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Probe set for IL28B gene polymorphism detection, gene chip and kit

A gene polymorphism and gene chip technology, which is applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of restricting market application and promotion, increasing application cost, weak signal, etc. To achieve the effect of improving detection efficiency and accuracy

Active Publication Date: 2016-04-20
珠海赛乐奇医学检验有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its signal is weak and must be excited to emit light, requiring expensive laser scanning equipment
In the process of clinical testing application, since the testing unit needs to invest in the purchase of laser scanning equipment, on the one hand, it significantly increases the investment cost of related testing, on the other hand, it also directly increases the application cost, which limits the market application and promotion of this technology

Method used

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  • Probe set for IL28B gene polymorphism detection, gene chip and kit
  • Probe set for IL28B gene polymorphism detection, gene chip and kit
  • Probe set for IL28B gene polymorphism detection, gene chip and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] [Example 1] Preparation of chip and detection kit

[0044] Gene chip: Coated by vacuum vapor deposition method, using a rotary vacuum coating machine on a silicon wafer (SiO 2 ) were plated with 475 angstroms of silicon nitride and 135 angstroms of TSPS films to prepare corresponding biosensors, and covered with 150 angstroms of polyphenylalanine-lysine coating, and finally passed through 1-10uM succinic hydrochloride Acid imide nicotinic acid salt treatment for 20 minutes, washed with water for chip production. The amino-modified probes were arranged according to Table 1, and the probes were respectively spotted on the processed chips, and the probe sequences were shown in Table 2; two parallel spotting points were set up for each group: react at room temperature, and prepare the samples containing Probes for gene chips.

[0045] Table 1 Gene Chip Probe Spotting Arrangement

[0046]

[0047]

[0048] Table 2 Spotted probe sequences

[0049] rs129798...

Embodiment 2

[0053] [Example 2] hybridization reaction process

[0054] The target gene fragments rs12979860 (C / T) and rs8099917 (T / G) in this example are shown in the sequence table SEQ ID NO.1 and SEQ ID NO.2. The plasmids 9860CC plasmid, 9860TT plasmid, 9917TT plasmid, and 9917GG plasmid containing the target fragment were synthesized by Nanjing GenScript Biotechnology Co., Ltd. Hot-Taq enzyme, dUTP, reverse transcription amplification reagent, and UNG enzyme were purchased from Shenzhen Faipeng Biological Co., Ltd. The gene chip was provided by Zhuhai Seleqi Biotechnology Co., Ltd., and the primers and probes were synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd. The plasmid was 2ug, and the plasmid was dissolved in 1ml TE buffer solution, and the plasmid concentration was 2ug / ml or 2000ng / ml. In this embodiment, the composition of the reaction system is shown in Table 4, and the primer sequences in this embodiment are shown in Table 5.

[0055] Table 4 PCR reaction system ...

Embodiment 3

[0072] [Example 3] Gene Chip Sensitivity Test Results

[0073] 1. Sample Preparation

[0074] Take the plasmids 9860CC plasmid, 9860TT plasmid, 9917TT plasmid, and 9917GG plasmid containing the target fragment, and dilute various high-concentration plasmids according to 10 times. The diluted plasmid concentration is shown in Table 6:

[0075] Table 6 Band detection plasmid concentration gradient

[0076]

[0077] 2. Detection

[0078] According to the method in Example 2, the above-mentioned samples were detected by using the prepared gene chip.

[0079] 3. Results

[0080] The 9860 system was added to the 9860CC plasmid to amplify the hybridization results as follows: figure 2 Shown: Among them, Figures A, B, C, D, and E correspond to 9860CC-4, 9860CC-5, 9860CC-6, 9860CC-7 and negative samples respectively.

[0081] The 9860 system was added to the 9860TT plasmid to amplify the hybridization results as follows: image 3 Shown: Among them, Figures A, B, C, D, and E c...

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Abstract

The invention relates to the technical field of biology, in particular to a probe set for IL28B gene polymorphism detection, a gene chip and a kit. The probe set for IL28B gene polymorphism detection comprises probes at the sites of rs12979860 and rs8099917; the sequences of the probes are: TATTTGTTGGGGTAATCAACTGTT SEQ ID NO.1 for the rs12979860-C probes; TATTTGCTGGGGAAACCACTTGTT SEQ ID NO.2 for the rs12979860-T probes; CATTTGTTGGGGTAACCAACTATT SEQ ID NO.3 for the rs8099917-T probes; TGTTTGCTGGCATAATCAATTATT SEQ ID NO.4 for the rs8099917-G probes. Through selection of specific primers and the probes in combination with the gene chip technology, detection efficiency and accuracy are improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a probe, a gene chip and a kit for detecting IL28B gene polymorphism. Background technique [0002] Hepatitis C virus is an infectious disease caused by hepatitis C virus (HCV). HCV infection is one of the main causes of global chronic liver disease. The HCV infection rate in my country is about 3.2%, and there are about 35,000 new cases of hepatitis C every year. Currently, there is no effective vaccine for hepatitis C to prevent infection. [0003] The human IL28B gene is located on the ACO11445.6 gene group of chromosome 19q13.13, belongs to the type III interferon family, and encodes IFN-λ3. The IL28B gene consists of 6 exons and 5 introns, containing [0004] Signal peptide composed of 22 amino acids. Studies have found that the treatment of hepatitis C is highly correlated with the host's IL28B gene polymorphism. The two polymorphic sites rs12979860 and rs8099917 of the IL...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C40B40/06
CPCC12Q1/6888C12Q2600/106C12Q2600/156
Inventor 宋家武王丽玲肖杰熊珊珊郝永芬康启鸣陈妙萍钟宇萍
Owner 珠海赛乐奇医学检验有限公司
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