{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T23:52:19Z","timestamp":1784073139853,"version":"3.55.0"},"reference-count":41,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2009,10,20]],"date-time":"2009-10-20T00:00:00Z","timestamp":1255996800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/3.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>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 \u201cA\u201d bioterrorism agents. The \u201cBio T\u201d DNA assay was developed to detect: Variola major (VM), Bacillus anthracis (BA), Yersinia pestis (YP), Francisella tularensis (FT) and Varicella zoster virus (VZV). The \u201cBio T\u201d 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 1\u00d7100~1\u00d7102 copies\/mL for BA, FT and YP. The LOD for VZV whole organism was 1\u00d710-2 TCID50\/mL. The LOD for recombinant controls ranged from 1\u00d7102~1\u00d7103copies\/mL for BA, FT, YP and VM. The RNA assay demonstrated LOD for RNA transcript controls of 1\u00d7104~1\u00d7106 copies\/mL without extraction and 1\u00d7105~1\u00d7106 copies\/mL with extraction for Ebola, RVF, Lassa and HSN. The LOD for dengue whole organisms was ~1\u00d710-4 dilution for dengue 1 and 2, 1\u00d7104 LD50\/mL and 1\u00d7102 LD50\/mL for dengue 3 and 4. The LOD without extraction for recombinant plasmid DNA controls was ~1\u00d7103 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 \u201cA\u201d Bioterrorism agents using a standard thermocycler.<\/jats:p>","DOI":"10.3390\/v1030441","type":"journal-article","created":{"date-parts":[[2009,10,20]],"date-time":"2009-10-20T11:02:24Z","timestamp":1256036544000},"page":"441-459","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":29,"title":["Simultaneous Detection of CDC Category \"A\" DNA and RNA Bioterrorism Agents by Use of Multiplex PCR &amp; RT-PCR Enzyme Hybridization Assays"],"prefix":"10.3390","volume":"1","author":[{"given":"Jie","family":"He","sequence":"first","affiliation":[{"name":"Department of Pediatric, Medical College of Wisconsin, Milwaukee, WI 53226, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrea J.","family":"Kraft","sequence":"additional","affiliation":[{"name":"Department of Pediatric, Medical College of Wisconsin, Milwaukee, WI 53226, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiang","family":"Fan","sequence":"additional","affiliation":[{"name":"Department of Pediatric, Medical College of Wisconsin, Milwaukee, WI 53226, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Meredith","family":"Van Dyke","sequence":"additional","affiliation":[{"name":"Department of Pediatric, Medical College of Wisconsin, Milwaukee, WI 53226, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lihua","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Pediatric, Medical College of Wisconsin, Milwaukee, WI 53226, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael E.","family":"Bose","sequence":"additional","affiliation":[{"name":"Department of Pediatric, Medical College of Wisconsin, Milwaukee, WI 53226, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marilyn","family":"Khanna","sequence":"additional","affiliation":[{"name":"National Institutes of Health, Bethesda, MD 20892-7616, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jacob A.","family":"Metallo","sequence":"additional","affiliation":[{"name":"Department of Pediatric, Medical College of Wisconsin, Milwaukee, WI 53226, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kelly J.","family":"Henrickson","sequence":"additional","affiliation":[{"name":"Department of Pediatric, Medical College of Wisconsin, Milwaukee, WI 53226, USA"},{"name":"Children\u2019s Research Institute, Children\u2019s Hospital of Wisconsin, P.O. Box 1997, Milwaukee, WI 53201-1997, USA"},{"name":"Children\u2019s Hospital of Wisconsin, P.O. Box 1997, Milwaukee, WI 53201-1997, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2009,10,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"323","DOI":"10.2165\/00066982-200106040-00013","article-title":"Biological agents: Weapons of warfare and bioterrorism","volume":"6","author":"Broussard","year":"2001","journal-title":"Mol. Diagn."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"259","DOI":"10.1016\/j.tim.2004.04.007","article-title":"The weapon potential of a microbe","volume":"12","author":"Casadevall","year":"2004","journal-title":"Trends Microbiol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1059","DOI":"10.1001\/jama.285.8.1059","article-title":"Working Group on Civilian Biodefense. 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