Primer composition and kit for detecting autophagy key gene expression level in Drosophila melanogaster, and use method of kit

Abstract

The present invention discloses a primer composition and a kit for detecting the autophagy key gene expression level in Drosophila melanogaster, and a use method of the kit. The primer composition comprises RT amplification primers and PCR amplification primers of 10 autophagy key genes and a RNA internal reference. The kit comprises DEPC water, a 5* RT buffer liquid, reverse transcription primers, reverse transcriptase, a Z solution, a 10* PCR buffer liquid, PCR primers, a 25 mM magnesium chloride solution, DNA polymerase, and a positive reference substance. According to the present invention, with the primer composition and the kit, the 10 autophagy key genes of Drosophila melanogaster can be simultaneously detected, and the detection of 192 samples can be completed in one day, such that the production cost and the detection cost can be saved, and the detection efficiency can be increased; and the RNA internal reference provides the control internal reference of the RNA integrity of the sample, such that the determination on the sample quality during the test process can be ensured, the false negative can be avoided, and the detection has the good sensitivity and the good specificity so as to avoid the problem of not high specificity of other detection methods.

Description

technical field [0001] The invention relates to a multiple gene detection kit and its application, in particular to a primer composition, a kit and its application for detecting the expression level of key autophagy genes in Drosophila melanogaster. Background technique [0002] Autophagy is an important process for the turnover of intracellular substances that is evolutionarily conserved in eukaryotes. In this process, some damaged proteins or organelles are wrapped by autophagic vesicles with a double-membrane structure, and sent to lysosomes (animals) or vacuoles (yeast and plants) for degradation and recycling, thereby realizing the cell itself. metabolic needs and the renewal of certain organelles. People have known the existence of autophagy for a long time, but only after the pioneering research of Japanese scientist Yoshinori Ohsumi, the winner of the 2016 Nobel Prize in Physiology and Medicine, did people realize its mechanism and understand its importance. At pre...

AI Technical Summary

Problems solved by technology

The advantages of fluorescent quantitative PCR technology: high sensitivity and accurate quantification, but there are also the following disadvantages: 1) low throughput: if only one fluorescent marker is used, only one gene can be detected at a time

When a sample needs to detect multiple genes at the same time, it must be tested one by one, the cost is relatively high, the efficiency is low, the cycle is long, and it is impossible to start with a large number of samples

2) The cost is relatively high: if multiple genes need to be detected at the same time, multiple fluorescent markers are required; at present, a maximum of 4 to 5 fluorescent markers are used in common to mark each gene segment to be detected, so multiple fluorescent markers are used relatively high cost

[0006] (2) As a relatively new technology, RNA sequencing has the advantages of high sensitivity and high throughput, but it is expensive

It has the advantages of high throughput, but the technology is expensive and complicated, and the synthesis and fixation of probes are complicated, and it cannot be accurately quantified, with poor repeatability and low sensitivity.

[0008] (4) The fluorescent in situ hybridization method is to hybridize fluorescently labeled gene probes to intracellular nucleic acids in situ, and detect the brightness and quantitative gene expression levels through a fluorescent microscope, which has the disadvantages of low throughput, low sensitivity, and high cost.

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