Method, primer, probe and kit for screening and identifying BCR-ABL unusual fusion types

Abstract

The invention discloses a method, primer, probe and kit for detecting six unusual fusion types of BCR-ABL fusion genes such as e6a2, e8a2, e19a2, e1a3, e13a3 and e14a3. The method comprises the following steps: performing primary screening on blood samples by adopting two tubes of fluorescent quantitative polymerase chain reaction (PCR), and quantitatively indentifying specific types of the blood samples belonging to the a2 or a3 group through primary screening judgment. Therefore, the screening cost is reduced, and the detection throughput is also improved. According to the detection method, primer, probe and kit, high sensitivity, high specificity and high detection throughput can be realized, and the method is a rapid and accurate type screening and identifying method.

Description

Technical field [0001] The present invention belongs to the field of life sciences and biotechnology, and particularly relates to a method, primers, probes and kits for clinically screening for unusual fusion types of BCR-ABL fusion genes. It adopts real-time fluorescent quantitative PCR technology to analyze human blood samples. The 6 rare types of BCR-ABL fusion genes e6a2, e8a2, e19a2, e1a3, e13a3, and e14a3 were initially screened and identified. Background technique [0002] The BCR-ABL fusion gene is produced by translocation of the long arm of chromosome 9 to the long arm of chromosome 22, resulting in the fusion of ABL proto-oncogene and BCR gene. Chromosome 22 after the t(9;22)(q34;q11) translocation is also called the Philadelphia chromosome (Ph chromosome). Ph chromosome and BCR-ABL fusion gene are the molecular basis of chronic myeloid leukemia (CML). In addition, the BCR-ABL fusion gene can also be found in Acute Lymphoblastic Leukemia (ALL), but rarely in Acute My...

AI Technical Summary

Problems solved by technology

This screening method judged by naked eyes has the following disadvantages: (1) There are cases of missed detection in the screening of uncommon types of BCR-ABL fusion gene

First of all, the premise of karyotype analysis is that the nucleated cells in the sample need to be cultured and have nuclear division phase; if the cell culture fails and there is no nuclear division phase, the analysis cannot be performed

Secondly, in the process of cell culture and proliferation, certain types of cells will overgrow, and if normal cells overgrow, the diseased cells will be "submerged" and cause the illusion of normal karyotype

In the case that the bone marrow cannot be extracted, the peripheral blood is extracted for karyotype analysis, and false negatives may also occur.

Furthermore, in some cases, the chromosomal translocation is extremely small, and the occult Ph chromosome appears, which cannot be found in the chromosome examination by naked eye analysis.

When the FISH method detects BCR-ABL fusion, although it does not need to culture cells, it can also detect the occult Ph chromosome, but the cost of the inspection is too high, and most patients will not choose this method for detection, so it is not true. The role of screening

(2) It is impossible to distinguish the specific type of BCR-ABL, so it is impossible to carry out targeted monitoring of minimal residual disease (Minimal Residua Disease, MRD)

(3) Low detection success rate and sensitivity

The disadvantages of multiplex PCR combined with agarose gel electrophoresis or sequencing are as follows: (1) there is non-specific amplification, and multiple PCR product bands will appear during electrophoresis, which is prone to false positives; (2) analysis by electrophoresis There is subjectivity in judging the results, especially in the monitoring of MRD, the critical results cannot be clearly analyzed and judged, and the cost will be increased through sequencing, and the time period will be greatly extended; (3) The sensitivity is lower than that of nested PCR and fluorescent PCR; (4) only qualitative detection; (5) time-consuming, if the PCR product is identified by electrophoresis, it will take 5-6 hours; if it is identified by sequencing, it will take 10-12 hours

Nested PCR combined with agarose gel electrophoresis or sequencing also has the following disadvantages: (1) The process is cumbersome and easy to pollute, and the judgment of critical results in electrophoresis identification is subjective; (2) There are many PCR reaction systems and high cost. Not suitable for high-throughput sample detection; (3) only qualitative detection; (4) time-consuming, if the PCR product is identified by electrophoresis, it takes 5-6 hours; if it is identified by sequencing, it takes 10-12 hours

Therefore, no matter whether multiplex PCR (mμltiplex PCR) or nested PCR (nested PCR) is used to amplify first, and then use agarose gel electrophoresis or sequencing to identify the uncommon type of BCR-ABL fusion gene, it cannot simultaneously meet the requirements. Advantages of high sensitivity and specificity

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