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Preparation method and application of gene chip for detecting important respiratory pathogenic viruses

A gene chip and respiratory technology, applied in the field of gene chip detection, can solve the problems of easy quenching, easy saturation of fluorescent signals, bulky volume, etc., and achieve the effects of good specificity, high specificity and high consistency rate.

Inactive Publication Date: 2012-07-18
深圳市普瑞康生物技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Disadvantages of this method: the fluorescence signal is easy to be saturated and quenched; there is autofluorescence phenomenon; more importantly, the detection instrument is expensive and bulky, which seriously limits the promotion and application of gene chips in China, especially in small and medium medical institutions And the promotion and application of on-site testing
[0007] Visual chip detection technology based on the principle of gold-labeled silver staining can greatly reduce the detection cost of biochips, but the detection sensitivity of single-step gold-labeled silver staining cannot meet the higher requirements in the field of molecular detection. Detection can completely replace fluorescence detection, which is of great significance for the promotion and application of biochip technology

Method used

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  • Preparation method and application of gene chip for detecting important respiratory pathogenic viruses
  • Preparation method and application of gene chip for detecting important respiratory pathogenic viruses
  • Preparation method and application of gene chip for detecting important respiratory pathogenic viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Development of a gene chip for visual detection of important respiratory pathogenic viruses

[0047] 1. Specific primer design and screening

[0048] In order to achieve specific amplification of 9 respiratory viruses, 9 pairs of specific primers were used to amplify in three tubes of multiplex RT-PCR system. The primer sequences and corresponding target virus information are shown in Table 1. The amplified products The results of agarose electrophoresis are attached figure 1 , it can be seen from the figure that all 9 kinds of respiratory viruses were specifically amplified.

[0049] 2. Screening of specific probes

[0050] The screening of various types of probes first excludes the non-specific crossover between the probes and other viruses in the respiratory tract and digestive tract, and then uses 9 kinds of viral RNAs as templates to hybridize the RT-PCR products with the alternative probes respectively to investigate the potential of each virus probe....

Embodiment 2

[0062] Example 2: Determination of Gene Chip Positive Judgment Criteria

[0063] The cutoff value is the standard for judging whether the signal value of the gene chip is positive. Each typing probe selects non-respiratory virus (i.e. negative strain) and blank control for gene chip hybridization, and through repeated experiments and data statistics, the negative virus The background statistical average value +2SD of strains and blank controls was used as the cutoff value of each probe, as shown in Table 6. The distinguishing ability of each respiratory virus detection probe is more than 2.5 times as the judgment standard of the virus positive.

[0064] Table 6 Determination of the Cutoff value of each probe

[0065]

Embodiment 3

[0066] Example 3: Specificity evaluation of gene chips for visual detection of important respiratory pathogenic viruses

[0067] Specificity is the most important assessment index of a diagnostic method. The present invention uses the optimized system and conditions to detect 9 strains of various types of common respiratory viruses. The chip detection results are shown in the attached image 3 . It can be seen from the figure that the present invention can correctly distinguish 9 common respiratory viruses with good specificity.

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Abstract

The invention relates to a gene chip for detecting important respiratory pathogenic viruses. A preparation method of the gene chip comprises the following steps of: preparing a specific primer; preparing a virus specific probe; preparing an oligonucleotide chip; establishing an RT-PCR (Reverse Transcription-Polymerase Chain Reaction) system; establishing a hybrid system; preparing a visual detection reagent; and establishing a developing method. The gene chip prepared by the invention can be used for simultaneously discriminating nine general respiratory pathogenic viruses comprising A and B type influenza viruses, parainfluenza viruses type 1 and 2, human metapneumovirus, respiratory syncytial virus, measles virus, rubella virus and mumps virus, provides a new solution for quickly detecting the general respiratory pathogenic viruses at high throughout and can provide guidance for monitoring, clinically diagnosing and treating the respiratory pathogenic viruses.

Description

technical field [0001] The invention relates to the preparation and application of a gene chip for detecting nine common respiratory pathogenic viruses, and belongs to the technical field of gene chip detection. Background technique [0002] Respiratory viruses refer to a large class of viruses that can invade the respiratory tract and cause local lesions of the respiratory tract or only use the respiratory tract as an invasion portal, mainly causing lesions of tissues and organs outside the respiratory tract. At present, there are at least 239 types of viruses in more than 10 genera of at least 7 virus families that can cause acute respiratory infections. Acute respiratory infection is one of the common clinical diseases and has become a public health problem that seriously threatens human health. According to the World Health Organization's statistical report in 2002, acute respiratory infection ranks third among the top ten causes of death in the world, and nearly four m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 王升启彭贤慧刘琪琦陈苏红张敏丽刘志红朱坤
Owner 深圳市普瑞康生物技术有限公司
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