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Kit for detecting TSHR (thyroid stimulating hormone receptor) gene mRNA expression through fluorescence quantitation RT-PCR (real-time polymerase chain reaction) and use method of kit

A thyroid stimulating hormone and receptor gene technology, applied in the field of gene detection, can solve the problems of high template requirements, high primer design requirements, low specificity of TSHR mRNA detection, etc., achieving strong anti-interference ability, rapid and economical detection, and elimination of false positives and the effect of false negative results

Inactive Publication Date: 2017-05-10
河北德路通生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using real-time fluorescence quantitative PCR (RT-PCR) technology to measure the relative expression of TgmRNA and TSHRmRNA in peripheral blood has high requirements for templates and primer design, and the detection specificity of relative expression of TSHRmRNA is not high

Method used

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  • Kit for detecting TSHR (thyroid stimulating hormone receptor) gene mRNA expression through fluorescence quantitation RT-PCR (real-time polymerase chain reaction) and use method of kit
  • Kit for detecting TSHR (thyroid stimulating hormone receptor) gene mRNA expression through fluorescence quantitation RT-PCR (real-time polymerase chain reaction) and use method of kit
  • Kit for detecting TSHR (thyroid stimulating hormone receptor) gene mRNA expression through fluorescence quantitation RT-PCR (real-time polymerase chain reaction) and use method of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] This example is the design and screening of primers and probes for amplifying thyroid stimulating hormone receptor mRNA reverse transcription cDNA.

[0051] According to the expression, cleavage of TSHR gene mRNA, and other similar sequences at the transcriptome level, the length and sequence of the primers were controlled to increase the specificity of the primers. Based on this, the full-length sequence of the TSHR gene CCDS (GenBank sequence number CCDS9872.1) was used as a template to design Six pairs of primers. The six pairs of primers were compared with the total mRNA sequence respectively, and the amplification specificity of the six pairs of primers was verified. Then, the amplification efficiency and specificity of the primers were further verified by specific experiments: DNA samples obtained by reverse transcription of total mRNA extracted from thyroid cancer tissues were used as templates, and six pairs of primers were used for SYBR Green-qPCR amplification...

Embodiment 2

[0056] This embodiment is the specificity test of primers and probes for amplifying thyroid stimulating hormone receptor mRNA reverse transcription cDNA, and the kit in this embodiment only includes Primers and probes for recording cDNA, with necessary reagents.

[0057] 1. Operation process

[0058] 1) Sample preparation

[0059] 5 ml of EDTA-anticoagulated fresh peripheral blood extracted was treated with red blood cell lysate to remove red blood cells, and then total RNA was extracted.

[0060] 2) Reverse transcription reaction

[0061] Take a certain amount of total RNA in the isothermal amplification reaction solution for reverse transcription reaction to obtain DNA samples.

[0062] 3) Detection of TSHR gene expression

[0063] Using the DNA sample obtained by reverse transcription as a template, using the primers and probes detected by TSHR, the expression level of TSHR was detected by qPCR.

[0064] 2. Test results

[0065] The amplification curve of TSHR detecte...

Embodiment 3

[0074] This embodiment is the design and screening of internal standard, internal standard primers, and detection internal standard probes. The detection kit finally determines the relative level of TSHR expression, which is compared with the amplification of the internal standard (GAPDH) and judged by Ct, Ct=Ct(GAPDH)-Ct(TSHR). GAPDH is a commonly used internal reference for the initial amount of the reaction template, and timely detection samples contain similar levels of TSHR, but after conversion of GAPDH, the expression of TSHR in unit sample size is different, as shown in Table 2 Internal standard, internal standard primers, internal standard for detection Probe TSHR sample detection and attached Figure 4 Internal standard, internal standard primers, detection internal standard probe TSHR sample detection results are shown. Therefore, the internal standard can effectively quantify two test samples from different sources, so that the expression of TSHR in the test sampl...

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Abstract

The invention provides a kit for detecting TSHR (thyroid stimulating hormone receptor) gene mRNA expression through a fluorescence quantitation RT-PCR (real-time polymerase chain reaction) and a use method of the kit. The kit detects the existence of expression of TSHR-mRNA in peripheral blood, tumor tissue samples and the like and the expression level with a Taqman-qPCR method. The kit has the advantages of high sensitivity, high accuracy, high detection rate and specificity, is matched with an internal standard and a positive reference substance, can effectively eliminate false positive and false negative results, is high in anti-jamming capacity and has the characteristics that detection is quick and economical, trauma of patients is small and the product stability is high.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a kit for detecting thyroid-stimulating hormone receptor gene mRNA expression by fluorescent quantitative RT-PCR and a use method thereof. Background technique [0002] Thyroid nodules are very common clinically, most of which are benign and a few are malignant. The early clinical manifestations of the two are similar, but the treatment methods and prognosis are different. Therefore, it is very important to distinguish between benign and malignant thyroid nodules. Although high-resolution ultrasound, scanning, CT, MRI, and thyroid fine-needle aspiration cytology (FNAC) can be used clinically to evaluate the benign and malignant properties of nodules, there are still certain limitations, which cause some patients to undergo inappropriate Necessary surgical treatment. Since thyroid cancer cells can secrete and express some thyroid-specific antigens, in recent years, some foreign scho...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q1/6886C12Q2600/158C12Q2600/166C12Q2600/178C12Q2531/113C12Q2561/101C12Q2521/107C12Q2545/101
Inventor 许宝芝白蛟腾秦颖李华文赵俊伟
Owner 河北德路通生物科技有限公司
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