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Primer pair and kit for detecting CYP3A4 genotyping by pyrosequencing

A technology of pyrosequencing and genotyping, which is applied in the field of primer pairs and kits for detecting CYP3A4 genotyping by pyrosequencing, which can solve the problems of difficulty in meeting clinical testing requirements, low accuracy of testing results, and long testing cycle To achieve real-time monitoring of the reaction process, high-throughput sample detection, and short reaction time

Inactive Publication Date: 2015-12-16
CHANGSHA 3G BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In order to solve the technical problems in the detection of CYP3A4 genotyping by the above method, such as low accuracy of detection results, long detection period, cumbersome operation and difficulty in meeting the requirements of clinical examination, the present invention provides a method with accurate detection results, high specificity, long detection cycle Primer pair and kit for detection of CYP3A4 genotyping by pyrosequencing method that is short, easy to operate and effectively meets the requirements of clinical testing

Method used

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  • Primer pair and kit for detecting CYP3A4 genotyping by pyrosequencing
  • Primer pair and kit for detecting CYP3A4 genotyping by pyrosequencing
  • Primer pair and kit for detecting CYP3A4 genotyping by pyrosequencing

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: the preparation of kit

[0041] 1. Design and synthesis of primers and probes

[0042] For the polymorphic site CYP3A4*1G detected in the human CYP3A4 gene, select specific mutation sites and use PyroMarkAssayDesign2.0 software to design primers; the forward amplification primers, reverse amplification primers and sequencing primers are first purified by PAGE , and then purified by HPLC, wherein the 5' of SEQ ID NO.1 is biotin-labeled.

[0043] Table 1. Mutation site and type:

[0044] Mutation

base change

CYP3A4*1G (rs2242480)

C>T

[0045] The amplified sequence is shown in Table 2:

[0046] Table 2. Specific amplification primers and primer sequences

[0047]

[0048]

[0049] 2. Selection of reference substance

[0050] A synthetic oligonucleotide chain TAYGGTTTGCAcontrololigo was used as the quality control substance; DNase / RNase-Free water was used as the blank control substance.

[0051] 3. Composition of PC...

Embodiment 2

[0054] Embodiment 2: the use of kit

[0055] 1. Sample testing

[0056] Dissolve the dry powder of the primers (the validity period of the primers is 1 month after dissolution). Prepare the system according to the number of templates: take the PCR reaction solution, add dissolved primers, uracil DNA glycosylase, and TaqDNA polymerase, aliquot the system, add sample DNA, blank control substance or positive control substance as templates, and form a PCR reaction system. Perform PCR amplification according to the PCR reaction procedure.

[0057] The main components of the CYP3A4*1G system are as follows:

[0058] Table 4. Main components of CYP3A4*1G system

[0059]

[0060]

[0061] The system reaction procedure is as follows:

[0062] Table 5. PCR reaction program

[0063]

[0064] After the amplification is completed, check the PCR results on agarose gel to proceed to the next step.

[0065] 2. Pyrosequencing

[0066] Sequencing was performed in accordance wit...

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Abstract

The invention relates to a primer pair and kit for detecting CYP3A4 genotyping by pyrosequencing, belonging to the technical field of in vitro nucleic acid detection. The primer pair comprises a forward amplification primer, a reverse amplification primer and a sequencing primer, wherein a 5' terminal of the forward amplification primer is subjected to biotin labelling. The kit comprises the forward amplification primer, a PCR (polymerase chain reaction) liquid containing the reverse amplification primer, the sequencing primer, uracil DNA (deoxyribonucleic acid)glycosylase and Taq polymerase. The kit provided by the invention has the advantages of accurate detection results, high specificity, short detection period, simplicity in operation, capability of effectively meeting the requirements of clinical examination, capability of monitoring the reaction process in real time, short reaction time, sequencing of PCR products on a pyrosequencing instrument after the PCR products are simply treated, high throughput sample detection and higher sensitivity than gold standard methods, namely capillary electrophoresis sequencing methods.

Description

technical field [0001] The invention relates to the technical field of in vitro nucleic acid detection, in particular to a pair of primers and a kit for detecting CYP3A4 genotyping by pyrosequencing. Background technique [0002] CYP450 enzyme is an important monooxygenase that plays an important role in the oxidation or reduction metabolism of many endogenous and exogenous compounds, including steroids, fatty acids, prostaglandins, drugs, carcinogens and toxins, etc. In recent years, nearly 200 CYP450 isoenzymes have been found in human liver microsomes abroad, among which CYP3A4 accounts for about 25% of the total CYP450 enzymes in the liver, and it participates in the metabolic reactions of various environmental toxins and commonly used chemotherapeutic drugs . [0003] So far, the CYP3A4 mutant alleles that have been found in Chinese are mainly intron 2, intron 10, CYP3A4*3, CYP3A4*4, CYP3A4*5, CYP3A4*6, CYP3A4*18 (among them, CYP3A4 *18B has the same mutation site as ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q2531/113C12Q2535/122C12Q2565/301
Inventor 滕祥云尹贞
Owner CHANGSHA 3G BIOTECH
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