LAMP (loop mediated isothermal amplification) detection primer combination and kit for GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene

Abstract

The invention provides a detection primer combination and kit for a GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene. The primer combination is designed in the sequence shown as SEQ ID No.1 as a target sequence and comprises F3, B3, FIP, BIP, LF and LB. When the primer combination is applied to an LAMP (loop mediated isothermal amplification) detection kit, results show that nonspecific amplification in primers and nonspecific binding amplification between the primers and a template are avoided for the primer combination, An LAMP amplified internal reference quality control system for a humanized sample is established successfully, and preliminary foundation is laid for widespread clinical use of the LAMP method in future.

Description

technical field [0001] The invention relates to a primer combination and a kit for GAPDH gene detection, belonging to the field of biotechnology. Background technique [0002] GAPDH or G3PDH is the English abbreviation of glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphatedehydrogenase). The enzyme is an enzyme in the glycolysis reaction, consisting of four subunits of 30-40kDa, with a molecular weight of 146kDa. The enzyme gene is a housekeeping gene, which is expressed at a high level in almost all tissues, and the protein expression in the same kind of cells or tissues is generally constant, and is not affected by the partial recognition sites contained, phorbol lipids, etc. Therefore, it is widely used as a standardized internal reference for PCR, extraction of total RNA, poly(A)+RNA, Western blot and other experimental operations. [0003] With the development of laboratory medicine, some molecular biology techniques, such as PCR and strand displaceme...

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Problems solved by technology

[0005] However, in recent years, the practical application of LAMP technology in the laboratory is relatively limited, and the progress is relatively slow. Based on the previous research, our research group found that there are two important reasons that limit its wide application, specifically: (1) the current lamp system It is difficult to achieve high-throughput detection in the true sense, and needs to be further improved; (2) Although the LAMP system does not need to increase or decrease the temperature during the detection process, it can greatly speed up the reaction speed and achieve efficient and rapid detection. However, compared with the traditional real-time PCR method Compared with the LAMP system, the stability of the LAMP system is poor, and there will often be amplification failures caused by sample nucleic acid extraction failures or excessive PCR inhibitors in the samples, resulting in false negative results. Therefore, in order to avoid false negatives, improve The reliability of the test results requires the introduction of an internal control amplification system to effectively monitor the process of sample processing and testing

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