ADVANCES IN THE AUTOMATED DETECTION AND SUBTYPING OF INFLUENZA VIRUSES THAT INFECT HUMANS

Objective:  Pandemic influenza remains a significant threat to public health. Early rapid detection and subtyping would be critical for recognition and control if a pandemic were to emerge. With this goal, we have developed an 8 and 12 analyte multiplex PCR assay that simultaneously distinguishes influenza A (FA) and B (FB) and subtypes FA (H1,3,5, N1,2) [8-plex] plus H2, H7, H9, and N7 [12-plex] with outstanding analytical and clinical sensitivities and specificities using electronic microarray (eMA) and enzyme hybridization (EHA) detection.  Critical and recurrent evaluation of primer sequences strengthens our design. The 12-plex assay includes detection of all influenza A subtypes known to have infected humans. The ultimate goal of this research is to develop a microfluidic rapid cycling device that detects all of the above analytes in an automated manner in under 2 hours with little technician involvement.

 Methods: Reverse transcription (RT) using random hexamers and then a multiplex PCR using specific primers targeting the M gene of FA, the NS1 and NS2 genes of FB, H1, H2, H3, H5, H7, H9, N1(H1), N1(H5), N2, N7 genes was performed  (8, 9, 10, 11, 12-plex). Positive controls for each gene were synthesized from cloned genes using T7 RNA polymerase. RNA was extracted from cultured virus (H1N1, H3N2, H5N1, influenza B, etc.) and clinical specimens using standard methods.  Primer design and in-silico coverage was determined by our newly created influenza website for primer design (www.ipdr.mcw.edu).  Amplicons were typed and subtyped on Nanogen’s NC400 eMA and in EHA using specific capture and detection oligonucleotides. The analytical sensitivity, cross reactivity, and specificity of the assays were evaluated using serial dilutions of RNA transcripts as well as RNA isolated from cultured virus.  Clinical sensitivity and specificity was evaluated on the 8-plex using clinical specimens, which had been previously tested by viral culture and/or other RT-PCR protocols.  RNA from a variety of types and subtypes of whole organisms (ATCC) and H5N1 RNA (Vietnam, China) was tested.

Results and summary: 1) 8-plex PCR-eMA assay: The analytical sensitivity of RT-PCR as analyzed on the eMA was shown to be ~100 copies(c)/reaction(rx) for each of the 8 genes.  RNA isolated from 28 different cultured types and subtypes (including 2 FB isolates, 1 H1N1, 3 H3N2, and 14 H5N1 from multiple clades) were correctly identified. 146 clinical specimens have been analyzed to date including 102 that were previously shown to be influenza positive and 44 that were shown to be positive for other respiratory viruses. This clinical study showed a sensitivity of 96% (95%CI, 90-99) and a specificity of 100% (95%CI, 92-100). 2) 8-plex and 12-plex PCR-EHA assays: Analytical sensitivities were shown to be 1.5 c/rx (10^3c/ml) for H3, H5, N2 and NS, 15 c/rx for N1H1, N1H5 and M genes and 150c/rx for H1 for the 8-plex, and 2.1 c/rx  (10^3c/ml) for H5, 2.1-21 (H2, H3, H7), 21 (H1, H1N1, N7, NS), 21-210 (N2, M), and 210 c/rx (H5N1, H9), for the 12-plex. A total of 5 whole organism strains of influenza viruses (2 H1N1, 1H3N2, and 2 FB strains) have been tested with the 12-plex so far, and correctly identified.  Further modifications to improve the 12-plex analytical sensitivity, additional clinical testing with the 12-plex, as well as development of the automated detection platform of the eMA is in progress.