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Genetic diagnosis reagent for Citrin deficiency disease and application of genetic diagnosis reagent

A technology for genetic diagnosis and diagnostic reagents, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc. It can solve the problems of high sample requirements, lack of SFDA certification for instrument detection, and limited popularity of instruments.

Inactive Publication Date: 2013-12-04
SHANGHAI LANMING BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Biochemical screening combined with gene sequencing detection technology has high requirements for samples, complex technical equipment, long time for detection, high cost, and is difficult to carry out on a large scale ("Chinese Journal of Medical Genetics" 2006 No. 6 655-658; BMC Med Genet .2013Feb10;14:24. doi:10.1186 / 1471-2350-14-24.)
Recently, researchers have tried to use HRM technology to screen several gene mutations involved in Citrin deficiency and have achieved some success, but the popularity of the corresponding instrument is limited, and the instrument has not yet obtained SFDA certification

Method used

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  • Genetic diagnosis reagent for Citrin deficiency disease and application of genetic diagnosis reagent
  • Genetic diagnosis reagent for Citrin deficiency disease and application of genetic diagnosis reagent
  • Genetic diagnosis reagent for Citrin deficiency disease and application of genetic diagnosis reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1: Preparation of SLC25A13 Haploid Genotype Standards

[0071] 1) Preparation of normal human genomic DNA

[0072] Normal human blood filter paper was extracted and prepared with Chlex100, amplified with gene-specific primers, routinely cloned into pMD18-T vector (Takara), sequenced with M13 universal primers, and confirmed that the SLC25A13 gene sequence was consistent with NT_079595. The concentration of normal human genome DNA was adjusted to 10ng / μl, or the concentration of T carrier containing SLC25A13 fragment was 1ng / μl as a 10-fold normal control template.

[0073] 2) Preparation of SLC25A13 mutant genotype standard

[0074] According to the SLC25A13 sequence and mutation, artificially synthesized 9 common Citrin-deficient mutant genes containing a single mutation: c.615+5G>A (IVS6+5G>A) (SEQ ID No.1), c.851_854del (851del4) (SEQ ID No.2 ), c.1177+1G>A (IVS11+1G>A) (SEQ ID No.3), c.1750+72_1751-4dup17ins (IVS16ins3kb) (SEQ ID No.4), c.615+1G>C (IVS6+1G...

Embodiment 2

[0075] Example 2: Design and synthesis of Citrin-deficient mutant gene amplification primers and MGB probes

[0076] Design synthetic primers according to the requirement that the length difference of the amplified gene sequence is not more than 10%, the extension time is not more than 45 seconds, and the difference in primer annealing temperature is not more than 5%. The wild type reporter probe is labeled Fam, and the mutant probe is labeled Tet (as shown in the table 1). Verify that fluorescent quantitative PCR primers specifically amplify target gene fragments, and TaqmanMGB site-specific probes report corresponding fluorescent signals. Artificially synthesized gene fragment-specific amplification primers and site-specific TaqmanMGB reporter probes. The sequences of gene fragment-specific primers and site-specific TaqmanMGB probes are shown in Table 1 (see SEQ ID No. 10 to SEQ ID No. 46).

[0077] Table 1

[0078]

[0079]

[0080]

Embodiment 3

[0081] Example 3: SLC25A13 Taqman quantitative PCR genotyping

[0082] 1) Standard genotype verification

[0083] Reaction system: primer mix 2.5 μl, 2x MultiplexPCRMix (Qiagen, Germany) 12.5 μl, standard template 2.5 μl, 5x EasyBuffer 5 μl, ddH 2 O2.5 μl.

[0084] Quantitative PCR instrument: AB7500 (AB, USA).

[0085] Reaction conditions: 95°C for 5 minutes; 94°C for 30 seconds-60°C for 30 seconds-72°C for 45 seconds, 40 cycles.

[0086] Result reading: If the signal is detected, it is determined as successful, and the existence of the gene locus is confirmed. No amplification was determined as negative and the locus was determined to be absent. The matching degree is required to be 100%. Signals were detected at all wild sites in the normal human genome, and no signals were detected at all mutations.

[0087] 2) Sample typing detection

[0088] Reaction system: 2.5 μl of primer mix, 12.5 μl of 2x MultiplexPCRMix (Qiagen, Germany), 2.5ul of sample genomic DNA (10 μg / μ...

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Abstract

The invention belongs to the technical field of biological products, and relates to a genetic diagnosis reagent for the Citrin deficiency disease and an application of the genetic diagnosis reagent. The quantitative PCR technology of Taqman MGB probes is used for detecting one or more of nine mutation sites of a Citrin deficiency disease gene SLC25A13, and therefore the majority of pathogenic mutations can be detected. According to the genetic diagnosis reagent for the Citrin deficiency disease, a quantitative PCR instrument and a common PCR reagent can be used; the nine Citrin deficiency disease mutant genetic loci are detected in an amplification mode so that a detection result can be obtained within 3-5 hours; the genetic diagnosis reagent can be used for detecting the Citrin deficiency disease in a single laboratory closed tube provided with the quantitative PCR instrument, thereby avoiding the situation that high concentration samples are contaminated; moreover, the genetic diagnosis reagent can be conveniently produced in a biotechnological company and used for detection in a biomedical detection mechanism, thereby meeting the requirement for industrialization promotion.

Description

technical field [0001] The invention belongs to the technical field of biological products, and in particular relates to a gene diagnostic reagent for Citrin deficiency and its application. Background technique [0002] Citrin is a mitochondrial calcium-binding aspartate / glutamate carrier (Aspartate / GlutamateCarrier, AGC) protein that plays an important role in the urea cycle and other metabolic processes. Citrin deficiency includes two different phenotypes, Adult Onset Type II Citrullinemia (CTLN2) and Neonatal Intrahepatic Cholestasis caused by Citrin Deficiency (NICCD), which are autosomal recessive. The heterozygous carrier rate of Citrin deficiency corresponding gene SLC25A13 gene mutation is common in East Asian countries, 1 / 69 in Japan, 1 / 79 in China, and 1 / 50 in South Korea. The incidence rate is about 1 / 17,000-34,000 (JInheritMetabDis. 2007Apr; 30(2): 139-44. MolAspectsMed. 2013Apr-Jun; 34(2-3): 465-84.). [0003] Citrin deficiency can be treated with food, medica...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 陈金中赵翊均
Owner SHANGHAI LANMING BIOTECH CO LTD
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