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Pyrophosphoric acid sequencing primer pair and kit for qualitatively detecting HLA-DQ genotyping

A technology of pyrosequencing and HLA-DQ, which is applied in the field of pyrosequencing primer pairs and kits, can solve the problems of low sensitivity, long detection cycle, cumbersome operation, etc., and achieve simple sample processing, short reaction time and high sensitivity high effect

Inactive Publication Date: 2016-12-21
CHANGSHA 3G BIOTECH
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  • Abstract
  • Description
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Problems solved by technology

[0011] In order to solve the technical problems of low sensitivity, long detection period, cumbersome operation and high cost in the process of detecting HLA-DQ genotyping by the above method, the present invention provides a method with high sensitivity, strong specificity, short detection period and easy operation. Pyrosequencing primer pair and kit for qualitative detection of HLA-DQ genotyping that is simple and effective to meet clinical testing requirements

Method used

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  • Pyrophosphoric acid sequencing primer pair and kit for qualitatively detecting HLA-DQ genotyping
  • Pyrophosphoric acid sequencing primer pair and kit for qualitatively detecting HLA-DQ genotyping
  • Pyrophosphoric acid sequencing primer pair and kit for qualitatively detecting HLA-DQ genotyping

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1: the preparation of kit

[0051] 1. Design and synthesis of primers and probes

[0052] Select specific mutation sites for the human HLA-DQ gene, and use PyroMark Assay Design2.0 software to design primers; the amplification primers and sequencing primers are first purified by PAGE, and then purified by HPLC. The 5' of SEQ ID NO.2 labeled with biotin.

[0053] Table 1. Mutation site and type

[0054] Mutation

Base change

HLA-DQ (rs9275319)

A>G

[0055] The amplified sequence is shown in Table 2:

[0056] Table 2. Specific amplification primers and primer sequences

[0057]

[0058] 2. Selection of reference substance

[0059] A synthetic oligonucleotide chain TAYGGTTTGCA control oligo was used as the quality control product; DNase / RNase-Free water was used as the blank control product.

[0060] 3. Composition of PCR reaction solution

[0061] Table 3. Composition of PCR reaction solution

[0062] raw material...

Embodiment 2

[0063] Embodiment 2: the use of kit

[0064] 1. Sample testing

[0065] 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.

[0066] The main components of the HLA-DQ system are as follows:

[0067] Table 4. Main components of the HLA-DQ system

[0068]

[0069] The system reaction procedure is as follows:

[0070] Table 5. PCR reaction program

[0071]

[0072] After the amplification is completed, check the PCR results on agarose gel to proceed to the next step.

[0073] 2. Pyrosequencing

[0074] Sequencing was performed in accordance with the standard...

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Abstract

The invention relates to a pyrophosphoric acid sequencing primer pair for qualitatively detecting HLA-DQ genotyping. The primer pair comprises a forward amplification primer, a reverse amplification primer and a sequencing primer, wherein the 5' terminals of the reverse amplification primer are respectively subjected to biotin labeling. The invention also relates to a pyrophosphoric acid sequencing kit for qualitatively detecting HLA-DQ genotyping. The kit comprises the amplification primers, a PCR (polymerase chain reaction) solution, the sequencing primer, a uracil DNA glycosylase and a Taq polymerase. The primer pair and kit have the advantages of accurate detection result, high specificity and short detection period, are simple to operate, and can effectively satisfy the requirements of clinical examination. Besides, the primer pair and kit can be used for monitoring the reaction process in real time, are short in reaction time, and can be used for sequencing and high-flux sample detection on a pyrophosphoric acid sequencing instrument after simply treating the PCR product. Compared with the gold standard method (capillary electrophoresis sequencing method), the primer pair and kit have the advantage of higher sensitivity.

Description

technical field [0001] The invention relates to the technical field of in vitro nucleic acid detection, in particular to a pair of pyrosequencing primers and a kit for qualitative detection of HLA-DQ genotyping. Background technique [0002] Liver cancer is the third most lethal malignant tumor in the world, and it is also a common malignant disease in China. About 700,000 people die of liver cancer every year worldwide. Hepatocellular Carcinoma (HCC) is the most common type of liver cancer. According to a survey by the International Agency for Research on Cancer in 2000, 80% of HCC patients are related to heterosexual hepatitis virus and hepatitis C virus. Medical history survey shows that more than 80% of Chinese patients with liver cancer have a history of hepatitis B. According to the data released by the Ministry of Health, China currently has a total of 93 million hepatitis B patients, accounting for more than one-third of the total number of hepatitis B patients in ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q1/6886C12Q2600/156C12Q2565/301C12Q2535/122
Inventor 滕祥云侯冬梅
Owner CHANGSHA 3G BIOTECH
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