Application Of Oligo Microarray Technology For Detection Of HIV

Aquired immunodeficiency syndrome (AIDS) caused by human immunodeficiency virus (HIV) had brought great attention worldwide. Early, rapid and accurate diagnostic approaches are very important for the quarantine of the source of infection. Currently, laboratory examinations were mainly immunological as exemplified by ELISA, colloidal gold indicator paper etc. PCR as well as the conventional virus identification method by tissue culture had also been employed. These methods, though practical, prove to be less optimal for early diagnosis. Oligonucleotide microarray technology was applied in this study in an attempt to find an early, rapid, accurate, sensitive and more efficient method for HIV detection and genotype.The gene chip technique is a new technology bridging both life sciences and electronic engineering. Its high speed, efficiency demonstrates the advantage in obtaining valuable information for comparison and analysis. The recently developed oligonucleotide microarray is adopted by laboratory and markets gradually, because itis economical and good specific. Our research adopts a labeling of whole gene technique to label with DNA and RNA at the same time, in combination with the gene chip technology, which could be applied to the early detection and genotyping of HTV manifestation.To begin with, optimization of oligonucleotide microarray were conducted various aspects ranging from substrate selection, printing solution selection, yielded satisfactory, spots uniformity and other hybridization parameters.Sequences of HIV were analyzed with the HIV database; 60mer oligonucleotide probes were designed by bio-informatics methods and aligned by BLAST to eliminate the sequences that were not specific to the HIV. Sixty-six oligonucleotides were originally selected and synthesized, to print into an oligo microarray for HIV detection and genotyping. DNA samples were labeled by RD fluorescence labeling method. At the same time, for shorting windows period for diagnosis to suit to detect RNA virus, RNA samples were labeled by random PCR labeling technique.The optimized microarray was applied for clinical studies. Thirty-one clinical samples were collected by transfusion department of Liu Hua Qiao hospital, among which 11 serum samples were from screening positive patients, while 20 whole blood samples were from normal people or non-HIV subjects. All of the samples have been detected with DNA and RNA and Nested PCR as parallel comparison, clinical data analysis method and the hybridization results of DNA and RNA samples were compared. In our experiments, Japanese encephalitis virus, influenza virus and the gene of green fluorescent protein oligonucleotide probes were used as quality control probes. At the same time, total RNAs of K562 cell, Hepatitis B virus, Hepatitis C virus, Hepatitis Dvirus and influenza virus were extracted and used as negative controls, in testing the clinical samples of the AIDS patients. RNA and DNA samples were labeled and applied to the microarray for hybridization. The results were scanned by confocal scanner. The images were extracted by ArrayPro Analyzer, data collected were compared to evaluate the probes and for the final selection of candidate probes for further oligo microarray preparations. After the probe selection has been completed, the application of this microarray to clinical diagnosis was studied. Through experimental study, we reached the following conclusions:1. Printing solution of 3*SSC and acrylic amine modified glass slide was found to be most suitable for our experiments, as regards to the improved efficiency of reaction. The conditions of preparing 60mer oligo microarrays are optimized, yielding satisfactory results with spots uniformity and other hybridization parameters.2. Various probes and samples have been introduced to be the quality control during the process of the hybridization, which eliminated the false positive/negative results while not harming the microarray performance.3. Sixty-six oligonucleotides were selected and synthesized to print into an oligo microarray for HIV detection and genotyping. Laboratory primary test was completed.4. It has been demonstrated that the serum samples detection case was 8. Among them subtype B is two cases, subtype C is one case. The coherence results withNest-PCR and sequencing analyses are 100 %.5. The stability experiment result demonstrated that the quality period of the microarray which our laboratory system had been made was at least three month. During the produce of microarray, seal completely was its important guideline.To summary, DNA microarray technology is characterized as mass scale, high throughput, high sensitivity and high; - specifics Appling the 60-mer oligos as microarray probes, the efficiency was further enhanced, while avoiding the false positive results. Small-scale study indicated that the 60mer oligo microarray has great advantages in the early diagnosis of infectious diseases. When multiple disease probes integrated into a single microarray, it would present even greater virtues in the clinical diagnosis of groups of diseases.