A fluorescent PCR detection kit for human interleukin 28b gene polymorphism

A gene polymorphism and kit technology, 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, The effect of reasonable technical scheme and reasonable design

Active Publication Date: 2018-04-13
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 operational technical requirements, low detection sensitivity, and inability to distinguish heterozygous types. In recent years, allele-specific PCR (AS-PCR), Literature reports on the detection of IL28Brs12979860 gene polymorphism by real-time fluorescent PCR technology, melting curve or high-resolution melting curve analysis (HRM) (J Molecular Diagnostics, 2011, 13(4): 446-51; JClin Microbiol, 2012: 50: 3353-55; Chinese Journal of Laboratory Medicine, 2013, 36(1): 59-62 and 36(8): 722-26), in which AS-PCR needs to use gel electrophoresis to observe the band size judgment results, the detection sensitivity is low, The operation is inconvenient; the fluorescent PCR detection reported in the literature either uses single-wavelength separate tubes to detect different rs12979860 gene subtypes, which is complicated to operate, or uses a single tube for detection but there is a problem of cross-effects between different subtypes, while HRM analysis has higher requirements for instruments. The results of morphism detection are affected by the instrument, and the shortcomings of these detection methods limit the clinical application of IL28B rs12979860 gene polymorphism detection

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  • A fluorescent PCR detection kit for human interleukin 28b gene polymorphism
  • A fluorescent PCR detection kit for human interleukin 28b gene polymorphism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: Design of IL28B gene polymorphism fluorescent PCR detection kit primer probe

[0028] According to the human IL28B rs12979860 gene sequence queried in the NCBI GenBank database, using VectorNTI, Oligo and other primer design software, the optimally obtained detection primer probe sequences are shown in Table 1, and the primer pair amplifies a 66 bp fragment on the IL28Brs12979860 gene.

[0029] Table 1 The designed kit EBV and internal reference detection primer probe

[0030]

[0031] The above primers and probes were synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd.

Embodiment 2

[0032] Example 2: Preparation of IL28B gene polymorphism fluorescent PCR detection kit

[0033]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.

[0034] Table 2 The volume of each component prepared by the reaction buffer of the kit

[0035] 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

...

Embodiment 3

[0039] Example 3: Application of the kit in the detection of IL28B gene polymorphism in whole blood samples of patients with hepatitis C

[0040] (1) Whole blood DNA extraction

[0041] Ten EDTA anticoagulated whole blood samples from patients with hepatitis C were collected clinically, and the whole blood DNA was extracted using the QIAamp DNABlood Mini Kit (Cat. No.51104) of QIAGEN Company, and 50 μl of eluted nucleic acids were extracted from each whole blood sample.

[0042] (2) Fluorescent PCR detection

[0043] 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, set ...

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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 about 3kb, combined with peginterferon and ribavirin has a significant effect on the treatment of hepatitis C (Tanaka Y, et al, Nat Genet, 2009, 41:1105-1109), wherein 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 type C Patien...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6858C12Q1/70
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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