title: Development of a real-time PCR Assay for identification of Coccidioides immitis by use of the BD Max system.
abstract: Rapid real-time PCR (RT-PCR) can be performed in a community hospital setting to identify Coccidioides species using the new Becton Dickinson molecular instrument BD Max. Following sample preparation, DNA extraction and PCR were performed on the BD Max using the BD Max extraction kit ExK-DNA-1 test strip and a master mix prepared by BioGX (Birmingham, AL). Sample preparation took 2 h, and testing on the BD Max took an additional 2 h. Method sensitivity and specificity were evaluated along with the limits of detection to confirm that this convenient method would provide medically useful information. Using serial dilutions, the lower limit of detection was determined to be 1 CFU/μl. Testing with this method was validated using samples from various body sites, including bronchial alveolar lavage (BAL) fluid; sputum and lung tissue samples; and pleural and spinal fluids. Safety protocols were established, and specimen preparation processes were developed for the various types of specimens. The range for the cycle threshold (CT) indicating adequate fluorescent signal to signify a positive result was established along with the acceptable range for the internal standard. Positive controls run with each batch were prepared by spiking a pooled BAL fluid specimen with a known dilution of Coccidioides immitis organism. Our experience with testing >330 patient samples shows that clinically relevant information can be available within 4 h using an RT-PCR method on the BD Max to identify Coccidioides spp., with sensitivity equivalent to culture.
authors.affiliation: Microbiology Department, Community Medical Centers-Fresno, Fresno, California, USA mmitchell@communitymedical.org.
authors.forename: Marilyn
authors.initials: M
authors.lastname: Mitchell
authors.affiliation: University of California-San Francisco, Fresno, California, USA.
authors.forename: Dominic
authors.initials: D
authors.lastname: Dizon
authors.affiliation: University of California-San Francisco, Fresno, California, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Libke
authors.affiliation: University of California-San Francisco, Fresno, California, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Peterson
authors.affiliation: Microbiology Department, Community Medical Centers-Fresno, Fresno, California, USA.
authors.forename: David
authors.initials: D
authors.lastname: Slater
authors.affiliation: Microbiology Department, Community Medical Centers-Fresno, Fresno, California, USA.
authors.forename: Akashdeep
authors.initials: A
authors.lastname: Dhillon
mesh_terms: D001826:Body Fluids / Q000382:microbiology; D003045:Coccidioides / Q000235:genetics / Q000302:isolation & purification*; D003047:Coccidioidomycosis / Q000175:diagnosis*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors