Rapid detection kit for CYP3A5*3 genotype based on POCT mode

A kit and genotype technology, applied in the field of molecular biology, can solve the problems of affecting the accuracy of results, environmental pollution, unsuitability for clinical promotion, etc., and achieve the effects of avoiding medication errors, simple operation, and high detection sensitivity.

Inactive Publication Date: 2019-01-22
重庆京因生物科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This kind of open operation is easy to cause environmental pollution and affect the accuracy of the results, so it is not suitable for clinical promotion
The current convent

Method used

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  • Rapid detection kit for CYP3A5*3 genotype based on POCT mode
  • Rapid detection kit for CYP3A5*3 genotype based on POCT mode
  • Rapid detection kit for CYP3A5*3 genotype based on POCT mode

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1 Cell lysate performance test

[0084] PCR is an extremely sensitive technique for the detection of trace amounts of DNA. At present, the main samples of painless and non-invasive detection methods in clinical practice include oral swabs, hair, nails, oral saliva and body cavity fluid, etc. After the contained DNA is extracted and purified, it can be added to the PCR reaction system for reaction, which requires a lot of manpower, material resources, and financial resources. In order to solve this problem, and considering that the amplification effect after directly adding cells is not ideal, and it is not suitable for clinical testing, the inventors tried to add a certain concentration of cell lysate. And the comparative experiment of adding cell lysate and not adding cell lysate was carried out. (Note: Exfoliated cells are used as templates)

[0085] The reagent formula is shown in Table 1-1, and 100 replicates were prepared for each of the three different ...

Embodiment 2

[0102] Example 2 LNA modified probe improves typing accuracy

[0103] LNA is an oligonucleotide derivative that has a similar structure to DNA / RNA, so it can effectively recognize and bind DNA and RNA. After LNA is used to modify oligonucleotides, it can increase the thermal stability of primers or probes and increase their annealing temperature by 3-8°C. The probes developed in this kit are modified with LNA. According to the software prediction, the Tm values ​​of the modified wild-type probes and mutant probes combined with the template are all increased by about 4°C. In order to fully demonstrate the difference between LNA-modified probes and non-LNA-modified probes, the following comparative experiments were carried out. The PCR system is shown in Table 2-1, and wild homozygotes, heterozygotes and mutant homozygotes were detected respectively, and three sets were made for each genotype. Repeat, see Table 1 for the reaction procedure.

[0104] The primers and probe seque...

Embodiment 3

[0124] Example 3 Primer Probe Optimum Ratio Optimization Experiment

[0125] After the specific primers and probes are screened and confirmed, the concentration of primers and probes in the PCR reaction system needs to be optimized (using oral cells as templates). The PCR reaction system corresponding to the primer and probe concentration is shown in Table 3-1 below.

[0126] Table 3-1 Primer Probe Concentration Experiment PCR Reaction Overall System Formula Table

[0127]

[0128] Experimental results:

[0129] (1) Primer concentration gradient experiment (concentration one, concentration two, concentration three)

[0130] a. The statistical results of Ct value are shown in Table 3-2 and Figure 9 :

[0131] Table 3-2 Statistical table of Ct values

[0132]

[0133] b. The statistical results of EPF value are shown in Table 3-3 and Figure 10 :

[0134] Table 3-3 Statistical table of endpoint fluorescence value (EPF) of three genotypes

[0135]

[0136] c. Th...

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Abstract

The invention provides a rapid detection kit for CYP3A5*3 genotype based on a POCT mode. The kit comprises at least a fluorescent quantitative PCR primer (SEQ ID NO: 1-4) and probe for detecting CYP3A5*3 (rs776746) genotype and a cell lysate. In addition, the kit may comprise dNTPs, DNA polymerase, Mg<2+>, a reaction buffer, a standard positive template, a sampling rod, a sample collection tube and the like. The kit can realize real-time detection without DNA purification. A sample can be directly added into a reagent for a PCR reaction, the kit is especially suitable for rapid accurate detection of samples with low DNA content (such as oral exfoliated cells), and the detection accuracy is 99% or more. The detection sensitivity is high, and the detection limit of genomic DNA as low as 0.125 ng can be accurately detected. The whole detection takes a short time, and a detection result can be obtained within one hour and provides a medicine use basis for a doctor at the first time, thereby reducing the risk of medicine administration.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a POCT mode-based rapid detection kit for CYP3A5*3 genotype. Background technique [0002] CYP3A5 is a member of the cytochrome P450 (CYP) gene superfamily, mainly expressed in the liver, prostate, small intestine and large intestine, and is a monooxygenase involved in the metabolism of foreign compounds, cholesterol and steroid synthesis. [0003] Studies have shown that the genetic diversity of the CYP3A5 gene is the main cause of the functional changes of the CYP3A5 protein. There are multiple mutation sites in the CYP3A5 gene. Among them, the single nucleotide mutation named CYP3A5*3 (6986A>G, ​​rs776746) will cause the gene to be cut prematurely when it is transcribed into mRNA, and eventually form an incomplete protein , to disable it. The data provided by the 1000 genome project phase 3 shows that the frequency of CYP3A5*3 alleles in Dai population in Xishuangbanna, C...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2531/113C12Q2563/107
Inventor 贺庭祯罗德朋向霄熊伟黎帮勇钟越刘黎董锐崔奇新杨园
Owner 重庆京因生物科技有限责任公司
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