Sylvia A. Norman

Multiplex Assay for Simultaneously Typing and Subtyping Influenza Viruses by Use of an Electronic Microarray

ABSTRACT We report on the use of an electronic microarray to simultaneously type influenza A and B viruses and to distinguish influenza A virus subtypes H1N1 and H3N2 from the potentially pandemic avian virus subtype H5N1. The assay targets seven genes: the H1, H3, H5, N1, and N2 genes of influenza A virus; the matrix protein M1 gene of influenza A virus; and the nonstructural protein (NS) gene of influenza B virus. By combining a two-step reverse transcription-multiplex PCR with typing and subtyping on the electronic microarray, the assay achieved an analytical sensitivity of 102 to 103 copies of transcripts per reaction for each of the genes. The assay correctly typed and subtyped 15 different influenza virus isolates, including two influenza B virus, five A/H1N1, six A/H3N2, and two A/H5N1 isolates. In addition, the assay correctly identified 8 out of 10 diluted, archived avian influenza virus specimens with complete typing and subtyping information and 2 specimens with partial subtyping information. In a study of 146 human clinical specimens that had previously been shown to be positive for influenza virus or another respiratory virus, the assay showed a clinical sensitivity of 96% and a clinical specificity of 100%. The assay is a rapid, accurate, user-friendly method for simultaneously typing and subtyping influenza viruses.

Evaluation of the NanoChip 400 System for Detection of Influenza A and B, Respiratory Syncytial, and Parainfluenza Viruses

ABSTRACT The NanoChip400 system uses multiplex PCR chemistry and electronic microarray detection of influenza A and B viruses; respiratory syncytial viruses A and B; and human parainfluenza virus types 1, 2, and 3. The results obtained with the NanoChip 400 system were compared with those obtained by direct fluorescent-antibody staining (DFA) and real-time PCR with 122 and 130 specimens, respectively. Concordance between DFA and NanoChip 400 system was obtained for 106 of 122 (86.9%) specimens. On the basis of discrepancy analysis with specimens available for confirmatory real-time PCR testing, the sensitivity and specificity of the NanoChip 400 were 98.6% and 100%, respectively. With respect to specimens previously tested by real-time PCR, the NanoChip 400 system demonstrated a sensitivity of 91.1% and a specificity of 100%. The NanoChip 400 system provides clinical laboratories with a practical, rapid, and sensitive method for the detection of common respiratory viruses.