Demonstration of the Pre-Emergency Use Authorization Path Using 3 Minor Groove Binder-Hydrolysis Probe Assays to Detect Escherichia coli O104:H4.

BACKGROUND: The Department of Defense (DoD) and the Food and Drug Administration (FDA) have collaboratively worked on a pre-Emergency Use Authorization (pre-EUA) process for in vitro diagnostic (IVD) devices, using FDA's regulatory flexibilities under the EUA authorities. The pre-EUA process enables FDA review of data in anticipation of a request for an EUA, advancing US government public health emergency preparedness efforts. METHODS: The IVD device developed to detect Escherichia coli O104:H4, for which an EUA has not been issued, serves as an example to illustrate that process. Specifically, DoD designed real-time PCR assays to target the virulent E. coli strain O104:H4 (etiological agent of the 2011 German outbreak) including: fliC (flagellin), Agg3C (AAF), and rfb (wbwC) on the basis of the published sequences. RESULTS: After development and optimization of these 3 specific assays, a defined protocol was followed to determine and document the sensitivity and specificity of each assay analytically. CONCLUSIONS: FDA reviewed these data and returned commentary on additional required experiments to complete the pre-EUA process and expedite the use of the device should there be an emergency need for an IVD device to detect this virulent E. coli strain before such a test is cleared by FDA.

Differentiation of Variola major and Variola minor variants by MGB-Eclipse probe melt curves and genotyping analysis.

Smallpox, caused by the Variola major virus, is considered to be one of the most lethal of all potential biological weapons and has far-reaching consequences. Real-time polymerase chain reaction (PCR) assays are available as a reliable diagnostic tool to detect members of the genus Orthopoxvirus. In addition real-time PCR assays specific for the variola virus have been developed that distinguish it from other orthopoxviruses. However, a positive identification of variola spp. does not classify the virus as the one that causes smallpox (V. major) or as the variant (Variola minor) that causes a much less severe form of the disease. This study reports the development of a real-time PCR minor groove binder (MGB)-Eclipse probe assay utilizing a sequence within the variola B9R/B10R gene complex that reliably differentiates V. major from V. minor by specific probe melting temperatures (T(m)s) and genotyping analysis. The MGB-Eclipse probe assay is an important step beyond the standard TaqMan-MGB assay and we feel this is a significant addition to our current variola species identification algorithm with TaqMan-MGB assays that target the B9R and B10R genes. The probe T(m)s for V. major and V. minor were 62.71 (+/-0.05) and 53.97 (+/-0.44) degrees C, respectively (P=<0.001). We also used the identical sequence to develop a TaqMan((R))-MGB assay that specifically detected V. minor but not V. major variants by qualitative analysis.

Comparative analysis of the Schleicher and Schuell IsoCode Stix DNA isolation device and the Qiagen QIAamp DNA Mini Kit.

Efficient, rapid, and reproducible procedures for isolating high-quality DNA before PCR gene amplification are essential for the diagnostic and molecular identification of pathogenic bacteria. This study evaluated the Qiagen QIAamp DNA Mini Kit and the Schleicher and Schuell IsoCode Stix DNA isolation device for isolating nucleic acid. Buffer, serum, and whole-blood samples were spiked with Bacillus anthracis Sterne vegetative cells and Yersinia pestis, while water was spiked with B. anthracis Sterne spores. Although minimal variations in limit of detection occurred among matrices, both the IsoCode Stix extraction method and the Qiagen procedure have comparable detection limits.