Preparation and application of gene chip for detecting important enteric causative viruses

A gene chip and enterovirus technology, applied in the direction of microbe-based methods, biochemical equipment and methods, microbiological measurement/inspection, etc., can solve the problems of easy quenching, easy saturation of fluorescent signal, bulky volume, etc., and achieve specificity The effect of good sex, high specificity and high consistency rate

Inactive Publication Date: 2013-08-07
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 fluorescent signal is easily saturated and quenched; there is autofluorescence; and the biggest disadvantage is that the detection instrument is expensive and bulky, which seriously limits the popularization and application of gene chips, especially in small and medium medical institutions and on-site detection Promoted application of
[0008] Visual 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. It 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 and application of gene chip for detecting important enteric causative viruses
  • Preparation and application of gene chip for detecting important enteric causative viruses
  • Preparation and application of gene chip for detecting important enteric causative viruses

Examples

Experimental program
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Effect test

Embodiment 1

[0046] Example 1: Development of gene chip for visual detection of important enteropathogenic virus nucleic acid

[0047] 1. Specific primer design and screening

[0048] In order to achieve specific amplification of 10 enteropathogenic viruses, 5 pairs of screened specific primers were used to amplify in three-tube multiplex RT-PCR system. The primer sequences and corresponding target virus information are shown in Table 1. The agarose electrophoresis results of the amplified products are shown in the appendix figure 1 , it can be seen from the figure that all 10 enteropathogenic viruses were specifically amplified.

[0049] 2. Screening of specific probes

[0050] The screening of various types of probes first excludes non-specific crossover between the probes and other viruses in the respiratory tract and digestive tract, and then uses RT-PCR products of 10 kinds of viral RNA as templates to hybridize with alternative probes respectively, and investigates the potential of...

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-enteroviruses (ie negative strains) and blank controls for gene chip hybridization. Through repeated experiments and data statistics, the negative The background statistical average value +2SD of the virus strain and the blank control was used as the cutoff value of each probe, as shown in Table 6. The discrimination ability of each enteropathogenic 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 chip for visual detection of important enteropathogenic virus nucleic acid

[0067] Specificity is the most important assessment index of a diagnostic method. The present invention uses optimized systems and conditions to detect 10 strains of various types of common enteroviruses. 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 10 common enteroviruses with good specificity.

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Abstract

The invention relates to a gene chip for detecting important enteric causative viruses. The preparation method for the gene chip for detecting important enteric causative viruses comprises the following steps: preparing a specific primer; preparing a virus-specific probe; preparing an oligonucleotide chip; building an RT-PCR (Reverse Transcription-Polymerase Chain Reaction) system; building a hybrid system; preparing a visual detection reagent; and building a color developing method. According to the gene chip prepared by the invention, 10 types of common enteric causative viruses can be simultaneously screened, including poliomyelitis virus I, II and II, enterovirus 71 type, coxsackie virus A16type, coxsackie virus B3, B4 and B5 types, echovirus 30 type and Rotavirus. According to the invention, a new solution for high-flux and quick detection of common enteric causative viruses can be provided, and guidance for monitoring, clinically diagnosing and treating the enteric causative viruses can be provided.

Description

technical field [0001] The invention relates to the preparation and application of a visualized gene chip for nucleic acid detection of ten common intestinal pathogenic viruses, and belongs to the technical field of gene chip detection. Background technique [0002] Enteroviruses are a large group of viruses transmitted through the fecal-oral route and infected through the digestive tract. Although its infection begins in the gastrointestinal tract, it rarely causes disease in these areas. Enteroviruses belong to the picornaviridae family of RNA viruses, including poliovirus, Coxsackievirus, enterocytopathic human orphan virus (ECHO for short), and There are 71 serotypes of new enteroviruses, and enteroviruses are infectious diseases caused by viruses. Mild clinical manifestations include fatigue, fatigue, and low-grade fever. Severe cases can cause systemic infection, damage to important organs such as the brain, spinal cord, heart, and liver, and poor prognosis, leaving ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C40B40/06C40B50/14C12R1/93
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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