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Kit for detecting polymorphism of interleukin 28B gene by utilizing fluorescence PCR (Polymerase Chain Reaction) technology

A kit and interleukin technology, which is applied in the field of medical molecular biology, can solve the problems of polymorphism detection results being affected by instruments, high operational technical requirements, and inability to distinguish heterozygous types, so as to avoid false positive results and reasonable technical solutions , to avoid inaccurate results

Active Publication Date: 2014-03-26
SHANGHAI XINGYAO MED TECH DEV CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003]Gene sequencing is the gold standard method for detecting IL28B rs12979860 gene polymorphism, but it has the disadvantages of high technical requirements, low detection sensitivity, and inability to distinguish heterozygous types. There are also reports in the literature on the detection of IL28B rs12979860 gene polymorphism using allele-specific PCR (AS-PCR), real-time fluorescent PCR technology, melting curve or high-resolution melting curve analysis (HRM) (J Molecular Diagnostics, 2011, 13(4):446-51; J Clin Microbiol, 2012:50:3353-55; Chinese Journal of Laboratory Medicine, 2013, 36(1):59-62 and 36(8):722-26), among which AS- PCR needs to use gel electrophoresis to observe the band size judgment results, the detection sensitivity is low, and the operation is inconvenient; the fluorescent PCR detection reported in the literature either uses a single wavelength tube to detect different rs12979860 gene subtypes, and the operation is complicated, or uses a single tube detection but there are differences. The problem of cross-effect of subtypes, while HRM analysis has high requirements for instruments, and the results of polymorphism detection are affected by instruments. The shortcomings of these detection methods limit the clinical application of IL28B rs12979860 gene polymorphism detection

Method used

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  • Kit for detecting polymorphism of interleukin 28B gene by utilizing fluorescence PCR (Polymerase Chain Reaction) technology
  • Kit for detecting polymorphism of interleukin 28B gene by utilizing fluorescence PCR (Polymerase Chain Reaction) technology

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Embodiment 1: Design of IL28B gene polymorphism fluorescent PCR detection kit primer probe

[0030] According to the human IL28B rs12979860 gene sequence queried in the NCBI GenBank database, Vector NTI, Oligo and other primer design software were used to optimize the obtained detection primer probe sequences as shown in Table 1. The primer pair amplified a 66 bp fragment of the IL28B rs12979860 gene.

[0031] Table 1 The designed kit EBV and internal reference detection primer probe

[0032]

[0033] The above primers and probes were synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd.

Embodiment 2

[0034] Example 2: Preparation of IL28B Gene Polymorphism Fluorescent PCR Detection Kit

[0035] The reaction buffer of the kit is self-prepared. According to the concentration and volume of each component in Table 2, the reaction buffer of the kit is prepared for 32 people. The prepared reaction buffer is divided into 20 μl for each reaction. After adding 10 μl of the template, the total reaction volume is 30 μl.

[0036] Table 2 The volume of each component prepared by the reaction buffer of the kit

[0037] components The initial concentration Reaction final concentration (30μl) Volume for 32 servings (μl) Tris-HCl (pH8.3) 1000mM 10mM 9.6 KCl 500mM 50mM 96 dATP 100mM 0.2mM 1.92 dGTP 100mM 0.2mM 1.92 dCTP 100mM 0.2mM 1.92 dUTP 100mM 0.2mM 1.92 MgCl 2 50mM 3.0mM 57. 6 CCpf 10μM 0.8μM 76.8 TTpf 10μM 0.8μM 76.8 pr 10μM 0.8μM 76.8 CCprobe 5μM 0.2μM 38.4 ...

Embodiment 3

[0041] Example 3: Application of the kit in the detection of IL28B gene polymorphism in whole blood samples of patients with hepatitis C

[0042] (1) Whole blood DNA extraction

[0043] Ten EDTA anticoagulated whole blood samples from patients with hepatitis C were collected clinically, and the whole blood DNA was extracted using the QIAamp DNA Blood Mini Kit (Cat. No.51104) of QIAGEN Company, and 50 μl of eluted nucleic acids were extracted from each whole blood sample.

[0044] (2) Fluorescent PCR detection

[0045] Take out the amplification part (reaction buffer) of the kit in Example 2 from the -20°C refrigerator, freeze-thaw, mix well, and centrifuge briefly, divide the reaction buffer into PCR reaction tubes at 20 μl / person, and then add Kit negative control, CC positive control, TT positive control and 10 μl of extracted sample templates, the total liquid volume of each reaction tube is 30 μl, put the above reaction tubes on the ABI7500 fluorescent PCR instrument, s...

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Abstract

The invention relates to a kit for detecting the polymorphism of an interleukin 28B (IL28B) gene by utilizing a fluorescence PCR (Polymerase Chain Reaction) technology. The kit is characterized in that three primers are adopted in a single reaction tube to augment sequences of CC type and TT type of an rs12979860 gene, and the CC type and the TT type of the gene are detected by two MGB fluorescence probes with different wavelengths. The kit is simple in operation and accurate and reliable in detection result, can be applied to detection of the polymorphism of the IL28B rs12979860 gene of a patient with clinical hepatitis C and further predicts the treatment effect of antiviral drugs.

Description

technical field [0001] The invention relates to the field of medical molecular biology, in particular to a human interleukin 28B gene polymorphism fluorescent PCR detection kit. Background technique [0002] Human interleukin 28B gene (Interleukin 28B, IL28B) is located on the short arm of human chromosome 19 (19q13), encoding interferon λ3 (INF-λ3) and participates in human antiviral immune response. IL28B can stimulate the release of interferon and increase cytotoxic T lymphocytes. The number of cells, the current research shows that the single nucleotide polymorphism (SNP) rs12979860 near the upstream of the IL28B gene is about 3kb, combined with pegylated interferon and ribavirin has a significant effect on the treatment of hepatitis C (Tanaka Y, et al , Nat Genet, 2009, 41:1105-1109), among which the rs12979860 polymorphism CC type has a 2-3 times higher viral clearance rate than CT heterozygous type, TT type hepatitis C or hepatitis C, and CC type is higher than non-CC...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q1/6883C12Q2600/106C12Q2600/156C12Q2563/107C12Q2561/101
Inventor 吴大治夏懿吴梅
Owner SHANGHAI XINGYAO MED TECH DEV CO LTD
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