Improvement of the specificity of a pan‐viral microarray by using genus‐specific oligonucleotides and reduction of interference by host genomes

Kang, Xiaoping; Qin, Chengfeng; Li, Yongqiang; Liu, Hong; Lin, Fang; Li, Yuchang; Li, Jing; Zhu, Qingyu; Yang, Yinhui

Rapid detection of viral pathogens is crucial for antiviral therapy. High‐density 60–70‐mer oligonucleotide microarrays have been explored for broad detection of many viruses. However, relatively low specificity and the complex analytical processes are the major limitations when pan‐viral oligonucleotide microarrays are used to detect viral pathogens. In this study, genus‐specific oligonucleotides were used as probes and modified sample preparations were carried out to improve the specificity and accuracy of the pan‐viral oligonucleotide microarray. Genus‐specific 63‐mer oligonucleotide probes were used for screening human pathogenic RNA viruses. A total of 628 oligonucleotide probes covering 32 RNA viral genera from 14 viral families were used. The number of oligonucleotide probes was decreased to simplify the analytical process of hybridization and to minimize cross‐hybridization. Host genomes were removed by DNase I/RNase T1 digestion before viral nucleic acid extraction, and non‐ribosomal hexanucleotides were used for reverse transcription to minimize interference of host genomes. Cultured viruses were used for microarray validation. The microarray was validated by cultured isolates that belonged to five viral genera. By using DNase I/RNase T1 digestion before viral nucleic acid extraction and non‐ribosomal hexanucleotides for reverse transcription, the specificity of the microarray was improved. Furthermore, the analytical process of hybridization results was simplified. The specificity of pan‐viral microarray could be improved by using genus‐specific oligonucleotides as probes and by using non‐ribosomal hexanucleotides for reverse transcription. Combined with subsequent degenerate reverse transcriptase‐polymerase chain reaction and sequencing processes, this improved genus‐specific oligonucleotides microarray provides a relatively flexible strategy for diagnosis of RNA virus diseases. J. Med. Virol. 83:1624–1630, 2011. © 2011 Wiley‐Liss, Inc.