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Fluorescent quantitative PCR probe primer group and kit for detecting high-frequency pathogenic variation of SLC22A5 gene

A SLC22A5-F1, fluorescence quantitative technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., to achieve clear pathogenicity, convenient and accurate interpretation of results, and low cost

Pending Publication Date: 2021-08-06
北京华诺奥美医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no fluorescent quantitative PCR primers, probes and kits specifically targeting this variant site

Method used

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  • Fluorescent quantitative PCR probe primer group and kit for detecting high-frequency pathogenic variation of SLC22A5 gene
  • Fluorescent quantitative PCR probe primer group and kit for detecting high-frequency pathogenic variation of SLC22A5 gene
  • Fluorescent quantitative PCR probe primer group and kit for detecting high-frequency pathogenic variation of SLC22A5 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] A fluorescent quantitative PCR kit for detecting high-frequency pathogenic variants of the SLC22A5 gene, including a probe primer set designed for the detection site, and the probe primer set includes for amplifying the SLC22A5 gene NM_003060.3:exon8: c. The upstream and downstream primers of the 1400C>G variation site and the MGB probe for the variation site, the sequence of the probe primer set is shown in Table 1-2:

[0056] Table 1 Primer Sequence

[0057]

[0058] Table 2 MGB probe sequence

[0059]

[0060] The method for detecting the sample to be tested by using the above kit is as follows:

[0061] (1) Prepare reagents according to Table 3;

[0062] Table 3 Reagent preparation

[0063] components Volume ul Final concentration primer F (10μM) 1.8 0.9μM primer R (10μM) 1.8 0.9μM TaqMan Probe 1 (10μM) 0.4 0.2μM TaqMan Probe 2 (10μM) 0.4 0.2μM qPCR reaction buffer 10 Template (DNA solution) 2 ...

Embodiment 2

[0068] The kit described in Example 1 was used to detect high-frequency pathogenic variants of the SLC22A5 gene. When wild-type DNA was used as a template, the detection results were shown in Table 5:

[0069] Table 5 wild-type DNA detection results

[0070] Reaction well position sample name allele Reporting group Ct value A2 235-3 / 2 G FAM 31.54 A2 235-3 / 2 C VIC 24.31 B2 237-3 / 2 G FAM 29.56 B2 237-3 / 2 C VIC 23.72 C2 611-3 / 2 G FAM 29.93 C2 611-3 / 2 C VIC 24.18 D2 3 / 2-BLANK G FAM Undetermined D2 3 / 2-BLANK C VIC Undetermined

[0071] Among them, samples 235, 237 and 611 are wild-type human genomic DNA, and 3 / 2-BLANK is a blank control using purified water. The threshold line was set to 0.1. It can be seen from Table 5 that the average Ct value of the VIC signal is 24.07 with a standard deviation of 0.3106; while the average Ct value of the FAM signal is 30.34 with a standard deviati...

Embodiment 3

[0073] The kit described in Example 1 was used to detect high-frequency pathogenic variants of the SLC22A5 gene. When using heterozygous DNA as a template, the detection results are shown in Table 6:

[0074] Table 6 Heterozygous DNA detection results

[0075] Reaction well position sample name allele Reporting group Ct value A1 SLC22A5-1-1 G FAM 24.65 A1 SLC22A5-1-1 C VIC 25.12 B1 SLC22A5-2-2 G FAM 24.60 B1 SLC22A5-2-2 C VIC 25.20 C1 SLC22A5-3-2 G FAM 24.69 C1 SLC22A5-3-2 C VIC 25.25 D1 SLC22A5-BLANK FAM FAM Undetermined D1 SLC22A5-BLANK VIC VIC Undetermined

[0076] Among them, the samples SLC22A5-1-1, SLC22A5-2-2 and SLC22A5-3-2 are heterozygous human genomic DNA of the mutation site NM_003060.3:exon8:c.1400C>G, and SLC22A5-BLANK was purified using Water blank. The threshold line was set to 0.1. It can be seen from Table 6 that the average Ct value of the VIC signal i...

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Abstract

The invention discloses a fluorescent quantitative PCR probe primer group and a kit for detecting high-frequency pathogenic variation of an SLC22A5 gene. The probe primer group comprises an upstream primer, a downstream primer and an MGB probe, wherein the upstream primer and the downstream primer are used for amplifying NM_003060.3:exon8:c.1400C>G variation sites of the SLC22A5 gene, and the MGB probe aims at the variation sites. The kit comprises the probe primer group aiming at a detection site, efficient DNA polymerase suitable for a high CG sequence, dNTPs, a buffer solution system suitable for a high GC sequence and ROX reference fluorescent dye used for calibrating the volume of a reaction solution. The probe primer group and the kit disclosed by the invention can be used for rapidly and accurately detecting c.1400C > G variation on an NM_003060 transcript of the SLC22A5 gene in a human whole blood sample in vitro, and a detection result is used for auxiliary diagnosis of primary carnitine deficiency.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a fluorescent quantitative PCR probe primer set and kit for detecting high-frequency pathogenic variation of SLC22A5 gene. Background technique [0002] Primary Carnitine Deficiency (PCD) is an autosomal recessive genetic disease characterized by abnormal carnitine metabolism. The causative gene of the disease is the SLC22A5 gene encoding organic cation transporter type 2 (OCTN2). The age span of onset of PCD is relatively large, but most children develop from 1 month to 7 years old, and the average age of onset is about 2 years old. The clinical manifestations of different patients are quite different, mainly including: (1) acute energy metabolism disorder, manifested as low ketone hypoglycemia, hyperammonia, and metabolic acidosis, etc.; (2) cardiomyopathy, manifested as ventricular hypertrophy, cardiac Insufficiency, arrhythmia, elevated creatine kinase, etc.; (...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6883C12Q1/6858C12Q2600/156
Inventor 张可欣王慧魏星
Owner 北京华诺奥美医学检验实验室有限公司
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