DNA can be extracted from clinical specimens such as blood, cerebrospinal fluid, and bacterial isolates. N. meningitidis can be detected by targeting two genes: ctrA and sodC. CtrA is the capsule transport to sell surface gene ad=and is located within the capsule locus. There are, however, 16% of meningococci that lack the ctrA gene or a capsule. Therefore, as assay was developed to detect all meningococci whether or not it has a capsule. This is the sodC assay and it targets the copper, zing superoxide dismutase gene and is not usually linked to the capsule locus. SodC detects both encapsulated and non-encapsulated meningococci that do not contain ctrA. H. influenza, on the other hand, utilizes the protein D gene, hpd. It is a lipoprotein found on the surface of all capsulated and non-encapsulated strains of H. influenza. Among the assays for S. pneumonia is the autolysin, lytA gene. The lytA gene is very specific to S. pneumonia and best separates it from other genotypically similar species such as S. mitis, S. oralis, and s. pseudopneumoniae. Its high specificity will rarely lead to false-positive or false-negatives.
During this research, we will validate new real-time PCR assays for more sensitive methods to detect meningococcal pathogens using sodC or ctrA, hpd, and lytA. Although PCR serotyping is needed for further typing, time did not permit further testing in the allotted timeframe.
II. Materials and Methods
Microcentrifuge (Eppendorf 5424 Hamburg, Germany)
Accu Block Digital Dry Bath (Labnet International, Inc Woodbridge, NJ)
Plate centrifuge (Fisher-Scientific)
PCR Hood (C.B.S. Scientific Co. Model P-036-02, Del Mar, CA)
7500 Fast Dx Real-Time PCR Instrument (Applied Biosystems)
7500 Fast System with 21CFR Part 11 Software
10% bleach (10:1, water: concentrated bleach)
1.7 ml microcentrifuge tubes (sterile, Avant: lot- 392200-W22998)
1 set of micropipettors (1-10 μl, 2-20 μl, 20-200 μl, and 100-1000 μl)
Pre-sterilized filter tips (10 μl, 20 μl, 200 μl, and 1000 μl)
MicroAmp Fast Optical 96-well Reaction plate with Barcode (Applied Biosystems, lot-4346906)
MicroAmp optical 8 cap strips (lot- P25A9 QA42)
TE buffer (10 mM Tris HCl, pH 8.0, 1 mM EDTA)
Lysostaphin (lot-011116, exp 011117)
0.85% NaCL (lot-070616, exp 070617)
HyClone HyPure Molecular Grade Water (lot-AB216652, exp 0518)
HotStarTaq Master Mix Kit containing dNTP, DNA polymerase, and reference dye (Qiagen, Hilden, Germany)
Working concentration of Neisseria meningitides, Haemophilus influenza, and Streptococcus pneumonia for primers and dual-labeled hydrolysis probes containing 5’ fluorophore and 3’ internal quencher
Preparing Reagent solutions
EDTA, 0.1 M, pH 8.0 (100ml)
1. Dissolve 3.7 g EDTA in 70 ml distilled deionized H2O (ddH2O).
2. Adjust pH to 8.0 with 10 M NaOH.
3. Add ddH2O to 100 ml.
4. Autoclave at 121°C for 20 minutes.
5. Store and room temperature
Tris-HCl 0.1 M, pH 8.0
1. Dissolve 1.2 g Tris base in 80 ml ddH2O.
2. Adjust to pH 8.0 with concentrated HCl.
3. Mix and add ddH2O to 100 ml.
4. Autoclave at 121°C for 20 minutes.
5. Store at room temperature.
TE Buffer (10mM Tris HCl, pH 8.0, 1 mM EDTA)
1. Add 10 ml of 0.1 M Tris-HCl, pH 8.0
2. Add 1 ml of 0.1 M EDTA, pH 8.0
3. Add sterile ddH2O to 100 ml and mix well
4. Store at room temperature
Fast preparation of DNA template from clinical isolates
A. N. meningitides and H. influenza (gram-negative)
1. Dispense 1.0 ml of TE buffer into labeled ? tubes.
2. Harvest colonies from 18-24 hour pure cultures of H. influenza or N. meningitides using a sterile polyester swab and swirl in the TE buffer to make a 0.1 McFarland suspension.
3. Transfer suspension into 1.7 ml microcentrifuge tubes
4. Vortex briefly and boil suspension at 100°C for 10 minutes
5. Proceed immediately with PCR or store at -20°C (may be stored at 4°C for a short period of time).
B. S. pneumonia (gram-positive)
1. Dispense 1.0 ml of 0.85% NaCl into labeled ? tubes.
2. Harvest colonies from 18-24 hour pure cultures of S. pneumonia using a polyester swab and swirl in the 0.85% NaCl to make a 0.1 McFarland suspension.
3. Transfer into 1.7 ml microcentrifuge tubes
4. Vortex briefly and incubate at 70°C for 15 minutes.
5. Microcentrifuge at 12,000 x g for 2 minutes and remove supernatant.
6. Re-suspend in 50 μL of TE Buffer and add 10 μL of lysostaphin
7. Incubate at 37 °C for 30 minutes up to 18 hours.
8. Heat-inactivate the enzymes in the suspension by boiling at 100 °C for 10 minutes.
9. Microcentrifuge at 12,000 x g for 4 minutes and remove supernatant for use as DNA template
6. Proceed immediately with PCR or store at -20 °C (may be stored at 4°C for a short period of time).
Preparation of Primers and Probes
Table 1 lists the nucleotide sequences, working concentrations, and chemical modifications of the primers and probes for N. meningitidis, H. influenza, and S. pneumonia species detection targeting the ctrA or SodC, hpd, and lytA genes, respectively. Primers and probes were diluted from concentrated stocks to working stocks using as demonstrated in Table 2. These primers and probes were stored at 4-8°C (may be stored at -20 °C if used less frequently.
Target Primer or Probe Name Real-time Primers and Probes Nucleotide Sequence (5’ to 3’) Working Stock Conc (μM) Final Conc (μM) Suggested Probe Modifications
ctrA F753 TGTGTTCCGCTATACGCCATT 3.75 300
R846 Gccatattcacacgatatacc 11.25 900
Pb820i AACCTTGAGCAA”T”CCATTTATCCTGACGTTCT 1.25 100 5’ FAM, BHQ1 on “T”, 3’ SpC6
sodC F351 GCACACTTAGGTGATTTACCTGCAT 3.75 300
R478 CCACCCGTGTGGATCATAATAGA 7.5 600
Pb387 CATGATGGCACAGCAACAAATCCTGTTT 1.25 100 5’ FAM, 3’ BHQ1
Hpd hpdF822 GGTTAAATATGCCGATGGTGTTG 1.25 100
HPDR952 TGCATCTTTACGCACGGTGTA 3.75 300
Pb896i TTGTGTACACTCCGT”T”GGTAAAAGAACTTGCAC 1.25 100 5’ FAM, BHQ1 on “T”, 3’ SpC6
lytA F373 ACGCAATCTAGCAGATGAAGCA 2.5 200
R424 TCGTGCGTTTTAATTCCAGCT 2.5 200
Pb400i TGCCGAAAACGC”T”TGATACAGGGAG 2.5 200 5’ FAM, BHQ1 on “T”, 3’ SpC6
Table 1. Primers and Probes for detection of bacterial meningitis pathogens
Concentrtion (μM) To make working stock
ctrA F753 3.75 18.75μL stock to 481.25 μL mgH2O
R846 11.25 56.25μL stock to 443.75 μL mgH2O
Pb820i 1.25 6.25 μL stock to 493.75 μL mgH2O
sodC F351 3.75 18.75μL stock to 481.25 μL mgH2O
R478 7.5 37.5 μL stock to 462.5 μL mgH2O
Pb387 1.25 6.25 μL stock to 493.75 μL mgH2O
Hpd F822 1.25 6.25 μL stock to 493.75 μL mgH2O
R952 3.75 18.75μL stock to 481.25 μL mgH2O
Pb896i 1.25 6.25 μL stock to 493.75 μL mgH2O
lytA F373 2.5 12.5 μL stock to 487.5 μL mgH2O
R424 2.5 12.5 μL stock to 487.5 μL mgH2O
Pb400i 2.5 12.5 μL stock to 487.5 μL mgH2O
Table 2. Primer and probe working stock from working concentration
Performing real-time PCR
1. Fill out a PCR template worksheet as demonstrated in Figure 1.
2. Turn on the real-time PCR machine to allow the lamp to warm-up.
3. Remove DNA preparations and positive control DNA from the -20 °C or 4 °C to the dirty room or hood to thaw.
4. Remove laboratory coat and discard gloves. Enter the clean workspace and put on clean laboratory coat and gloves.
5. In the clean room, gather reagents: HotStar commercial master mix, working stock primers and probes, and PCR grade water. If primers and probes were frozen, allow to thaw completely before use.
6. Vortex or flick each tube prior to use. Make one master mix per primer and probe set. The master mix per one sample should contain:
12.5 μL of master mix
4.5 μL of sterile, PCR-grade water
2 μL of forward primer
2 μL of reverse primer
2 μL or probe
23 μL total before adding 2 μL of DNA
When calculating master mix total volume, add enough master mix reagents for 2 extra reactions to make sure there is enough mix.
7. Pipette 23 μL of the prepared master mix into each appropriate well a 96-well plate, according to the plate template worksheet.
8. Add 2 μL of PCR-grade water to the clean NTC wells and then cap only that row until ready to add the DNA samples in the dirty room. May alternately cover the plates with a one-time adhesive film.
9. Wipe down the clean workspace area and used micropipettes with 10% bleach, then 70% ethanol and turn on UV light for one hour, if available.
10. Remove laboratory coat and discard gloves.
11. Put on a fresh pair of gloves and carefully transport the plate to the dirty room or hood.
12. According to the DNA template worksheet, add 2 μL of DNA to the appropriate well. Change gloves frequently between samples.
13. Cap columns of wells as you go. May use a roller tool to secure caps tightly.
14. Wipe down the workspace with 10% bleach, then 70% ethanol and turn on the UV light for one hour, if available.
15. Remove the laboratory coat and discard gloves.
16. Observe the bottom of the plate to ensure there are not any bubbles at the bottom of the wells. If there are, gently flick the well to allow the bubble to rise to the top.
17. If possible, spin the plate at 500 x g for a few seconds to bring down any droplets and to mix.
18. Observe the wells from the bottom of the plate once again to ensure there are not any bubbles at the bottom of the wells that could potentially give a false negative.
19. Transport the plate directly to and place it in the PCR machine.
Operating the PCR Instrument
1. Once in the PCR application, click “create a new document”.
2. Change the run mode to “standard”. Click “next”
3. Select the appropriate target for the appropriate wells according to the DNA template worksheet.
4. Change the reference dye to “none”. Click “Finish”
5. Once the system initializes, label each well with the appropriate name or sample number.
6. Click on the instrument tab to set the cycle parameters.
7. The cycle parameters set for the primers and probes given in the table are:
1 cycle of 50 °C for 2 minutes
1 cycle of 95 °C for 10 minutes
50 cycles of 95 °C for 15 seconds + 60 °C for 1 minute
8. Turn off the machine lamp when the assay is complete.