Primary Study Of Oligonucleotide Microarray For Typing And Subtyping Influenza Viruses

Influenza is an acute respiratory tract infections caused by a virus. It is highly contagious in the populations and can cause wordwide epidemic.which had caused hundreds of thousands casualty. There are three types of influenza viruses, type A, type B and type C, and the type A is further subtyped according to reactivity of one of fifteen different hemagglutinin (HA) and one of nine different neuraminidase (NA). Commonly the two primary human influenza A virus subtypes H1N1 and H3N2 viruses and influenza B are responsible for disease in humans.Influenza viruses are segmented,negative-stranded RNA virues. The influenza A and B virus genome consists of eight single stranded RNA segments,and influenza C consists of seven, lacking one that encoding NA protein. Of the eight RNA segments, segments encoding nucleoprotein(NP)and matrix protein(MP)are highly conserved in all three types of influenza viruses and the characteristics of the NP and MP antigens are the basis of typing of influenza viruses. The antigens of HA and NA are easy to change,based on which influenza A are further subtyped.A variety of techniques are available for the detection and identification of influenza viruses. Standard laboratory methods are based on virus isolation, serologic detection, antigen detection and multiplex reverse transcription-PCR(RT-PCR), however none of which can simultaneouly detect and identify all the types and subtypes of influenza. It is therefore necessary to develop a new efficient, high-througput and rapid method for detecting and subtyping influenza viruses. The applications of genechip technology as a diagnostic tool show great promise, since genechip theoretically permit a simultaneous screen for any of tens of thousands of nucleic acid sequences.According to the characteristics of probes, genechip is classified into three types: DNA chip, cDNA microarray and oligonucleotide microarray. Recent research showed that oligonucleotide microarray had a higher specificity than DNA microarray and cDNA microarray, and also own enough sensitivity for pathogen detection.Oligonucleotide chip can be fabricated by both in-situ synthesizing short oligonucleotide probes and synthesizing 15~80mer probes in length with a DNA synthesizer followed by microarray printing on the substrates. Of both methods, the later is easy to be achieved, as it is an open and flexible system. Researches on length selection of probes suggest that 60mer oligonucleotide probes can achieve the balance of sensitivity and specificity, and one 60mer probe can represent a gene.In our researches, oligo 6.0 software was used to design oligonucleotide probes targeting the gene sequences of NP, MP, HA and NA of influenza viruses and 77 probes were chosen. To get high specificity and sensitivity, all the selected probes meet the following principles:melting temperatures of probes are between 85±5°C, GC contents vary from 40% to 60%, no steady second structure exists, sequence blast results show that each oligo has less than 70% homology with all other genes, Contiguous bases matching to any other gene are no more than 18 bases. Followingthe synthesizing and purification, oligo probes were printed on glass substrate.The RNA extracted from human H1N1, H3N2 and influenza B viruses were reversely transcripted and labeled by restriction display(RD) method, and then were applied to the oligonucleotide microarray. Hybridization results were analyzed to cross out those probes with low specificity and sensitivity, of which the signal to noise ratios(SNR) were less than 2 when hybridized to the fluorescent products from corresponding kind of influenza viruses , or of which the SNRs were more than 1.5 fold different from other organisms sequences. 33 oligonucleotide probes remained, which were comparatively more specific and sensitive.To improve the specificity and sensitivity of oligonucleotide chip, labeling methods, substrates and hybridization conditions were optimized.1. Optimization of labeling methods. In this part, we report a new method of Fluorescent labeling technique in microarray studies: Universal Primer Labeling (UPL), of which the efficiency was compared with that of other three different labeling methods,namely restriction display direct labeling(RD-direct), restriction display incorporation labeling (RD-incorporation) and the reverse transcription coupled random primer spiking labeling (RT-PSL). influenza viruses RNA were respectively labeled with the four labeling methods mentioned above, and applied to influenza virus oligonucleotide microarray. Signals extracted from the microarrays were analyzed with SPSS 10.0. Results showed that the fluorescent intensity, signal-to-noise ratio (SNR), true positive ratio (TPR) of probes and labeling reproducibility of UPL were comparable with those of the RD-labeling methods, and higher than those of the RT-PSL. Moreover, UPL reduced the complexity of the procedures in comparison with the other labeling techniques.2. Selection of substrates for printing the microarray. In this part the characteristics of three different substrates UltraGAPS? aminosilane slide,poly-L-lysine-coated slide DAKO and poly-L-lysine modified microscope slide were evaluated. The methods are as following: Oligonucleotide microarrays were printed on the three different kinds of substrates mentioned above. These slides were hybridized with corresponding Cy3 labeled oligonucleotide probes 0 Following the standard washing procedures, the slides were scanned by GenePix 4100A Scanner and spot sizes , fluorescent intensities and background intensities of three kinds of substrates were analyzed by Genepix6.0 software to evaluate the appropriateness of the substrates. Influences of the substrates on hybridization efficiency were evaluated after Cy3-labeled DNA samples of green fluorescent protein were hybridized to the corresponding 60mer oligonucleotide probes. Good homogeneities were observed on all the substrates, however, slide Corning or DAKO had more stable immobilization and showed higher hybridization efficiency than microscope slide.3. Optimization of the timing and buffer compositions. Results showed that hybridizations at 40 °C for 4 to 8h in hybridization buffer containing 30% formamide,5 X SSC and 0.1%SDS were better than those in other conditions for oligonucleotide chip for influenza viruses.4. Quality control of microarray processes. To monitor the process of labeling and reduce the false positive ratio and false negative ratio, oligonucleotide probes targeting Green Fluorescence Protein ( GFP ) gene were designed and GFP gene was cloned into G-T plasmid and G-S plasmid, from which the RNA or DNA of GFP gene could be obtained and used as positive sample. The methods were as following: Four oligonucleotide probes, one positive probe and influenza diagnostic probes were printed on DAKO slides. The gene encoding the GFP was subcloned into a cloning vector for in vitro expression of the GFP mRNAs. The RNA or DNA of GFP encoding sequence obtained from the recombined plasmids were respectively labeled by the restriction display (RD). Control DNA from blood of healthy humanvolunteers were also labeled. The labeled DNA samples and cy3- universe primers U were applied to oligonucleotide microarray on the slides. The fluorescent signals were scanned by Genepix 4000B and analyzed with the statistics software SPSS 11.0. Results showed that all the oligo probes complement to GFP gene and hybridized with the corresponding labeled samples gave strong signals, The signals of positive control probes were also strong in all hybridizations while the blank control was completely negative. Conclusionly the established quality control microarray has perfect sensitivity and specificity and can be used to monitor the process of labeling, hybridization and scanning.5. Sample validation with microarray. 21 clinical thoat swabs from influenza-like patients were used to validate the oligonucleotide chip for influenza viruses. The methods were as following: RNA extracted from the throat swabs were reversely transcripted and labeled with fluorescence using RD method, then the fluorescent products were hybridized to oligo microarray, on which were printed 33 selected probes comparatively with higher specificity and sensitivity, four GFP positive control probes and five negative conrol probes complement the gene sequence encoding luciferase protein. Following washing and scanning, the hybridization results were analyzed with GenePix Pro 6.0 software. Simultaneously, 10 pairs of primers were designed and synthesized, which respectively targeted the RNA segments encoding NP, MP, HA and NA protein, and were divided into four groups in multiplex RT-PCR reactions used to detect clinical throat swabs. Results showed that oligonucleotide chip had good specificity and sensitivity, and both the positive probes and negative ones performed satisfactorily. Of 21 clinical specimens, five were human H1N1 viruses infections, one H3N2 and influenza B viruses respectively, which were consistent with the results using mutiplex RT-PCR.In summary, the oligonucleotide microarray for influenza viruses has goodspecifity and sensitivity, which provides a sound basis for its implementation in clinical diagnosis. Moreover, GFP positive control is a feasible quality control, while UPL provides a promising labeling method for increasing the efficiency of oligonucleotide microarray.