Detection of Biological Threat Agents by Real-Time PCR - Comparison of Assay

Transcription

1 Detection of Biological Threat Agents by Real-Time PCR - Comparison of Assay Performance on the Idaho Technology, Inc. R.A.P.I.D., the Roche LightCycler, and the Cepheid Smart Cycler Supplemental Data File MATERIALS AND METHODS DNA Extraction The DNA used in this study was from reference material obtained from recognized culture collections, commercial vendors, clinics, or unique entries from previous United States Army Medical Research Institute of Infectious Diseases (USAMRIID) collections. Pure cultures of each agent were grown on appropriate media at optimal temperatures and harvested for DNA isolation. The DNA was extracted using either Bactozol Kits (Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer s recommendations or Qiagen (Valencia, CA) kits as previously described (1). Extracted DNA was quantified and checked for quality with a spectrophotometer by measuring absorbance at 260, 280, and 320 nm. The ability of the DNA to be amplified was established with a universal primer set to the 16s rrna gene (2). The presence of high molecular weight DNA was confirmed by running 400 ng of each genomic DNA on 0.7% agarose gels. Identity was confirmed for each DNA by sequence analysis of the entire 16s rrna gene and comparison with published data

2 Assay Design and Optimization The real-time PCR assay primer and probe sequences are listed in Data Supplement Tables 2a-2g. Target sequences were selected for agent specificity. The specific primer and TaqMan or TaqMan -MGB probe sequences were designed using Primer Express Version 2.0 for Windows (Applied Biosystems, Foster City, CA). All primer and probe sequences were analyzed using BLASTN to ensure specificity for their respective targets. All primers were synthesized using standard phosphoramidite chemistry with an ABI 394 DNA/RNA synthesizer (Applied Biosystems). The probes were synthesized by Applied Biosystems, and contained 6-carboxyfluorescein (FAM) at the 5' end and either 6-carboxy-tetramethyl rhodamine (TAMRA) or a non-fluorescent quencher with the MGB protein at the 3' end. Primer candidates were used to amplify 1 pg of target template in the presence of SYBR green dye. Melt curves and agarose gels were analyzed to eliminate inefficient and/or dimer producing primer pairs. Probe concentrations were standardized by diluting the probes so that fluorescence background was with a gain setting of 16 on the R.A.P.I.D., with a gain setting of 8 on the LightCycler version 1.2 using LC Software version 3.3, or on the Smart Cycler. MgCl 2 and primer concentrations were optimized sequentially, as follows, on a single instrument; MgCl 2 was optimized in 1 mm increments from 3 mm to 7 mm, and primers were optimized symmetrically in 0.1 μm increments from 0.1 μm to 1.0 μm. The combinations that exhibited the earliest crossing threshold value (C T ) and generated the highest endpoint fluorescence value (EPF) were chosen as the optimal conditions for each assay

3 Idaho Technology, Inc. Wet Chemistry Reactions were carried out in either 20 µl (R.A.P.I.D. and LightCycler ) or 25 µl (Smart Cycler ) volumes. Each assay contained 1X PCR buffer (50 mm Tris ph 8.3; 250 µg/ml of bovine serum albumin [BSA]) (Idaho Technology, Inc.) and 0.2 mm dntp mix (Idaho Technology, Inc.). Eight tenths (0.8) Unit (R.AP.I.D. and LightCycler ) or one (1.0) Unit (Smart Cycler ) of Platinum Taq DNA polymerase (Invitrogen) was added per reaction. Each Smart Cycler reaction also included 1X SCAR buffer (0.2 mm Tris ph 8.0; 0.2 mg/ml BSA; 150 mm Trehalose; and 0.2% Tween 20). Smart Cycler assays incorporating TaqMan -MGB probes were not supplemented SCAR buffer. Optimal concentrations of primers, probe, and MgCl 2 were added and the master mix was distributed to reaction tubes. Five µl of control/template DNA was added just before analysis on the instruments. Thermal cycling conditions were standardized on all three instruments and were performed as follows: 1 cycle at 95 o C for 2 min, followed by 45 cycles of 95 o C for 0 sec (R.A.P.I.D. and LightCycler ) or 1 sec (SmartCycler ) and 60 o C for 20 sec. Fluorescence readings were taken at the end of each 60 o C step. Limit of Detection Experiments Tenfold serial dilutions were made in TE buffer (Roche Molecular Biochemicals) from 10 pg to 1 fg along with a 50 fg standard of measured genomic DNA were prepared. The standards were run in triplicate on the R.A.P.I.D. and Smart Cycler to determine an estimate of assay sensitivity. The reported detection limit was subsequently - 3 -

4 established by testing a minimumof 60 replicates at a single concentration. The lowest concentration that produced a sensitivity of 97% (58 of 60 positive, 59 of 61 positive, or 60 of 62 positive) was considered the assay limit of detection. A minimum of 60 replicates was used to establish satistical confidence (>95%) in the limit of detection as well as to provide a minimum number of replicates for eventual measurement of operator, instrument, and day to day variability (20 data points for each variable). Inclusivity and Exclusivity Experiments A general panel of 60 organisms was analyzed using the optimized conditions to establish inclusivity and exclusivity (Data Supplement Table 1). In addition, panels of nearest genetic neighbors were also used when available for the organisms of interest. Panels were constructed to include: threat organisms, nearest genetic neighbors to threat organisms, organisms sharing an environmental niche with a threat organism and thus likely to be found in environmental samples, organisms sharing a clinical niche with a threat organism, especially respiratory pathogens, opportunists, and normal respiratory flora, and organisms observed repeatedly in clinical and environmental samples. In all cases 100 pg of genomic DNA was used to determine if the assays cross-reacted with nucleic acids from other organisms. Inclusivity and exclusivity testing was performed on the R.A.P.I.D. and qualitative results were obtained using Detector Software version Data Analysis For the R.A.P.I.D., each reaction capillary tube was read in Channel 1 (F1) at a gain setting of 16 with data analyzed with the LightCycler Data Analysis (LCDA) - 4 -

5 software LCDA software version Qualitative calls were made using the R.A.P.I.D. Detector Software version For the LightCycler, each reaction capillary tube was read in Channel 1 (F1) at a gain setting of 8 with data analyzed with the LCDA software version Sample curves were analyzed using the Second Derivative Maximum with the baseline adjustment set to Arithmetic. For the Smart Cycler, data analysis was performed with the Cepheid Smart Cycler software version 1.2d or 2.0b. Smart Cycler settings consisted of a primary curve analysis with a manual threshold setting of 10, background subtraction turned on, boxcar average set to five cycles, background minimum cycle set to 5, and background maximum cycle set to 45. References 1. Coyne SR, Craw PD, Norwood DA, Ulrich MP. Comparative analysis of the Schleicher and Schuell IsoCode Stix DNA isolation device and the Qiagen QIAamp DNA Mini Kit. J Clin Microbiol 2004;42: Bricker BJ, Halling SM. Differentiation of Brucella abortus bv. 1, 2, and 4, Brucella melitensis, Brucella ovis, and Brucella suis bv. 1 by PCR. J Clin Microbiol 1994;32: