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Preparation and application of gene chip capable of detecting seven diarrhea viruses

A technology for detecting gene chips and diarrhea viruses, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection. Time length and other issues, to achieve the effect of good specificity, high specificity, and accurate screening

Inactive Publication Date: 2015-09-30
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, cell culture methods are time-consuming and complicated to isolate viruses; PCR and immunological methods can only detect one or a few pathogens, which cannot fully meet the needs of detecting various types of diarrhea viruses

Method used

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  • Preparation and application of gene chip capable of detecting seven diarrhea viruses
  • Preparation and application of gene chip capable of detecting seven diarrhea viruses
  • Preparation and application of gene chip capable of detecting seven diarrhea viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Development of Gene Chip for Diarrhea Virus Screening and Detection

[0040] 1. Primer probe design and screening

[0041] First, download the diarrhea virus target gene sequence from the NCBI gene database. After the sequence download is complete, use the AlignX program in the Vector NTI Advance 10 (invitrogen) software package to perform global alignment of each pathogen gene sequence according to the default parameter settings. Design specific oligonucleotide probes, universal and specific primers at conserved positions in the gene sequence according to the alignment results. After screening, a total of 14 upstream and downstream primers were finally determined, and the reverse primer was bio-labeled at the 5' end as the primer used in the chip; 12 specific detection probes were determined, and the 3' end NH 2 retouch.

[0042] 2. Oligonucleotide chip preparation and probe array

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

Embodiment 2

[0051] Example 2: Evaluation of specificity of gene chip for detection of seven diarrhea viruses

[0052] Specificity is the most important assessment index of the diagnostic method, and the gene chip of the present invention uses an optimized system and conditions to detect poliovirus type 1, poliovirus type 2, coxsackie virus A16, echo virus, Enterovirus type 71, hepatitis A virus, adenovirus type 55, adenovirus type 1, adenovirus type 3, enterovirus type 70, by appendix Figure 4 It can be seen that the above virus detection results are all negative for diarrhea virus, with good specificity. The present invention detects 7 kinds of diarrhea viruses, image 3 It can be seen that all 7 kinds of diarrhea viruses can be clearly distinguished, indicating that the present invention has good specificity.

Embodiment 3

[0053] Example 3: Evaluation of Sensitivity of Gene Chip for Detection of Seven Diarrhea Viruses

[0054] Construction of seven diarrhea virus plasmid DNA as detection reference, from 10 5 copies / μl serial dilution to 10 1 copies / μl, the chip test is carried out, and the results are shown in the attached Figure 5 . attached by Figure 5 It can be seen that the detection limit of the present invention: the detection limits of the seven diarrhea viruses are all 3×1000 copies / reaction.

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Abstract

The invention relates to preparation and application of a gene chip capable of detecting seven diarrhea viruses. The preparation method comprises the following steps: preparing specific primers and probes for seven diarrhea viruses, preparing an oligonucleotide chip, establishing a multiplex PCR (polymerase chain reaction) system, and establishing a hybridization system. The gene chip provided by the invention can simultaneously detect seven important enteroviruses, including Group A rotavirus, Group B rotavirus, Type I norovirus, Type II norovirus, astrovirus, Sapovirus and enteric adenovirus. The gene chip has the advantages of high speed, high accuracy, high flux and high specificity, and can provide a new detection means for clinical diagnosis and epidemiological survey of diarrhea virus infection.

Description

technical field [0001] The invention relates to the preparation and use of seven kinds of diarrhea virus detection gene chips, and belongs to the technical field of gene chip detection. Background technique [0002] Infectious diarrhea is the second most frequently-occurring disease in children after respiratory tract infection, and it is distributed worldwide, especially in children under 5 years old in developing countries. The most common pathogens of infectious diarrhea are bacteria and viruses. With the change of sanitary conditions and the use of antibiotics, the proportion of bacterial diarrhea in infectious diarrhea in children has gradually decreased, while the proportion of viral diarrhea has increased. [0003] Pediatric viral diarrhea is an important public health problem in both developing and developed countries. Rotavirus, for example, studies have shown that almost all children under the age of 5 have rotavirus infection. Viral diarrhea has a rapid onset, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C40B40/06C12R1/93
CPCC12Q1/70C12Q1/6837C40B40/06
Inventor 王升启周标刘琪琦陈苏红张敏丽
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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