He Jie, Kraft Andrea J, Fan Jiang, Van Dyke Meredith, Wang Lihua, Bose Michael E, Khanna Marilyn, Metallo Jacob A, Henrickson Kelly J
Department of Pediatric, Medical College of Wisconsin, Milwaukee, WI 53226, USA;
Viruses. 2009;1(3):441-459. doi: 10.3390/v1030441.
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.
同时检测多种生物恐怖主义潜在病原体的检测方法有限。我们开发了两种多重PCR和逆转录PCR酶杂交检测方法（mPCR-EHA、mRT-PCR-EHA），以同时检测多种美国疾病控制与预防中心（CDC）A类生物恐怖主义病原体。“Bio T”DNA检测方法用于检测：天花病毒（VM）、炭疽芽孢杆菌（BA）、鼠疫耶尔森菌（YP）、土拉弗朗西斯菌（FT）和水痘带状疱疹病毒（VZV）。“Bio T”RNA检测方法（mRT-PCR-EHA）用于检测：埃博拉病毒（Ebola）、拉沙热病毒（Lassa）、裂谷热病毒（RVF）、汉坦病毒辛诺柏病毒株（HSN）和登革病毒（血清型1-4）。通过使用基因组DNA、重组质粒阳性对照、RNA转录本对照、替代（加标）临床样本和常见呼吸道病原体，对这两种检测方法的灵敏度和特异性进行了测试。DNA检测方法对基因组DNA的分析灵敏度（检测限（LOD）），对于BA、FT和YP为1×10(0)1×10(2)拷贝/毫升。VZV全病毒的LOD为1×10(-2) TCID(50)/毫升。重组对照的LOD，对于BA、FT、YP和VM为1×10(2)1×10(3)拷贝/毫升。RNA检测方法显示，对于RNA转录本对照，埃博拉病毒、裂谷热病毒、拉沙热病毒和汉坦病毒辛诺柏病毒株在未提取时的LOD为1×10(4)1×10(6)拷贝/毫升，提取后为1×10(5)1×10(6)拷贝/毫升。登革全病毒的LOD，登革1型和2型约为1×10(-4)稀释度，登革3型和4型为1×10(4) LD(50)/毫升和1×10(2) LD(50)/毫升。埃博拉病毒、裂谷热病毒、拉沙热病毒和汉坦病毒辛诺柏病毒株的重组质粒DNA对照在未提取时的LOD约为1×10(3)拷贝/毫升（每次反应1.5个输入拷贝）。两种检测方法中使用的引物和探针，与常见呼吸道病原体或目标分析物之间均未检测到交叉反应。使用BioT DNA检测方法检测的264份替代临床样本和使用BioT RNA检测方法检测的549份样本，对临床灵敏度进行了评估。BioT DNA检测方法和BioT RNA检测方法的临床特异性分别为99.6%和99.8%。使用加标临床标本时，这两种检测方法的替代灵敏度，对于FT、BA（pX02）, YP, VM, VZV, 登革2、3、4型为100%（95%CI 83-100），对于BA（pX01）和登革1型为95%（95%CI 75-100）。两种BioT多重检测方法对加标标本的特异性均为100%（95% CI 99-100）。与其他现有检测方法（培养、血清学、PCR等）相比，BioT DNA mPCR-EHA和BioT RNA mRT-PCR-EHA这两种检测方法，使用标准热循环仪检测多种A类生物恐怖主义病原体时，均具有快速、灵敏和特异的特点。
