TITLE: Standard Operating Procedure for Real-time PCR Detection and Identification of Bordetella pertussis, B. parapertussis, and B. holmesii using AB7500
AUTHORS: Adria D. Lee, Pamela K. Cassiday, Lucia C. Pawloski, Kathleen M. Tatti, Monte D. Martin, Elizabeth C. Briere, M. Lucia Tondella, Stacey W. Martin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The appropriate use of clinically accurate diagnostic tests is essential for the detection of pertussis, a poorly controlled vaccine-preventable disease. The purpose of this study was to estimate the sensitivity and specificity of different diagnostic criteria including culture, multi-target polymerase chain reaction (PCR), anti-pertussis toxin IgG (IgG-PT) serology, and the use of a clinical case definition.  An additional objective was to describe the optimal timing of specimen collection for the various tests. </div><div class = "text-block">Clinical specimens were collected from patients with cough illness at seven locations across the United States between 2007 and 2011. Nasopharyngeal and blood specimens were collected from each patient during the enrollment visit.  Patients who had been coughing for ≤ 2 weeks were asked to return in 2-4 weeks for collection of a second, convalescent blood specimen.  Sensitivity and specificity of each diagnostic test were estimated using three methods—pertussis culture as the “gold standard,” composite reference standard analysis (CRS), and latent class analysis (LCA). </div><div class = "text-block"><span>Overall, 868 patients were enrolled and 13.6% were </span><span style = "font-style:italic;">B. pertussis</span><span> positive by at least one diagnostic test.  In a sample of 545 participants with non-missing data on all four diagnostic criteria, culture was 64.0% sensitive, PCR was 90.6% sensitive, and both were 100% specific by LCA.  CRS and LCA methods increased the sensitivity estimates for convalescent serology and the clinical case definition over the culture-based estimates.  Culture and PCR were most sensitive when performed during the first two weeks of cough; serology was optimally sensitive after the second week of cough.</span></div><div class = "text-block">Timing of specimen collection in relation to onset of illness should be considered when ordering diagnostic tests for pertussis. Consideration should be given to including IgG-PT serology as a confirmatory test in the Council of State and Territorial Epidemiologists (CSTE) case definition for pertussis.</div></div>

[STEPS]
?. Aliquot undiluted primers and probe and store for up to 6 months at -20ºC. Cover probe aliquot tubes with foil or use dark colored tubes.
?. Prepare IS481 primers and probe: Prepare 30 μM (10X solution) concentrations of primers and 90 μM (10X solution) probe using sterile PCR grade water. Dispense 10X working concentrations into 10-reaction aliquots and store at -20ºC. Prepare 3 μM working concentrations of primers and 9 μM working concentration probe by diluting the 10X solution to 1:10 using sterile PCR grade water just before running the assay.
?. Prepare pIS1001 primers and probe: Prepare 90 μM (10X solution) concentrations of primers and 30 μM (10X solution) concentrations probe using sterile PCR grade water. Dispense 10X working concentrationsinto 10-reaction aliquots and store at -20ºC. Prepare 9 μM working concentrations of primers and 3 μM working concentration of probe by diluting the 10x solution to 1:10 using sterile PCR grade water just before running the assay.
?. Prepare hIS1001 primers and probe: Prepare 30 μM (10X solution) concentrations of primers and probe using sterile PCR grade water. Dispense 10X working concentrations into 10-reaction aliquots and store at -20ºC. Prepare 3 μM working concentrations of primers and probe by diluting the 10X solution to 1:10 using sterile PCR grade water just before running the assay.
?. Thaw working concentrations of primers and probe and mix gently with a pipette.
?. Quick spin for ~5 sec.
?. Add 0.84 μl per reaction of each working concentration of each primer and probe to 1.5 ml tube (PCR mix tube).
?. Add 1 μl of PCR-grade water per reaction to the PCR mix tube.
?. Mix PerfeCTa ® qPCR ToughMix, UNG by pipetting up and down, then add 12.5 μl of the 2X master mix per reaction to the PCR mix tube.
?. Mix by pipetting and quick spin.
?. Dispense 23 μl of master mix to each test well.
?. Add 4 μl of water and 21 μl of master mix to NTC well.
?. Add 4 μl of sample DNA to the appropriate wells and close with optical caps. All specimens should betested in duplicate. A third reaction should be performed with 1:5 dilution of the DNA extract.
?. Add 4 μl of positive control dilutions and seal with the optical cap.
?. Briefly centrifuge 96-well plate and confirm that reaction mixes are at the bottom of the wells, with no bubbles present.
?. Place the 96-well plate in the AB7500 and close tray. Start run.
?. ptxS1 Primer and Probe Reagent preparation: Prepare 70 μM (10X solution) concentrations of primers and 30 μM (10X solution) concentrations of probe using sterile PCR grade water. Dispense 10X working concentrations into 10-reaction aliquots and store at -20ºC. Cover probe aliquot tubes with foil or use dark colored tubes. Do not store aliquots in frost-free freezers and do not re-freeze thawed aliquots. Prepare 7 μM working concentrations of primers and 3 μM working concentration of probe by diluting the 10X solution to 1:10 using sterile PCR grade water just before running the assay.
?. Thaw working concentrations of primers and probe and mix gently with a pipette.
?. Quick spin for ~5 sec.
?. Add 2.5 μl per reaction of each working concentration of each primer and probe to a 1.5 ml tube (PCR mix tube).
?. Add 1 μl of PCR-grade water per reaction to the PCR mix tube.
?. Add 1 μl of PCR-grade water per reaction.
?. Mix PerfeCTa ® qPCR ToughMix, UNG by pipetting up and down, then add 12.5 μl of the 2X master mix per reaction to the PCR mix tube.
?. Mix by pipetting and quick spin.
?. Dispense 21 μl of master mix to each well.
?. Add 4 μl of water to NTC well.
?. Add 4 μl of sample DNA to the appropriate well and close well with an optical cap. All specimens should be tested in duplicate. A third reaction should be performed with 1:5 dilution of the DNA extract.
?. Add 4 μl of positive control to final well and reseal with the optical cap.
?. Briefly centrifuge 96-well plate and confirm that reaction mixes are at the bottom of the wells, with no bubbles present.
?. Place the 96-well plate in the AB7500 and close tray. Start run.
?. RNaseP Primer and Probe Reagent Preparation: Prepare 4 μM working concentrations of primers and 1 μM working concentration of probe using sterile PCR grade water. Dispense working concentrations into 10-reaction aliquots and store in dark for up to 6 months at -20ºC.
?. Thaw working concentrations of primers and probe and mix gently with a pipette.
?. Quick spin for ~5 sec.
?. Add 2.5 μl per reaction of each working concentration of each primer and probe to a 1.5 ml tube (PCR mix tube).
?. Add 1 μl of PCR-grade water per reaction.
?. Mix PerfeCTa ® qPCR ToughMix, UNG by pipetting up and down, then add 12.5 μl of the 2X master mix per reaction to the PCR mix tube.
?. Mix by pipetting and quick spin.
?. Dispense 21 ul of master mix to each well.
?. Add 4 μl of water to NTC well.
?. Add 4 μl of sample DNA to the appropriate well and close wells with an optical cap.
?. Add 4 μl of positive control to final well and close with an optical cap.
?. Briefly centrifuge 96-well plate and confirm that reaction mixes are at the bottom of the wells, with no bubbles present.
?. Place the 96-well plate in the AB7500 and close tray. Start run.
?. Interpretation: The NTC reactions should not exhibit background. A cycle threshold (Ct) value in the NTC control indicates sample contamination during PCR set up. The B. pertussis DNA positive control (A639) should be positive for both IS481 and ptxS1and negative for pIS1001 and hIS1001. A negative result for either IS481 or ptxS1 means that either the control DNA is old or the PCR was not set up correctly. A positive result for either pIS1001 or hIS1001 means that contamination of the control occurred or the PCR was not set up correctly. In either case, the assay must be repeated. The human genomic DNA positive control should be positive for RNaseP. A negative result means that either the control DNA is old or the PCR was not set up correctly. The blank swabs or sterile water should be negative. If they are not, this indicates that there was contamination during the DNA extraction step and all positive specimens must be re-extracted. All clinical specimens (not isolates) should be positive for RNaseP. When the above controls are met, any specimen that is positive for two of the three replicates (two reactions with undiluted DNA extract and one reaction with DNA diluted 1:5) for the IS481 assay and that is positive for one of the three replicates (two reactions with undiluted DNA extract and one reaction with DNA diluted 1:5) for ptxS1 assay is considered positive for the presence of B. pertussis. If a specimen is negative in the IS481 assay, positive in both the ptxS1 assay and the pIS1001 assay the combined result is considered positive for B. parapertussis. If a specimen is positive in the IS481 assay in at least two of the three replicates, positive for hIS1001 and negative in the ptxS1 assay it is positive for B. holmesii. Note: Care should be taken when using this algorithm to interpret high Ct values (35 cycles or greater) IS481 positive / ptxS1 negative clinical specimens. Since IS481 is present in many copies and ptxS1 is only present in one copy, a clinical specimen that is late cycle IS481 positive (Ct values 35 or greater) and ptxS1 negative may indicate the presence of low levels of B. pertussis. Note: In outbreak settings, in particular, when many specimens are handled in a short period of time, low levels of B. pertussis DNA detected by high cycle IS481 positive and ptxS1 negativemay be due to DNA contamination during specimen collection, DNA extraction, or PCR set up. In this case the PCR result should be interpreted as indeterminate if not confirmed by another laboratory test such as culture or serology. Epidemiological and clinical relevance of high IS481 positive PCR tests should also be taken into consideration. It is also important to keep a record of the Ct values of all reactions in a database in order to facilitate results interpretation.
?. Limitations: (1) If inhibitors are present in a DNA extraction, PCR assays may produce a false negative result. If inhibition of the RNaseP assay is noted for a clinical specimen, extracted DNA should be tested at 2 or more dilutions (e.g., 1:5 and 1:10) to verify the result. (2) Data suggest that clinical specimens collected subsequent to initiation of antimicrobial treatment may not be positive for Bordetella spp. due to reduction of organisms. Whenever possible, specimens collected prior toadministration of antimicrobial agents should be used to determine infection with Bordetella spp. (3) RnaseP should not be performed if specimen was transported in Regan Lowe transport medium containing horse blood, as cross-reactivity has been observed.