Primers and kit used in loop-mediated isothermal amplification detection method of PML-RAR alpha fusion gene

Abstract

The invention discloses primers and a kit used in loop-mediated isothermal amplification detection method of PML-RAR alpha fusion gene and the primers and the kit are used for solving the problems in conventional detection of leukemia minimal residual disease that false positive easily appears, the detection cost is high, the operation is complex and the detection time is long. According to the sequence of the PML-RAR alpha gene, multiple groups of primers are designed by virtue of PrimerExplorer V4 software, a pair of specific inner primers F3 and B3 and a pair of specific outer primers FIP and BIP are screened and the kit for detection of PML-RAR alpha gene in the acute promyelocytic leukemia minimal residual disease is established. The kit has the advantages of high amplification efficiency, good specificity and low requirement on instruments and equipment, a constant-temperature water bath pot is only needed, which is easily realized in clinic, the detection time is not more than one hour, which is less than a half of the detection time of the conventional PCR.

Description

technical field [0001] The invention relates to a rapid gene detection method, in particular to a primer and a kit for a loop-mediated isothermal amplification detection method of a PML-RARα fusion gene. Background technique [0002] Acute promyelocytic leukemia (APL) is the most dangerous type of non-lymphocytic leukemia clinically. Due to the application of all-trans retinoic acid and arsenic trioxide, the complete remission rate of APL has reached more than 80%. However, leukemia relapse has always been the main obstacle to clinical consolidation after remission, maintenance therapy and overall survival of patients, and the root cause of relapse is mainly from residual leukemia cells in the body, that is, minimal residual disease (MRD) in acute leukemia. Leukemia patients after remission need to detect minimal residual cells regularly to prevent relapse. Simple and accurate rapid detection will greatly facilitate patients' reexamination, improve the reexamination rate, se...

AI Technical Summary

Problems solved by technology

PCR takes a long time, low sensitivity and specificity, high instrument requirements

Although the specificity and sensitivity of the real-time fluorescent quantitative PCR method have been improved, it needs fluorescent primers and probes, expensive detection equipment, and takes 2-3 hours

[0004] Loop-mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) is a novel constant temperature nucleic acid amplification method developed in 2000. Compared with conventional PCR, it does not require thermal denaturation of the template, temperature cycling, electrophoresis and ultraviolet light. Observation and other processes are simple, fast, and specific, and can achieve on-site high-throughput rapid detection without relying on any special equipment. The detection cost is much lower than that of fluorescent quantitative PCR. The promotion period of this method is mainly for microbial detection. Now it has been widely used in the field of microbial detection, and many related patents have been authorized. However, due to insufficient understanding of researchers and the fact that human genes are more complex than microorganisms, there are no reports of using LAMP method to detect leukemia PML-RARα gene at home and abroad.

[0005] LAMP primers designed for the same gene sequence have many differences in amplification efficiency and specificity. LAMP primers can be automatically generated by software, but whether fast and specific primers can be screened from many primer sets is the key to the detection of genes by the LAMP method. The essential

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