Primer group for identifying three kinds of spliceosomes of PML-RAR[alpha] fusion gene and test kit and application of primer group

Abstract

The invention discloses a primer group for identifying three kinds of spliceosomes of a PML-RAR[alpha] fusion gene and a test kit and application of the primer group. The primer group for identifyingthree kinds of spliceosomes of the PML-RAR[alpha] fusion gene comprises a specific forward primer, a reverse primer, a specific fluorescent probe and a competitive primer which are used for detectingPML-RAR[alpha] fusion genes bcr1, bcr2 and bcr3 spliceosomes; according to the invention, a real-time fluorescent quantitative PCR technology is applied, multiple PCR primers and the specific fluorescent probe are adopted, and the three spliceosomes bcr1, bcr2 and bcr3 of the PML-RAR[alpha] fusion gene in a sample can be simultaneously detected in one reaction through the competitive primer; the primer group can be widely applied to multiple fields such as clinical early diagnosis, drug curative effect, after-healing monitoring and scientific research of PML-RAR[alpha] fusion gene related diseases, and has the characteristics of semi-automatic detection, high detection speed, high sensitivity and wide sample application range.

Description

technical field [0001] The present invention relates to the technical field of gene detection, and more specifically, relates to a primer set for identifying three spliced ​​bodies of PML-RARA fusion gene, a kit thereof and applications thereof. Background technique [0002] More than 90% of patients with acute promyelocytic leukemia (APL, M3 type) are caused by the karyotype t(15;17)(q22;q21) translocation, which combines the PML gene in 15q22 with the PML gene in 17q21. The RARα gene is fused to produce two fusion genes, PML-RARα and RARα-PML. The PML-RARα fusion gene has heterogeneity. During its formation, the breakpoint on RARα is always located in the second intron within the 17kb length range, but there are many variations on the breakpoint on PML, mainly concentrated in 3 A breakpoint cluster (bcr): the breakpoint bcrl is in the sixth intron of PML, forming a long transcript (L); the breakpoint bcr2 is in the sixth exon of PML, producing a variable transcript ( V);...

AI Technical Summary

Problems solved by technology

Southern blotting technology has a wide range of applications in the qualitative and quantitative detection of specific fusion genes, gene mutation analysis, restriction map analysis, gene rearrangement and loss detection, and has the characteristics of high sensitivity, but it also has complex operations, radioactive contamination, and short half-life. , instability and other shortcomings, it is difficult to use in large scale clinically; fluorescence in situ hybridization technology uses fluorescently labeled specific nucleic acid probes to hybridize with target DNA molecules, and the specificity is determined by observing the fluorescent signals under a fluorescent microscope or confocal laser scanner. The amount of stained target DNA after probe hybridization is currently a commonly used clinical method for detecting fusion genes, but the interpretation of the results depends on the subjective interpretation ability of the reader, which requires relatively high requirements for relevant experience and interpretation techniques, and is highly subjective. High cost, low throughput

[0005] As for the use of fluorescent PCR technology to detect the three spliced ​​bodies of the PML-RARα fusion gene, the patent CN102925558A discloses a kit for detecting the mRNA expression of the PML-RARα fusion gene. The detection process requires three PCR reaction systems to detect PML at the same time. -There may be three spliced ​​forms of the RARα fusion gene, and the purpose of identifying and distinguishing the three spliced ​​forms of the PML-RARα fusion gene in one reaction system cannot be achieved, which has the disadvantages of time-consuming, laborious, and high reagent costs

Chen Z et al. (2015) disclosed a RT-qPCR method for multiple detection of acute promyelocytic leukemia PML-RARa fusion gene 3 splice bodies in a single tube (Chen, Zhanguo, et al. "Development and Validation of a 3- PlexRT-qPCR Assay for the Simultaneous Detection and Quantitation of the ThreePML-RARa Fusion Transcripts in Acute Promyelocytic Leukemia."Plos One10.3(2015):e0122530.), RARA is detected by 3 primers and a probe, and PML primer 5 is Primers for bcr1 and bcr2; PML primer 6 is a primer for bcr3. These primers share one probe (RARA probe). Although they can simultaneously detect three spliced ​​forms of the PML-RARa fusion gene in one reaction system, they still Three types of PML-RARα cannot be effectively distinguished

Images