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Citrin deficiency pathogenic gene detection primer and kit

A technology for detection kits and disease-causing genes, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., and can solve problems such as cumbersome operations, high detection costs, and insufficient sensitivity and specificity. Achieve the effect of simple operation, high sensitivity and high specificity

Active Publication Date: 2019-01-11
江门市妇幼保健院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current genetic detection methods have disadvantages such as high detection cost, cumbersome operation, insufficient sensitivity and specificity, etc.

Method used

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  • Citrin deficiency pathogenic gene detection primer and kit
  • Citrin deficiency pathogenic gene detection primer and kit
  • Citrin deficiency pathogenic gene detection primer and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] An embodiment of the Citrin deficiency disease-causing gene detection kit of the present invention comprises an upstream primer shown in SEQ ID No.1, a downstream primer shown in SEQ ID No.2 and a probe shown in SEQ ID No.3. needle; the 5' end of the probe is connected with a fluorescent reporter group FAM, and the 3' end is connected with a fluorescent quencher group BHQ1.

[0039] SEQ ID No.1: 5'-GGAGGAGGGCAGCAATCAG-3';

[0040] SEQ ID No.2: 5'-GGCCTCAGCCAAGTTAAAGG-3';

[0041] SEQ ID No.3: 5'FAM-TTTGTTTTTCCCCTACAGACGTATGACCTTAGC

[0042] AGACATTGAAC-BHQ1 3'.

[0043] The primers were synthesized by a biological company and configured according to the instructions.

Embodiment 2

[0045] In this example, the kit described in Example 1 is used to detect the causative gene of Citrin deficiency.

[0046] 1. Reaction system configuration

[0047] Configure the reaction system according to the components and component final concentrations shown in Table 1:

[0048]Table 1 reaction system

[0049]

[0050] The DNA template is DNA extracted from peripheral blood cells of the patient.

[0051] 2. Reaction procedure

[0052] The PCR reaction program is shown in Table 2:

[0053] Table 2 Reaction program

[0054]

[0055]

[0056] 3. Judgment criteria for results

[0057] The kit of the invention can be used to detect the wild type, heterozygous mutation and homozygous mutation of SLC25A13 gene c.851_854del (rs80338720), so as to diagnose Citrin deficiency. The criteria for judging the test results are as follows:

[0058] (1) Wild type of the causative gene of Citrin deficiency: Ct value is less than 35; Tm value: 70.94±0.55°C, single peak;

[0...

Embodiment 3

[0067] In this example, the pathogenic gene of Citrin deficiency was detected in 15 cases of clinical samples. Take the peripheral blood sample of the subject to be tested, extract the DNA, use the kit described in Example 1, and perform detection according to the detection method described in Example 2, and at the same time perform the first-generation sequencing on the sample. The test results are shown in Table 3:

[0068] Table 3 Test results

[0069]

[0070]

[0071] The above results show that the kit of the present invention has good accuracy when used in the detection of Citrin deficiency disease-causing genes, and the detection results are completely consistent with the sequencing results.

[0072] Table 4Mg 2+ Final concentration optimization reaction system

[0073]

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Abstract

The invention discloses a Citrin deficiency gene detection primer, comprising an upstream prim as shown in SEQ ID No. 1, a downstream primer as shown in SEQ ID No. 2 and a probe as shown in SEQ ID No.3. The invention also discloses the use of the detection primer in preparing a Citrin defect detection reagent and a Citrin defect detection reagent kit. The detection primer provided by the invention can realize the simultaneous detection of the wild type of the c.851_854del (rs80338720) of the disease-causing gene SLC25A13 of the Citrin deficiency in the same tube PCR reaction, Heterozygous mutation and homozygous mutation have the advantages of simple operation, high sensitivity, high specificity and low cost, which are very suitable for the detection and diagnosis of clinical Citrin deficiency.

Description

technical field [0001] The invention belongs to the technical field of gene mutation detection, and in particular relates to a detection primer and a kit for Citrin deficiency disease-causing genes. Background technique [0002] Citrin protein is a calcium-binding transmembrane solute carrier protein located on the inner mitochondrial membrane, which is responsible for transporting aspartate in the mitochondria to the cytoplasm, and transporting glutamate in the cytoplasm to the interior of the mitochondria. This process is coupled with malate shuttling and reoxidation of reduced nicotinamide adenine dinucleotide (NADH) in the cytoplasm to NAD + , thereby maintaining the stability of the oxidation / reduction state in the cytoplasm. Citrin protein is encoded by the SLC25A13 gene and is mainly expressed in the liver. The SLC25A13 gene is a nuclear gene, located on human chromosome 7q21.3, containing 18 exons, the length of its open reading frame is 2028bp, and the code contai...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/686C12N15/11
Inventor 曾钦龙唐佳孙淑湘孙铁兰
Owner 江门市妇幼保健院
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