title: Comparison of multiplex PCR hybridization-based and singleplex real-time PCR-based assays for detection of low prevalence pathogens in spiked samples.
abstract: Molecular diagnostic devices are increasingly finding utility in clinical laboratories. Demonstration of the effectiveness of these devices is dependent upon comparing results from clinical samples tested with the new device to an alternative testing method. The preparation of mock clinical specimens will be necessary for the validation of molecular diagnostic devices when a sufficient number of clinical specimens is unobtainable. Examples include rare pathogens, some of which are pathogens posing a biological weapon threat. Here we describe standardized steps for developers to follow for the culture and quantification of three organisms used to spike human whole blood to create mock specimens. The three organisms chosen for this study were the Live Vaccine Strain (LVS) of Francisella tularensis, surrogate for a potential biothreat pathogen, Escherichia coli, a representative Gram-negative bacterium and Babesia microti (Franca) Reichenow Peabody strain, representing a protozoan parasite. Mock specimens were prepared with blood from both healthy donors and donors with nonspecific symptoms including fever, malaise, and flu-like symptoms. There was no significant difference in detection results between the two groups for any pathogen. Testing of the mock samples was compared on two platforms, Target Enriched Multiplex-PCR (TEM-PCR™) and singleplex real-time PCR (RT-PCR). Results were reproducible on both platforms. The reproducibility demonstrated by obtaining the same results between two testing methods and between healthy and symptomatic mock specimens, indicates the standardized methods described for creating the mock specimens are valid and effective for evaluating diagnostic devices.
authors.affiliation: Diatherix Laboratories, LLC, Huntsville, AL, USA.
authors.forename: Donna
authors.initials: D
authors.lastname: Hockman
authors.affiliation: Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research (CBER), Food and Drug Administration (FDA), Silver Spring, MD, USA.
authors.forename: Ming
authors.initials: M
authors.lastname: Dong
authors.affiliation: Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research (CBER), Food and Drug Administration (FDA), Silver Spring, MD, USA.
authors.forename: Hong
authors.initials: H
authors.lastname: Zheng
authors.affiliation: Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research (CBER), Food and Drug Administration (FDA), Silver Spring, MD, USA.
authors.forename: Sanjai
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Diatherix Laboratories, LLC, Huntsville, AL, USA.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Huff
authors.affiliation: Diatherix Laboratories, LLC, Huntsville, AL, USA.
authors.forename: Elena
authors.initials: E
authors.lastname: Grigorenko
authors.affiliation: Office of Biodefense, Research Resources, and Translational Research, Division of Microbiology and Infectious Diseases (DMID), National Institute of Allergy and Infectious Diseases (NIAID), Bethesda, MD, USA.
authors.forename: Maureen
authors.initials: M
authors.lastname: Beanan
authors.affiliation: Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research (CBER), Food and Drug Administration (FDA), Silver Spring, MD, USA. Electronic address: robert.duncan@fda.hhs.gov.
authors.forename: Robert
authors.initials: R
authors.lastname: Duncan
mesh_terms: D041001:Babesia microti / Q000302:isolation & purification; D003470:Culture Media / Q000737:chemistry; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004926:Escherichia coli / Q000302:isolation & purification; D005604:Francisella tularensis / Q000302:isolation & purification; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013048:Specimen Handling
chemical_list: D003470:Culture Media; D004269:DNA, Bacterial
keywords: Blood-borne pathogens; Real-time PCR; Spiked specimens; Target Enriched Multiplex PCR