Multiplex assay for simultaneously typing and subtyping influenza viruses by use of an electronic microarray.

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 10(2) to 10(3) 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.

Simultaneous Detection of CDC Category "A" DNA and RNA Bioterrorism Agents by Use of Multiplex PCR & RT-PCR Enzyme Hybridization Assays.

Assays to simultaneously detect multiple potential agents of bioterrorism are limited. Two multiplex PCR and RT-PCR enzyme hybridization assays (mPCR-EHA, mRT-PCR-EHA) were developed to simultaneously detect many of the CDC category "A" bioterrorism agents. The "Bio T" DNA assay was developed to detect: Variola major (VM), Bacillus anthracis (BA), Yersinia pestis (YP), Francisella tularensis (FT) and Varicella zoster virus (VZV). The "Bio T" RNA assay (mRT-PCR-EHA) was developed to detect: Ebola virus (Ebola), Lassa fever virus (Lassa), Rift Valley fever (RVF), Hantavirus Sin Nombre species (HSN) and dengue virus (serotypes 1-4). Sensitivity and specificity of the 2 assays were tested by using genomic DNA, recombinant plasmid positive controls, RNA transcripts controls, surrogate (spiked) clinical samples and common respiratory pathogens. The analytical sensitivity (limit of detection (LOD)) of the DNA asssay for genomic DNA was 1x10(0)~1x10(2) copies/mL for BA, FT and YP. The LOD for VZV whole organism was 1x10(-2) TCID(50)/mL. The LOD for recombinant controls ranged from 1x10(2)~1x10(3)copies/mL for BA, FT, YP and VM. The RNA assay demonstrated LOD for RNA transcript controls of 1x10(4)~1x10(6) copies/mL without extraction and 1x10(5)~1x10(6) copies/mL with extraction for Ebola, RVF, Lassa and HSN. The LOD for dengue whole organisms was ~1x10(-4) dilution for dengue 1 and 2, 1x10(4) LD(50)/mL and 1x10(2) LD(50)/mL for dengue 3 and 4. The LOD without extraction for recombinant plasmid DNA controls was ~1x10(3) copies/mL (1.5 input copies/reaction) for Ebola, RVF, Lassa and HSN. No cross-reactivity of primers and probes used in both assays was detected with common respiratory pathogens or between targeted analytes. Clinical sensitivity was estimated using 264 surrogate clinical samples tested with the BioT DNA assay and 549 samples tested with the BioT RNA assay. The clinical specificity is 99.6% and 99.8% for BioT DNA assay and BioT RNA assay, respectively. The surrogate sensitivities of these two assays were 100% (95%CI 83-100) for FT, BA (pX02), YP, VM, VZV, dengue 2,3,4 and 95% (95%CI 75-100) for BA (pX01) and dengue 1 using spiked clinical specimens. The specificity of both BioT multiplex assays on spiked specimens was 100% (95% CI 99-100). Compared to other available assays (culture, serology, PCR, etc.) both the BioT DNA mPCR-EHA and BioT RNA mRT-PCR-EHA are rapid, sensitive and specific assays for detecting many category "A" Bioterrorism agents using a standard thermocycler.

The Influenza Primer Design Resource: a new tool for translating influenza sequence data into effective diagnostics.

BACKGROUND: Recent outbreaks of highly pathogenic avian influenza and multiple occurrences of zoonotic infection and deaths in humans have sparked a dramatic increase in influenza research. In order to rapidly identify and help prevent future influenza outbreaks, numerous laboratories around the world are working to develop new nucleotide-based diagnostics for identifying and subtyping influenza viruses. While there are several databases that have been developed for manipulating the vast amount of influenza genetic data that have been produced, significant progress can still be made in developing tools for translating the genetic data into effective diagnostics. DESCRIPTION: The Influenza Primer Design Resource (IPDR) is the combination of a comprehensive database of influenza nucleotide sequences and a web interface that provides several important tools that aid in the development of oligonucleotides that may be used to develop better diagnostics. IPDR's database can be searched using a variety of criteria, allowing the user to align the subset of influenza sequences that they are interested in. In addition, IPDR reports a consensus sequence for the alignment along with sequence polymorphism information, a summary of most published primers and probes that match the consensus sequence, and a Primer3 analysis of potential primers and probes that could be used for amplifying the sequence subset. CONCLUSIONS: The IPDR is a unique combination of bioinformatics tools that will greatly aid researchers in translating influenza genetic data into diagnostics, which can effectively identify and subtype influenza strains. The website is freely available at http://www.ipdr.mcw.edu.

Current methods for the rapid diagnosis of bioterrorism-related infectious agents.

Bioterrorism is the calculated use of violence against civilians to attain political, religious, or ideologic goals using weapons of biological warfare. Bioterrorism is of particular concern because these weapons can be manufactured with ease and do not require highly sophisticated technology. Moreover, biologic agents can be delivered and spread easily and can effect a large population and geographic area. The terrorist attacks occurring around the world necessitate society's continued investment in adequate defense against these unpredictable and irrational events.