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Polygene sequencing primer and detecting method for detecting congenital hypothyroidism

A technology for thyroid function and detection method, applied in the fields of molecular biology and clinical laboratory science, can solve the problems of difficult clinical application of diseases, long manual operation time and high cost, and achieve the effect of reducing the cost of sequencing, low cost and high accuracy

Inactive Publication Date: 2014-10-29
MATERNAL & CHILD HEALTH HOSPITAL OF GUANGXI ZHUANG AUTONOMOUS REGION GUANGXI ZHUANG AUTONOMOUS REGION
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

One-generation sequencing, also known as "Sanger sequencing", is the "gold standard" of DNA sequencing because of its intuitive results. Difficulties in clinical application of diseases caused by polygenes

Method used

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  • Polygene sequencing primer and detecting method for detecting congenital hypothyroidism
  • Polygene sequencing primer and detecting method for detecting congenital hypothyroidism
  • Polygene sequencing primer and detecting method for detecting congenital hypothyroidism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: CH polygenic PCR primer design

[0041] Use oligo software to design PCR amplification primers for seven CH-related genes such as DUOX2, TG, and TSHR (Table 1). The primer design follows the following principles: the length of the primer is suitable in the range of 19-23bp; the primer should have no dimers, Especially the possibility of dimer formation at the 3' end; no hairpin structure; the difference between upstream and downstream primer Tm values ​​should not exceed 5°C; GC content should be 45-55%. After the upstream and downstream primers are determined, use Blast to compare and analyze the upstream and downstream primers in the NCBI database to ensure the specificity and amplification efficiency of the primers. According to the distance between the exons, some exons were merged to design primers together. A total of 83 pairs of primers were designed, and the average length of the amplified product was about 1.5kb.

Embodiment 2

[0042] Example 2: Collection of blood samples and extraction of genomic DNA:

[0043] A total of 192 unrelated CH patients from Guangxi (age: 9 days to 6 years old, median age 18 days) were selected according to the diagnostic criteria of the "Technical Standards for Newborn Disease Screening" issued by the Ministry of Health. All subjects or family members signed a written informed consent form. This study was approved by the Ethics Committee of our hospital and complied with the World Medical Association Declaration of Helsinki: Ethical Principles for Human Medical Research.

[0044] 192 cases of blood samples were prepared according to the following method, using the kit: Tiangen Biochemical Technology (Beijing) Co., Ltd., Blood Genome Extraction Kit (DP318)

[0045] 1. Add 1000 μl of ligsisbuffer to 500 μl of anticoagulated blood, and mix well until clear. Centrifuge at 4000rpm for 5min. Discard the supernatant.

[0046] 2. Add 1500 μl of ligsis buffer to the precipitat...

Embodiment 3

[0055] Example 3: Target Region Amplification

[0056] All the primers designed in Example 1 were sent to Shanghai Sangong for synthesis. After the primers were synthesized, they were centrifuged at 12,000g for 2 minutes to precipitate the dry powder. According to the instructions, add high-pressure sterilized double-distilled water to dilute to a final concentration of 10uM, and use them after they are fully dissolved. PCR experiment. The annealing temperature of all primers was explored using the American AB Veriti gradient PCR instrument, and the PCR amplification system was 25ul. For target fragments with high GC content, DMSO was added to the system to a final concentration of 2%. For primers with low specificity for amplified bands, consider changing the annealing temperature or redesigning the primers to ensure that the amplified target bands are clear and specific.

[0057] Using TAKARA high-fidelity enzyme, according to the 25 μl system, using the American AB Veriti...

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Abstract

The invention discloses a polygene sequencing primer and a detecting method for detecting congenital hypothyroidism, belonging to the fields of molecular biology and clinical examination. A group of polygene PCR primer sequences for detecting congenital hypothyroidism is composed of one or more primer sequences selected from a PCR primer sequence of a Pax-8 gene, a PCR primer sequence of a TG gene, a PCR primer sequence of an IYD gene, a PCR primer sequence of a DUOX2 gene, a PCR primer sequence of an NIS gene, a PCR primer sequence of a TSHR gene and a PCR primer sequence of a TPO gene. The polygene sequencing primer has the advantages that a plurality of disease-causing genes capable of inducing congenital hypothyroidism are simultaneously detected through new generation sequencing, so that the polygene sequencing primer is low in time consumption, high in detecting efficiency, low in cost, relatively high in sensitivity and specificity and capable of rapidly and comprehensively realizing gene mutation detection and disease classification of clinical patients.

Description

technical field [0001] The invention relates to a congenital hypothyroidism multi-gene sequencing primer and detection method, in particular to a multi-gene sequencing detection and analysis method for congenital hypothyroidism (Congenital hypothyroidism, CH), which belongs to molecular biology field and field of clinical laboratory science. Background technique [0002] Congenital hypothyroidism (CH for short, Congenital hypothyroidism, CH) is one of the most common endocrine diseases that cause mental and physical developmental disorders in newborns, and its prevalence varies in different regions, races, and environments. There may be differences. The national average prevalence of CH is 1 / 2050, but it can reach 1 / 835 in Guangxi. Early diagnosis and treatment are the main measures to reduce the harm of the disease. [0003] At present, the diagnosis of CH is mainly divided into general diagnosis and thyroid radionuclide scan diagnosis. The general diagnosis is mainly to...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q1/6886C12Q2600/156C12Q2531/113C12Q2535/122
Inventor 陈少科顾学范陈荣誉付春云罗静思范歆李川
Owner MATERNAL & CHILD HEALTH HOSPITAL OF GUANGXI ZHUANG AUTONOMOUS REGION GUANGXI ZHUANG AUTONOMOUS REGION
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