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Real-time fluorescent quantitative PCR kit for detecting TRECs and KRECs genes, and application thereof

A technology of real-time fluorescence quantification and kits, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. Advanced problems, to achieve accurate and reliable test results, reduce false positive results, and achieve high copy number effects

Inactive Publication Date: 2018-12-14
上海捷易生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This patent adopts a two-step method including nested PCR amplification and real-time fluorescent quantitative PCR amplification. The operation is complicated and time-consuming, and there is no clear reference threshold. Amplification errors caused by PCR, sample contamination caused by pipetting operations after nested PCR, and accumulated errors in many links will eventually lead to a high possibility of missed detection
If a missed test occurs during the screening process, the child will miss the best opportunity for diagnosis and treatment

Method used

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  • Real-time fluorescent quantitative PCR kit for detecting TRECs and KRECs genes, and application thereof
  • Real-time fluorescent quantitative PCR kit for detecting TRECs and KRECs genes, and application thereof
  • Real-time fluorescent quantitative PCR kit for detecting TRECs and KRECs genes, and application thereof

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Experimental program
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Effect test

Embodiment 1

[0048] Embodiment 1: the preparation of kit

[0049] 1. Design and synthesis of primers and probes

[0050] According to the UCSC website query TRECs, KRECs and TRAC gene sequence, use Primer 3.0 to design fluorescent quantitative PCR forward and reverse primers and probes on TRECs, KRECs and TRAC genes. The selected primers have high PCR amplification efficiency, and the selected probes have good specificity for gene binding. Both primers and probes were entrusted to Suzhou Jinweizhi Biotechnology Co., Ltd. for chemical synthesis, in which primers were purified by PAGE, and probes were purified by HPLC. The 5' end of the TRECs detection probe is labeled with a HEX fluorescent group, and the 3' end is a TAMRA quencher group; the 5' end of the KRECs detection probe is labeled with a HEX fluorescent group, and the 3' end is a TAMRA quencher group; The 5' end of the TRAC detection probe is labeled with a FAM fluorescent group, and the 3' end is a TAMRA quencher group. Primer s...

Embodiment 2

[0069] Example 2: Verification and use of the kit

[0070] 1. Collection of verification samples after kit development

[0071] The clinically confirmed positive SCID and XLA children's dry blood samples (donated by Beijing Obstetrics and Gynecology Hospital) were used as the positive control group for the kit verification, and the expected test results should be positive.

[0072] Dried blood samples (donated by Beijing Obstetrics and Gynecology Hospital) from children with no clinical phenotype and clinically judged to be normal were used as samples to be tested, and the expected test results should be negative. The dried blood samples of the positive control group and the samples to be tested were stored at 4°C. 2. Extraction of DNA from dried blood samples

[0073] The operation steps are as follows:

[0074] A. Use a clean puncher to take a dried blood piece with a diameter of 3mm, put it into a 1.5ml EP tube, add 100μl 0.5% (V / V) Triton X-100, shake at 800rpm for 10mi...

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Abstract

The invention relates to a real-time fluorescent quantitative PCR kit for detecting TRECs and KRECs genes. The kit comprises: 1) a DNA extraction reagent; 2) a standard product containing a TRECs geneinsertion sequence, a KRECs gene insertion sequence and a TRAC gene insertion sequence; 3) a fluorescent PCR reaction solution I containing fluorescent PCR primers and detection probes of the TRECs gene and the TRAC gene; and 4) a fluorescent PCR reaction solution II containing fluorescent PCR primers and detection probes of the KRECs gene and the TRAC gene. The kit has the advantages of high sensitivity, good specificity, simple and rapid detection method, reliable experimental result, realization of screening of immunodeficiency diseases mainly including SCID and antibody deficiency, provision of clinically relevant indications for other primitive immunodeficiency diseases related to T cell and B cell development or other systemic diseases, and facilitation of early diagnosis and treatment.

Description

technical field [0001] The invention belongs to the field of in vitro nucleic acid diagnosis, and relates to a real-time fluorescent quantitative polymerase for detecting T cell receptor excision circles (T-cell receptor excision circles, TRECs) and K-deleting recombination excision circles (KRECs) genes Chain reaction kit and its application. Background technique [0002] Severe combined immunodeficiency disease (severe combined immunodeficiency disease, SCID) is the most serious type of combined immunodeficiency disease, and patients often die within one year after birth. According to large-scale screening abroad, the incidence of SCID is about 1 / 58,000 live births, which is significantly higher than the previously thought 1 / 100,000. SCID is the deficiency and / or dysfunction of T lymphocytes, B lymphocytes, and NK cells caused by a variety of genetic factors, resulting in severe defects in both humoral immunity and cellular immunity. This primary immunodeficiency is often...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6883C12Q2600/158C12Q2561/113C12Q2563/107C12Q2545/114C12Q2545/101
Inventor 朱智李艳艳欧恩智陈珺
Owner 上海捷易生物科技有限公司
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