for detecting human APOE and SLCO1B1 gene polymorphisms. Background technique [0002] Statins are currently the most effective drugs for lowering low-density lipoprotein cholesterol (LDL-C) levels and thereby reducing the risk of cardiovascular disease. However, different individuals respond differently to different statins because of differences in the genetic characteristics of statin metabolism and transport in the liver. In particular, key transporters involved in the hepatic metabolism of statins, such as anion transporting polypeptide (OATP1B1) (encoded by the SLCO1B1 gene) and apolipoprotein E (Apolipoprotein E, ApoE) gene polymorphisms can affect the plasma and liver concentration, thereby affecting the efficacy and safety of statins. Changes in the risk of myopathy or the ability to lower LDL-C of statins will change the benefit-risk ratio of th...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883
CPCC12Q1/6883C12Q2600/156C12Q2600/106
Inventor 贾慧珍苗保刚李明李红东王小舟
Owner 苏州天隆生物科技有限公司
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