Dvelopment Of A DNA-based Microarray For Detection Of Human Adenovirus Serotypes

Human adenovirus(HAdV)originally found in the tonsil tissues of the body,is a kind of no coated three-dimensional symmetric icosahedron double-stranded DNA virus particles.Adenovirus pathogenic spectrum is related to serum type.Different serotype can cause different diseases.So we need a detection method to determine the type.At present there are 68 adenovirus type according to a report in the literature.The International Committee on Taxonomy of Viruses(ICTV)divided HAdV 1-52 into 7 subgenus(A-G).B subgenus can cause respiratory disease,mainly including 3,7,14,11,and 55 type.In recent years,the sometimes outbreak of respiratory tract infection caused by adenovirus.The epidemic developed quickly and is strong infection.The severe cases can be life-threatening illness.Its early detection diagnosis and classification has become a important problem faced by medical workers and researchers.Among the present detection and classification method,the virus separation process is too long and the immunological method does not distinguish serotype.PCR amplification method by amplification specific genes,combining with the gene sequencing method to determine the type,but the cost of gene sequencing method is higher,it is mainly used for laboratory research and determination of new adenoviruses.Ring mediated isothermal amplification is high sensitivity and good specificity,but it,s primer design have a higher demand.So it can not achieve the requirement of high flux.Therefore,we urgently need to establish a rapid and simple detection method of adenovirus type.The purpose of this paper is to establish a adenoviriral serotype detection method of chemiluminescence gene chip todetection methods,including adenovirus 3,7,14,11,and 55.The gene chip can simultaneously detect 5 kinds of adenovirus and determine its type.This method shortens the testing time and improves the detection efficiency.It provides a new classification method for adenovirus serotype detection.Based on the specific gene sequences in conserved region of adenovirus from GeneBank,oligonucleotide primers and probes were desigened by Primer5.0 or Clustalx1.8.msw Alignmen and synthesized to prepare the oligonucleotide microarray.The specific genomic sequences were amplified by multiplex PCR method.The multiplex PCR amplification products were hybridized with the specific probes of microarray that was scanned after washing and chemiluminescence,and then analysed the result.After optimizing of the multiple PCR system,hybridization reaction and condition of chemiluminescence,the specificity,sensitivity and reproducibility of the chip were evaluated.Testing 38 cases of clinical samples assess the practicability of gene chip.In this study the hexon gene of adenovirus is target gene.Four pairs of primers and five probes,were screened finally.HAdV3?7and 14 have respective primers.HAdV 11 and 55 by adenovirus share a primer.Five kinds of adenovirus have respective probes.The geng chip was examined by plasmid of adenovirus for the specificity,sensitivity and repeatability.PCR amplification was conducted with plasmid as a template.After amplification products hybridizedwith gene chip probes,the signals of gene chip was right.The lowest detection limit of the gene chip is 3×103 copies/?l.The variation coefficient are less than 15% within the same chip or between different chips.The microarray detection results of 38 clinical samples were approximately consistent with the direct sequencing method(37/38).This paper has established a adenoviriral serotype detection method of chemiluminescence gene chip to detection methods.This method can detect five of adenovirus type simultaneously,namely HAdV3,7,11,14 and 55.The gene chip can meet the requirement in the specificity,sensitivity and repeatability.This study provides a new high-throughput detection method for the clinical classification diagnosis of adenovirus and epidemiological investigation.