Paper-based micro-fluidic chip enhancement type chemiluminescence gene sensing method

Abstract

The invention discloses a paper-based micro-fluidic chip enhancement type chemiluminescence hlyA gene detection method. The method comprises the steps that (1) a screen printing board is pressed on chromatography paper, coated with wax and heated, and the chromatography paper is naturally disengaged from the screen printing board and aired; (2) a catching probe and a signal probe are designed; (3) a paper-based micro-fluidic chip is pretreated; (4) a DNA sample to be tested and the catching probe are incubated, the signal probe is added to conduct a hybridization reaction with the DNA sample to be tested, and HRP-SA is added for incubation; finally a base solution is added to trigger enhancement type chemiluminescence, and an luminescence signal is collected by a CCD digital imaging device in an imaging mode. Compared with an expensive and complex optical imaging system, the simple CCD device is adopted for imaging detection, an enhancement type chemiluminescence system is combined with a biotin-streptavidin affine magnification system, the detection flexibility of paper-based micro-fluidic chip enhancement type chemiluminescence is greatly improved, and the detection limit can reach 6.3*10<-2>pmol / L.

Description

technical field [0001] The invention belongs to the field of gene detection, and in particular relates to a paper-based microfluidic chip-enhanced chemiluminescent method for detecting Listeria monocytogenes hlyA gene. Background technique [0002] A gene is a functional DNA molecular fragment, the basic unit of genetic information, and the most basic determinant of all species. Genetic detection refers to a technology that detects target nucleic acid molecules through certain detection methods, and analyzes the target nucleic acid molecule disease-causing genes, disease susceptibility genes, etc. Listeria monocytogenes (abbreviated as "Listeria monocytogenes" in some places in this paper), as an important food-borne pathogen, can cause human and animal infection diseases, and it is composed of a variety of virulence factors. Since Listeria lysin was reported by the Parrisius group in 1985, there has been much evidence that Listeria monocytogenes LLO, which is encoded by th...

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Problems solved by technology

[0008] (1) The target DNA length is less than 50bp; the longer the target DNA length, the wider the application range, and the application range is obviously narrower than 50bp;

[0009] (2) The heating process in the wax screen printing method is 130°C (150s). If the temperature is too high and the time is too long, the properties of the paper will change, which is not conducive to the subsequent detection reaction;

[0010] (3) Rinsing is carried out in the reaction pool, which will lead to unclean rinsing, and multiple rinsing will lead to cross-contamination of reagents;

[0011] (4) The signal substance used - nanoporous gold adsorbed by carbon quantum dots, its synthesis process is relatively complicated, and the process of marking the signal probe with the signal substance is complicated and takes a long time;

[0012] (5) A weak luminescence analyzer is used to collect and analyze chemiluminescence signals. Although it can achieve low sensitivity detection, the analyzer is expensive, resulting in high detection costs, and does not have the ability to detect high-throughput

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