Malignant hyperpyrexia multiplex PCR-LDR molecular diagnosis kit and application thereof

A technology of PCR-LDR and molecular diagnosis, which is applied in the field of multiple PCR-LDR molecular diagnosis kits for malignant hyperthermia, which can solve the problems of cumbersome operation, poor repeatability and high cost

Pending Publication Date: 2020-11-20
诚谨医学检验所(山东)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These methods have problems such as high cost, low a

Method used

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  • Malignant hyperpyrexia multiplex PCR-LDR molecular diagnosis kit and application thereof
  • Malignant hyperpyrexia multiplex PCR-LDR molecular diagnosis kit and application thereof
  • Malignant hyperpyrexia multiplex PCR-LDR molecular diagnosis kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 149

[0268] Example 1 PCR-LDR detection kit for 49 SNP sites and its use

[0269] 1. Kit composition:

[0270] PCR amplification reagents include: PCR buffer solution, MgCl2, the mixture Remi× of dNTPs, Ex-Taq enzyme, 18 pairs of primer mixtures (as shown in SEQ ID NO.1-36) and ultrapure water; LDR connection reaction reagent includes : 1 set of universal fluorescent probes and 49 sets of detection probe mixtures (as shown in SEQ ID NO.37-179), DNA ligase, 10× buffer and ultrapure water; genotyping reagents include: RO×-300 Internal standard, genotyping standards corresponding to 49 mutation sites used for genotyping; quality control products include: negative quality control product is sterilized double distilled water, wild type quality control product is 49 identified by sequencing The sites are all wild-type genomic DNA, and the positive quality control products are C.103T>C, C.130C>T, C.487C>T, C.488G>T, C.742G>A, C.742G>C , C.982C>T, C.1021G>C, C.1021G>A, C.1565A>C, C.1597C...

Embodiment 2

[0288] Embodiment 2 specificity test

[0289] Materials: 49 known positive mutant plasmid samples of SNP sites were taken, and 5 known negative samples were taken. All the above samples were verified by gene sequencing, and were tested with this kit at the same time. The results were analyzed and verified: 49 sites of PCR- The detection results of the LDR detection kit are completely consistent with the sequencing results, and the kit has strong specificity.

Embodiment 3

[0290] Example 3 Sensitivity test of mutation detection rate

[0291] With the wild-type mixed standard plasmid as the background, some mutant plasmids were added in proportions of 50%, 5%, 1%, 0.1%, and 0% to verify the sensitivity of mutation detection. The results show that the PCR-LDR-based 49 SNP site detection scheme can break through the limitations of traditional nucleic acid mutation detection technology, and detect multiple mutation sites with a detection sensitivity of up to 1%, see Figure 3A-C.

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Abstract

The invention discloses a malignant hyperpyrexia multiplex PCR-LDR molecular diagnosis kit. The malignant hyperpyrexia multiplex PCR-LDR molecular diagnosis kit comprises 18 pairs of specific primers(the nucleotide sequences are shown as SEQ ID NO. 1-36), one group of universal fluorescent probes and 49 groups of detection probes (the nucleotide sequences are shown as SEQ ID NO. 37-179). The kitalso comprises a PCR amplification reagent, an LDR ligation reagent, a genotyping reagent and quality control products, wherein the quality control products comprise a negative quality control product, a wild type quality control product and a positive quality control product. The kit can simultaneously detect 49 gene polymorphism sites, and can be used for genotyping, DNA polymorphism detection,malignant hyperpyrexia (MH) molecular diagnosis and the like. By the kit, the problems of false positive and false negative of results are solved due to directness of sequencing results. The kit hasthe advantages of high flux, high sensitivity, simplicity in operation, short flow, simpler result analysis, cost saving, low cost and the like.

Description

technical field [0001] The invention relates to a malignant hyperthermia multiplex PCR-LDR molecular diagnosis kit and application thereof, belonging to the technical field of DNA molecular detection. Background technique [0002] Malignant hyperthermia (MH) is a family hereditary subclinical skeletal muscle genetic pharmacology disease, often treated by halogen-containing volatile inhaled anesthetics (halothane, enflurane, isoflurane, etc.) Induced by depolarizing muscle relaxants (succinylcholine), manifested as hyperthermia, muscle rigidity, tachycardia, mixed acidosis, elevated muscle enzymes, myoglobinuria, etc. sign. The incidence of succinylcholine for general anesthesia is about 1 / 220,000. There are also domestic reports of suspected malignant hyperthermia induced by ketamine, lidocaine, haloperidol, and clozaza. In foreign literature, MH is called a terrible unstable metabolic disease. The disease develops dangerously and is unpredictable. Because the body is in ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6883C12Q1/6858C12Q2600/156C12Q2600/166
Inventor 步迅刘艳艳杨雪段文元马素军
Owner 诚谨医学检验所(山东)有限公司
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