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Multiplex PCR (polymerase chain reaction) kit for detecting leukemia fusion genes

A technology that integrates genes and leukemia, and is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc. It can solve the problems of affecting amplification efficiency, complex detection methods, and high professional requirements, so as to improve detection efficiency and save reagent usage. , The effect of high clinical detection rate

Inactive Publication Date: 2014-07-23
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

Since multiple pairs of primers are prone to interact in the same reaction system, such as the formation of hairpin structures, dimer structures, etc., thereby affecting the amplification efficiency, this is the main reason for dividing into eight tubes for detection, but it also leads to this problem. Such detection methods are complex and cumbersome, and have high requirements for operators related to the profession, so it is difficult to popularize

Method used

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  • Multiplex PCR (polymerase chain reaction) kit for detecting leukemia fusion genes
  • Multiplex PCR (polymerase chain reaction) kit for detecting leukemia fusion genes
  • Multiplex PCR (polymerase chain reaction) kit for detecting leukemia fusion genes

Examples

Experimental program
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Effect test

Embodiment 1

[0031] In this example, multiple primers were validated by clinically confirmed specimens of patients carrying fusion genes of AML1 / ETO, BCR / ABL e1a2, BCR / ABL e13a2, BCR / ABL e14a2, PML / RARα L type, PML / RARα S type, and E2A / PBX1 type I The feasibility of combining the working principle of public primers and the sensitivity of detection.

[0032] The multiplex PCR kit for detecting leukemia fusion genes used in this example includes: conventional multiplex PCR components, chimeric primers and public primer pairs for common leukemia fusion genes. The public primer pairs and chimeric primers (multiple primer sequences with public primers) for common leukemia fusion genes and the corresponding detected fusion genes are shown in Table 1.

[0033]Table 1

[0034]

[0035] The above conventional multiplex PCR components include: positive control primers, Taq DNA polymerase, dNTP, MgCl 2 , PCR buffer, deionized. The specific sequence of the positive control primer is: SEQ ID NO....

Embodiment 2

[0039] Embodiment 2, public primer sensitivity detection

[0040] The plasmids containing the common primer sequences at different concentrations were detected by the common primers SEQ ID No.1 and SEQ ID No.2. After extracting the plasmid containing the public primer sequence from Escherichia coli, use deionized water to dilute the gradient by 10 times, from 10 7 copy to 10 1 Copy and dilute the plasmid template with different concentrations as the DNA amplification template of the PCR reaction system; use 25 μL PCR reaction system: each 25 μL PCR reaction system includes: 5 U / μL Taq polymerase 0.15 μL, 25 mM MgCl 2 2 μL, 10 mM dNTP 0.5 μL, 10xTaqBuffer 2.5 μL, two public primers with a concentration of 10 μM each 1 μL, plasmid templates with different concentrations 1 μL, deionized water 16.85 μL; PCR reaction conditions are: denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, Anneal at 55°C for 30s, extend at 72°C for 30s, a total of 35 cycles; final...

Embodiment 3

[0041] Embodiment three, multiplex PCR system sensitivity detection

[0042] Using the cDNA plasmid templates of e1a2 and e14a2 types of BCR / ABL, L and S types of PML / RARα, AML1 / ETO, and E2A / PBX1 type I fusion genes, the public primers and multiplex primers were detected by two rounds of PCR reactions using multiplex PCR The specificity and sensitivity of the primers used are SEQ ID No.1-SEQ ID No.14. Its working principle is as attached image 3 As shown, in the first round of PCR reaction, chimeric primers were used for amplification, and lower concentrations of chimeric primers were used to reduce the probability of interaction between primers to reduce dimers and possible non-specific amplification. The product fragments with common primer sequences at both ends are obtained as templates for the second round of amplification; the second round of PCR reaction is based on the public primers for the first round of product amplification fragments, the purpose is to improve th...

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Abstract

The invention belongs to the field of gene detection, and in particular relates to a multiplex PCR (polymerase chain reaction) kit for detecting leukemia fusion genes. The kit comprises a conventional multiplex PCR component, and chimeric primer and common primer pairs aiming at the common leukemia fusion genes. Chimeric primers are sequences of adding common primers to the 5' ends of specific primers of seven shear isomers of the common leukemia fusion genes BCR / ABL, PML / RARalpha, AML1 / ETO and E2A / PBX1. By using the multiplex PCR kit for detecting the leukemia fusion genes, seven leukemia fusion genes including AML1 / ETO, BCR / ABLe1a2, BCR / ABLe13a2, BCR / ABLe14a2, PML / RARalphaL, PML / RARalphaS and E2A / PBX11 with high incidence can be simultaneously detected, so that the consumption of reagents can be saved, the detection cost can be reduced, and meanwhile, the detection efficiency is improved. Moreover, the highest detection sensitivity of the kit can reach 10 copies per 25mu L, and the kit is simple in operation and high in clinical detection rate, so that the kit has clinical popularization and application values.

Description

technical field [0001] The invention belongs to the field of gene detection, in particular to a multiplex PCR kit for detecting four common leukemia fusion genes. Background technique [0002] The working principle of multiplex PCR is to add multiple pairs of primers to the same PCR reaction system to simultaneously amplify multiple DNA fragments. As one of the important derivative technologies of PCR, this method is efficient, economical and simple, and can greatly save time and Reagent cost and many other advantages. At present, this technology has been widely used in biotechnology, quarantine and medical testing and other fields. [0003] In a multiplex PCR reaction, the following reagents are generally included: two or more primer pairs, dNTP, MgCl 2 , PCR buffer, template DNA, Taq DNA polymerase, and other adjuvants (DMSO, glycerol, BSA), usually in order to obtain better amplification results, the amount of reagents used, the reaction conditions of PCR amplification,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2535/125C12Q2531/113C12Q2537/143
Inventor 孙万平胡颖熹张舒羽张国兴姚利李凯陈子兴
Owner SUZHOU UNIV
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