Study On The Design And Preparation Of Human Parvovirus B19 Microarray

Human parvovirus B19 is a small DNA virus with single-stranded, linear genome of 5594 nucleotides (nt) in length. It has been found that the B19 is one of the major etiologic agents of erythematic infectiosum (EI) and transient aplastic crisis (TAC) in patients with hemolytic anemia, and has been identified to be associated with fetal death, arthritis and chronic anemia. In China, the earliest report of B19 infection dated back in 1990, numerous reports pertaining to the infections came forth ever since. As the virus poses threat to public health, a simple and sensitive method for its detection is urgently needed.The technique of DNA microarray provides a plausible alternative for detecting the infection of human parvovirus B19. DNA Microarray is an emerging technology that has been expected to provide good diagnostic sensitivity and specificity in the diagnosis of human diseases, especially virus infection. With such a system, all the genomic information of the virus can be integrated into a microarray of about 1cm2 in size, which is expected to provide dependable basis for the clinical diagnosis. Currently two methods for preparing the DNA microarray probe are employed, one is PCR amplification of the DNA fragments, and the other is to use artificially synthesized oligonucleotides. Both approaches have been tested in this study.Initially, specific PCR primers were designed with the Primer Premier 5.0 to amplify the conserved regions of human parvovirus B19 genome. The purified PCR products, which were also the selected microarray probes, were immobilized on a specialized slide substrates by Pixsys 5500 Arrayer. Testing samples were made from plasmid hosting the sequence of the human parvovirus B19. The plasmid fragments were labeled with fluorescent Cy3-dUTP by restriction display technique. After hybridization with target DNA, the microchips were scanned with ScanArray Lite scanner, to generatea database of hybridized signals simultaneously.Subsequently, an oligonucleotide microarray was also designed and prepared to be used in detecting the human parvovirus B19. This microarray was prepared by robotic deposition of oligonucleotides on glass slides. These oligonucleotides were from the human parvovirus B19 genome. Assay also was monitored by hybridization to the oligonucleotide microarrays with fluorescently labeled DNA fragments prepared from plasmid and human serum genome.In summary, we prepared the human parvovirus B19 microarray for the diagnosis of human parvovirus B19 with two formats of microarray probes. The samples labeled by RD technique were used to hybridize with the prepared microarrays. The results of hybridization showed that the microarray for diagnosis of human parvovirus B19 are capable of detecting the virus genomic sequences.