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Preparation and application of adenovirus parting gene chip

A technology of gene chip and gene chip detection, which is applied in the field of gene chip detection, can solve the problems of complex operation, time-consuming, inability to accurately determine the type, etc., and achieve the effect of high specificity, accurate screening, and good specificity

Inactive Publication Date: 2016-06-15
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the detection methods of adenovirus mainly include immunological methods (virus isolation test, serum neutralization test, antigen antibody detection, etc.), which are time-consuming, complicated to operate, and cannot accurately determine the type; PCR method can only detect one or several pathogens and cannot detect many serotypes; gene sequencing is mainly used to determine new types of viruses

Method used

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  • Preparation and application of adenovirus parting gene chip
  • Preparation and application of adenovirus parting gene chip
  • Preparation and application of adenovirus parting gene chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Development of five kinds of adenovirus typing gene chips

[0040] 1. Primer probe design and screening

[0041] First, the adenovirus target gene sequence was downloaded from the NCBI gene database. After the sequence was downloaded, the AlignX program in the VectorNTIAdvance10 (invitrogen) software package was used to perform a global alignment of the gene sequences of each type according to the default parameter settings. Design specific oligonucleotide probes, general and specific primers at the conserved positions of the gene sequence according to the comparison results. After screening, a total of 8 upstream and downstream primers were finally determined, and the reverse primer was bio-labeled at the 5' end as the primer for the chip; 5 specific detection probes were determined, and the NH at the 3' end 2 grooming.

[0042] 2. Preparation of oligonucleotide chip and probe array

[0043] After completing the probe screening, the final probe array was ...

Embodiment 2

[0051] Example 2: Specificity evaluation of adenovirus typing gene chip

[0052] Specificity is the most important assessment index of a diagnostic method. The gene chip of the present invention uses optimized systems and conditions to detect influenza virus, measles virus type 40 adenovirus, type 41 adenovirus and rubella virus. Figure 4 It can be seen that the above virus detection results were all negative for the five adenoviruses, with good specificity. The present invention detects five types of adenovirus by typing image 3 It can be seen that the five types of adenoviruses can be clearly distinguished, indicating that the specificity of the present invention is good.

Embodiment 3

[0053] Example 3: Sensitivity evaluation of adenovirus typing gene chip

[0054] Construct adenovirus plasmid DNA as a reference product for detection, starting from 10 5 copies / μl serially diluted to 10 1 copies / μl, for chip detection, see the attached results Figure 5 . attached by Figure 5 It can be seen that the detection limit of the present invention: the detection limit of adenovirus typing is 3×1000copies / reaction.

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Abstract

The invention relates to preparation and application of a gene chip capable of detecting adenoviruses in a parting mode.A preparation method comprises the steps of preparing specific primers and probes for different types of adenoviruses, preparing oligonucleotides chips, establishing a plurality of PCR systems, and establishing a hybridization system.The gene chip prepared through the method can screen different types of adenoviruses including a 3-type adenovirus, a 7-type adenovirus, a 14-type adenovirus, an 11-type adenovirus and a 55-type adenovirus.The gene chip has the advantages of quickness, accuracy, high throughput and high specificity.A new detection means is provided for clinical diagnoses of different types of adenovirus infections and epidemiological surveys.

Description

technical field [0001] The invention relates to the preparation and application of an adenovirus typing gene chip, and belongs to the technical field of gene chip detection. Background technique [0002] Adenovirus is one of the important pathogens causing human respiratory tract and digestive tract infection. It is a double-helical DNA genome virus without an envelope and has numerous serotypes. There are currently 57 serotypes identified, which are divided into 7 subgenera (A-G). Different serotypes cause different diseases. Among them, C, E and B subgenuses mainly cause respiratory diseases, and other B subgenuses mainly cause urinary tract diseases. Infection, A, F, and G mainly cause gastrointestinal infections, and D subgenus mainly causes keratitis and conjunctivitis. About 50% of human adenovirus infections cause related symptoms, such as epidemic keratitis and infantile enteritis can also cause the prevalence of pneumonia. Therefore, accurate determination of its...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C40B40/06C40B50/18C12R1/93
CPCC12Q1/701C12Q1/6837C12Q2565/519C12Q2563/103
Inventor 王升启陈晓飞刘琪琦陈苏红周标徐胜平
Owner ACADEMY OF MILITARY MEDICAL SCI
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