Quick detection method of pathogenic microbe diagnosis type gene chip

Abstract

The quick detection method of pathogenic microbe diagnosis type gene chip includes the following steps: preparing slide; preparing probe of length from several hundred to several thousand base and concentration of 0.25-0.5 microgram / microliter via PCR proliferation of genes and specific segments in pathogen genome; preparing chip via sample application with gene chip sample applying instrument; printing and processing; processing sample via incorporating marker Cy5-dUTP in PCR proliferation and introducing restrictive enzyme incision to obtain segment of 200-600 bps length; hybridizing in 2X hybridizing liquid comprising 60 % formamide, 12XSSC and 0.24 % SDS; cleaning after hybridization; and scanning detection. The present invention has the advantages of high marking efficiency, stable hybridizing detection result, greatly shortened hybridization time, etc. and is especially suitable for the diagnosis of infectious diseases clinically.

Description

technical field [0001] The invention relates to a detection method of a gene chip, in particular to a rapid detection method of a pathogenic microorganism diagnostic gene chip. Background technique [0002] The two main application areas of gene chip technology are expression profile detection and pathogenic microorganism diagnosis. situation or simultaneous detection of multiple pathogenic microorganisms or different subtypes of the same pathogenic microorganism. [0003] At present, the main bottlenecks restricting the application of gene chips in the diagnosis of clinical infectious diseases in terms of technology are as follows: [0004] (1) The labeling efficiency is not high and the effect is uncertain: There are many labeling methods for target DNA fragments, but there are mainly three types, one is the incorporation of fluorescently labeled dNTPs during the extension or amplification process; the other is The primers are labeled directly and then extended; the thir...

AI Technical Summary

Problems solved by technology

[0004] (1) The labeling efficiency is not high and the effect is not sure: there are many labeling methods for target DNA fragments, but there are mainly three types, one is the incorporation of fluorescently labeled dNTPs in the process of extension or amplification; the other is Labeling directly on the primers followed by extension; a third method involves incorporation of aa-dNTPs during extension or amplification, followed by indirect labeling of the aa-dNTPs with fluorescein

The former method involves the ratio relationship between each dNTP and the fluorescently labeled dNTP. At present, there is no unified standard, and an inappropriate ratio will seriously affect the incorporation efficiency and further affect the hybridization results.

In addition, the non-uniform incorporation efficiency makes it difficult to establish a correlation between the amount of target fragments and the fluorescence intensity after hybridization, which makes it difficult to use the chip for quantitative analysis.

Although the second method is more economical, because there are only fluorescent substances on the primers, the fluorescent intensity of the label is low, especially for the target fragment with low copy number, its sensitivity is not high

Although the third method can amplify the signal intensity, the operation is cumbersome and not easy to put into clinical application

[0005] (2) Poor control of hybridization kinetic conditions: The hybridization process of the gene chip is affected by various factors such as the length of the probe and the fluorescent molecule, Tm value, steric hindrance, etc. The current probes mainly include PCR products and Oligo , the labeling of the sample mainly includes the amplification of the whole genome or the amplification of a specific fragment

Poor control of hybridization kinetics between probes and sample molecules will seriously affect hybridization results

[0006] (3) The hybridization time is too long: Currently, the commonly used hybridization temperatures mainly include high temperature (55-65°C) and low temperature (42°C). The main feature is that formamide is added to the low-temperature hybridization buffer to reduce the annealing temperature. The usual hybridization time takes 16-20 hours, especially for high-temperature hybridization, because it is easy to evaporate and requires a large amount of sample solution, the hybridization time should not be too long

Too long hybridization time limits the industrialization process of diagnostic chips for infectious diseases to a certain extent

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