Newborn baby TRECs and KRECs gene copy number detection kit using digital PCR technology and application thereof

A technology of gene copy number and detection kit, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., to achieve the effects of reducing clinical detection costs and workload, strong design specificity, and strong tolerance

Inactive Publication Date: 2018-12-11
上海捷易生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Only when the gene copy numbers of TRECs and KRECs are lower than the set threshold at the same time, a second round of copy number detection of the internal reference gene TRAC is required to determine whether the detection is out of control caused by sample quality problems

Method used

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  • Newborn baby TRECs and KRECs gene copy number detection kit using digital PCR technology and application thereof
  • Newborn baby TRECs and KRECs gene copy number detection kit using digital PCR technology and application thereof
  • Newborn baby TRECs and KRECs gene copy number detection kit using digital PCR technology and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Design and synthesis of primers and probes for detecting TRECs gene, KRECs gene, and internal reference gene TRAC copy number by ddPCR

[0051] For the TRECs gene, KRECs gene, and the internal reference gene TRAC gene, use the UCSC gene sequence as the source, use the Primer 3.0 software to design PCR primers and Taqman probes, and select the best combination as follows:

[0052] Upstream and downstream primers used to detect TRECs genes:

[0053] Upstream primer SEQ ID NO:1: 5'-CCATGCTGACACCCTCTGGTT-3'

[0054] Downstream primer SEQ ID NO:2: 5'-TCGTGAGAACGGTGAATGAAG-3'

[0055] When using the above primers for PCR amplification, the amplified fragment is 138bp;

[0056] Upstream and downstream primers used to detect KRECs genes:

[0057] Upstream primer SEQ ID NO:4: 5'-TCCCTTAGTGGCATTATTTGTATCACT-3'

[0058] Downstream primer SEQ ID NO:5: 5'-AGGAGCCAGCTCTTACCCTAGAGT-3'

[0059] When using the above primers for PCR amplification, the amplified fragment i...

Embodiment 2

[0069] Example 2: Detection of TRECs gene, KRECs gene, and internal reference gene TRAC copy number in dried blood film samples

[0070] 1. The first round of testing

[0071] (1) Extraction of dried blood film DNA:

[0072] A. Use a clean puncher to take a 3mm diameter dried blood piece (donated by the Beijing Obstetrics and Gynecology Hospital in this specific case), put it into a 1.5ml EP tube, add 100μl containing 0.5% (V / V) Triton X-100 PBS (commercially available), shake at 800rpm for 10min, and absorb the solution as much as possible;

[0073]B. Add 100 μl PBS (commercially available), centrifuge at 12,000 rpm for 1 min, and absorb the solution as much as possible;

[0074] C. Add 100 μl Generation DNA Elution Solu II (commercially available), shake at 800 rpm for 10 minutes, and absorb the solution as much as possible;

[0075] D. Add 30μl Generation DNA Elution Solu II, bathe in a 99-degree water bath for 30 minutes, centrifuge at 12,000 rpm for 1 minute, and trans...

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Abstract

The invention relates to a newborn baby TRECs and KRECs gene copy number detection kit using a digital PCR technology. The kit comprises a forward primer, a reverse primer and a probe used for detecting a TRECs gene, a forward primer, a reverse primer and a probe used for detecting a KRECs gene, and a forward primer, a reverse primer and a probe used for detecting a reference gene TRAC. The detection of the project can be completed only through hole forming to drill a round dried blood spot (with the blood quantity about 3mul) with the diameter being 3mm; the blood consumption is low; the kitis very suitable for the newborn baby screening unsuitable for great blood sampling quantity. Two rounds of sequential detection strategy are used; in the first round of detection, only the synchronous detection on the TRECs and KRECs gene copy number by single hole is needed; the two results are quality control reference indexes for each other. The second round of reference gene TRAC copy numberdetection is needed only when the TRECs and KRECs gene copy numbers are lower than the threshold values at the same time; whether the detection control loss due to sample quality problem occurs or notis determined.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a neonatal TRECs and KRECs gene copy number detection kit using digital PCR technology and its application in the screening of severe combined immunodeficiency disease and agammaglobulinemia. 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 defined as Naive T cell deficiency. Without treatment, SCI...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6851
CPCC12Q1/6851C12Q1/6883C12Q2600/118C12Q2531/113
Inventor 陈珺李艳艳欧恩智朱智
Owner 上海捷易生物科技有限公司
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