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Gene chip for detecting various pathogens carried by mosquitoes and detection method

A gene chip and pathogen technology, applied in the field of gene chips, can solve problems such as no one has done it yet, and achieve the effects of high specificity, simple operation steps and good stability.

Inactive Publication Date: 2015-07-29
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, gene chips have been used to detect multiple viruses of a certain genus. For example, the patent No. CN 101781687A discloses a gene chip for detecting multiple viruses of the genus Flavivirus, but for simultaneous The gene chip for detecting pathogens of multiple genera (viruses, protozoa, worms, etc.) with far different taxonomic status has not yet been carried out.

Method used

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  • Gene chip for detecting various pathogens carried by mosquitoes and detection method
  • Gene chip for detecting various pathogens carried by mosquitoes and detection method
  • Gene chip for detecting various pathogens carried by mosquitoes and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Preparation of gene chip

[0033] 1. Design of gene probes

[0034] There are many ways to design gene probes, and the common principles followed by different methods are: the true melting temperature T of the probe m It should be consistent with the design temperature. In this example, Primer Primer 5.0 software was used to design all probes and primers. The sequences of each probe are shown in Table 1:

[0035] Table 1 Probe sequence list

[0036]

[0037]

[0038] 2. Preparation of gene chip

[0039] The synthesis of the gene probe was entrusted to Shanghai Sangon Bioengineering Service Co., Ltd., and the purification method was HPLC.

[0040] The equipment used for gene chip spotting was GMS417 gene chip spotting instrument, Affymetrix Company, USA, and the spotting reagents were provided by Shanghai Bio-Tech Co., Ltd.

[0041] The spotting mode of the gene chip is as follows: each probe repeats 3 spots to make a 6×12 DNA microarray. The speci...

Embodiment 2

[0053] Embodiment 2: Gene chip detection method for various pathogens carried by mosquitoes

[0054] 1. Construction of gene chip detection system

[0055] 1. Virus DNA extraction: If detecting mosquitoes, put the mosquitoes to be tested into a 1.5mL centrifuge tube and grind until they are completely crushed. Use Tiangen DNA Extraction Kit DP304 (Beijing Tiangen) to extract pathogen DNA from mosquitoes or patient blood according to the instructions.

[0056] 2. Establishment of PCR amplification system

[0057] It was carried out in the Pathogenic Biology Department of the Second Military Medical University, and the equipment and reagents used are as follows:

[0058] Equipment: TC-96 / G / H(b)A Gene Amplifier, Hangzhou Bioer;

[0059] Reagents: Reaction solution A, 10× buffer: provided by Beijing Tiangen; dNTP: provided by Shanghai Sangon Bioengineering Technology Service Co., Ltd.; primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. The ...

Embodiment 3

[0073] Embodiment three: the processing of gene chip kit

[0074] 1. Processing of kits

[0075] The gene chip kit was processed according to the system and conditions established in the above examples, and the composition of the kit is shown in Table 6.

[0076] Table 6 Composition of gene chip kit

[0077] component name

Specification

quantity

Gene detection chip (N)

1 serving / sheet, 50 sheets / box

1

Hybridization Chromogenic Kit

50 times / box

1

Amplification solution 1~3

50 times / box

1

Reaction A

100μL / tube

1

Reaction solution B

50μL / tube

1

[0078] Note: Amplification solutions 1-3 are Flavivirus amplification solution, Filaria amplification solution and Plasmodium amplification solution respectively.

[0079] 2. Instructions for the operation of the kit

[0080] (1) Amplification

[0081] Step 1: Take the reagent out of the kit, let it thaw at room temperature, centrifug...

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Abstract

The invention provides a gene chip and a detection method thereof capable of simultaneously detecting various pathogens of the important mosquito-borne disease epidemic in China. The gene chip comprises a substrate, and a flavivirus probe, a filaria probe and a plasmodium probe which are fixed on the substrate, so that the gene chip can detect various pathogens carried by mosquitoes simultaneously, meanwhile, the detection method provided by the invention is simple in operation step, high in specificity, good in stability and suitable for detection of mosquito-borne pathogens in mosquitoes, patients' blood and tissue fluid, a method for early detection and finding of popularization of the mosquito-borne disease epidemic efficiently at a low cost is provided for prevention of the mosquito-borne disease and an effective reference material is provided for prevention of the mosquito-borne disease.

Description

technical field [0001] The invention belongs to the field of gene chips, and in particular relates to a gene chip for detecting various pathogens carried by mosquitoes and a detection method. Background technique [0002] Mosquito-borne diseases occupy an important position in preventive medicine. Because they are transmitted by mosquitoes, they have strong transmission power, high incidence rate and wide range of harm, which pose a great threat to human health and life safety. The mosquito-borne diseases that are prevalent or have been prevalent in my country include malaria, lymphatic filariasis, Japanese encephalitis, and dengue fever. Detection of mosquito-borne pathogens is one of the effective measures for early detection of epidemics and prevention of mosquito-borne diseases. [0003] A simple and accurate detection method is an important technical guarantee for the discovery of mosquito-borne pathogens. Understanding its prevalence and trends can provide a scientifi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C40B40/06
CPCY02A50/30
Inventor 朱淮民周正斌彭恒李翔宇
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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