The Tm mapping method using IMLL Q-probes described here is an improvement of the Tm mapping method we previously reported9. Therefore, it should be noted that most of the methods are the same as the previously reported Tm mapping method.
Clinical specimens
A total of 18 whole blood samples were collected from patients with sepsis at Toyama University Hospital and Nagaresugi Geriatric Hospital. All procedures were performed under a protocol approved by the Ethics Committee at the University of Toyama and Nagaresugi Geriatric Hospital, and written informed consent was obtained from all patients. The methods were carried out in accordance with the approved guidelines.
Isolation of bacterial genomic DNA from whole blood
A total of 2 mL of venous blood or, as a negative control for DNA extraction, 2 mL of molecular-grade distilled water (water deionized and sterilized for molecular biology; Nacalai Tesque, Inc., Kyoto, Japan) was collected in EDTA-2K tubes (BD Biosciences Japan, Tokyo, Japan). The blood samples were then centrifuged at 100×g for 5 minutes to spin down the blood cells, and the resulting supernatant fractions (1 mL) were used. The supernatants were centrifuged again at 20,000×g for 10 minutes, and 950 μL of the supernatant fractions was carefully removed, taking to avoid disturbing the pellets. Next, 1 mL of molecular-grade distilled water (water deionized and sterilized for molecular biology; Nacalai Tesque, Inc.) was added to the pellets, and the mixture was gently turned upside down several times and subsequently centrifuged at 20,000×g for 5 minutes. Finally, 1 mL of the supernatant fractions was again carefully removed unless the pellet was resuspended before using the DNA extraction kit. DNA was isolated from the pellets using a DNA extraction kit (QIAamp UCP Pathogen Mini Kit; Qiagen) in accordance with the supplier’s instructions. Finally, bacterial DNA was eluted with 100 μL of elution buffer.
Isolation of bacterial genomic DNA from bacterial colonies
The bacterial colonies were selected with a sterile inoculating loop and suspended in 1 mL of molecular-grade distilled water (water deionized and sterilized for molecular biology; Nacalai Tesque, Inc.). The samples were subsequently centrifuged at 20,000×g for 10 minutes, and 950 μL of the supernatant was carefully removed while taking care to avoid disturbing the pellets. DNA was isolated from the resulting pellets using a DNA extraction kit (QIAamp UCP Pathogen Mini Kit; Qiagen) in accordance with the supplier’s instructions. Finally, bacterial DNA was eluted with 100 μL of elution buffer.
PCR assays
The VeritiTM Thermal Cycler (Applied Biosystems, USA) was used for the amplification, and LightCycler® Nano (Roche Applied Science, Germany) was used for the Tm value analysis of the IMLL Q-probes. When using the LightCycler® Nano, which has two independent thermal blocks, we recommend using the same thermal block for all seven PCR tubes for Tm mapping identification. All PCR assays were performed as single-tube assays (no multiplex PCR). We used 1.5-mL PCR-clean Eppendorf tubes that were RNase- and DNase-free (Eppendorf, Germany), 0.2-mL PCR tubes (Qiagen) for the first PCR and 0.1-mL Strip Tubes and Caps (Qiagen) for the second (nested) PCR. All oligonucleotide primers were designed using a multiple alignment software program (ClustalX; Science Foundation Ireland, Dublin, Ireland) and were synthesized by Life Technologies Japan, Ltd. (Tokyo, Japan). All Q-probes were designed using a multiple alignment software program (ClustalX) and were synthesized by NIPPON STEEL & SUMIKIN Eco-Tech Corporation (Tsukuba, Japan). Bacterial universal primers were designed to universally amplify the seven regions of the bacterial 16S ribosomal RNA gene (Fig. 1b).
The primers were as follows: The first PCR primers (forward: 5′-AGAGTTTGATCATGGCTCAG-3′, reverse: 5′-CCGGGAACGTATTCACC-3′, amplicon size: 1378 bp), Region 1 primers (forward: 5′-AGAGTTTGATCATGGCTCAG-3′, reverse: 5′-CGTAGGAGTCTGGACCGT-3′, amplicon size: 338 bp), Region 2 primers (forward: 5′-GACTCCTACGGGAGGCA-3′, reverse: 5′-TATTACCGCGGCTGCTG-3′, amplicon size: 199 bp), Region 3 primers (forward: 5′-AGCAGCCGCGGTAATA-3′, reverse: 5′-GGACTACCAGGGTATCTAATCCT-3′, amplicon size: 287 bp), Region 4 primers (forward: 5′-AACAGGATTAGATACCCTGGTAG-3′, reverse: 5′-AATTAAACCACATGCTCCACC-3′, amplicon size: 181 bp), Region 5 primers (forward: 5′-TGGTTTAATTCGATGCAACGC-3′, reverse: 5′-GAGCTGACGACAGCCAT-3′, amplicon size: 120 bp). The Q-probes were as follows: IMLL Q-probe 1-1 (5′- GCCATCGGATGTGCCCAGATAAGATTAGCTAGTAGGTG-3′, probe size: 38 bp, delta G value: -0.47 kcal/mol, binding site: 193-230 in E. coli 16S rRNA, Accession No. AB548582), IMLL Q-probe 1-2 (5′-AGGTAACGGCTTACTAAGGCAACGATCGTTAGCTGGTCTGAG-3′, probe size: 42 bp, delta G value: -0.33 kcal/mol, binding site: 231-272 in E. coli 16S rRNA), IMLL Q-probe 2 (5′-GAATCTTCGACAATGGGGGAAAGCCTGATGGAGCCATGCCGCGTG-3′, probe size: 45 bp, delta G value: -1.54 kcal/mol, binding site: 335-379 in E. coli 16S rRNA), IMLL Q-probe 3-1 (5′-GTAATACGGTGGGAGCTAGCGTTATTCGGAATTACAGGGCG-3′, probe size: 41 bp, delta G value: -0.62 kcal/mol, binding site: 503-543 in E. coli 16S rRNA), IMLL Q-probe 3-2 (5′-GGCGGTTTGTTAAGTCAGTAGTGAAAGGCCCGGGCTCAACTTGG-3′, probe size: 44 bp, delta G value: -0.63 kcal/mol, binding site: 557-600 in E. coli 16S rRNA), IMLL Q-probe 4 (5′-GTCCACGCTGTAAACGATGAGTATTAAGAGGTTGTGCC-3′, probe size: 38 bp, delta G value: -0.95 kcal/mol, binding site: 776-813 in E. coli 16S rRNA), IMLL Q-probe 5 (5′-GAACCTTACCTAATCTTGACATCCTTAGAACTTTGCAGAGAT-3′, probe size: 42 bp, delta G value: -0.99 kcal/mol, binding site: 949-990 in E. coli 16S rRNA), Q-probe for S. aureus (5′-GATCCGCGCTGCATTAGATA-3′, probe size: 20 bp, binding site: 211-230 in S. aureus 16S rRNA, Accession No. AB681291).
During the first PCR procedure, the PCR reaction mixture (20 µL) contained 2 µL of DNA template in 200 µM of each dNTP (CleanAmpTM Hot Start dNTP Mix; Sigma-Aldrich, USA) filtered using an Amicon Ultra 50K centrifugal filter (Merck Millipore, Germany), 50 mM KCl, 2.25 mM MgCl2, 10 mM Tris-HCl (pH 8.3), 0.3 µM of each primer, and 1.0 units (0.5 µL) of eukaryote-made thermostable DNA polymerase supplemented with stock buffer solution. The generation of eukaryote-made thermostable DNA polymerase using Saccharomyces cerevisiae has been described previously. In place of 2 µL of DNA template, the PCR reaction mixture contained 2 µL (8.0 ng/µL) of DNA extracted from Escherichia coli (ATCC 25922) as a positive control or 2 µL of molecular-grade distilled water (water deionized and sterilized for molecular biology; Nacalai Tesque, Inc.) as a negative control for the PCR step.
Each sample was incubated for five minutes at 95 °C to activate the Hot Start dNTPs and then denatured for 10 seconds at 94 °C, annealed for 10 seconds at 57 °C, and extended for 30 seconds at 72 °C for 40 cycles.The PCR product was diluted 100-fold with molecular-grade distilled water (water deionized and sterilized for molecular biology; Nacalai Tesque, Inc.) and then used as a template for the second (nested) PCR procedure.
For the second (nested) PCR procedure, the PCR reaction mixture (20 µL) contained 2 µL of DNA template of the diluted first PCR product in 200 µM of each dNTP (CleanAmpTM Hot Start dNTP Mix; Sigma-Aldrich) filtered using an Amicon Ultra 50K centrifugal filter (Merck Millipore), 50 mM KCl, 2.5 mM MgCl2, 10 mM Tris-HCl (pH 8.3), 0.75 µM of each forward primer, 0.25 µM of each reverse primer, and 1.0 units (0.5 µL) of eukaryote-made thermostable DNA polymerase supplemented with stock buffer solution. The 7 samples used to amplify Regions 1 to 5 were incubated for 5 minutes at 95 °C to activate the Hot Start dNTPs and then denatured for 10 seconds at 94 °C, annealed for 10 seconds at 57 °C, and extended for 10 seconds at 72 °C for 30 cycles.
Tm value analyses
A total of 8 µL each of the 20 µL of second PCR amplicons was mixed with 0.12 µM of IMLL Q-probes (Total 10 µL). For the Tm value analysis, the resulting 7 mixtures were heated at 95 °C for 5 minutes, decreasing at 4 °C/second, and then cooled at 40 °C for 1 minute. Tm value analyses were performed from 40 °C to 80 °C, increasing at 0.1 °C/step. The data profile was subsequently analyzed using the LightCycler® Nano software program (Roche Applied Science, Germany), and the Tm values were identified.
Analytical sensitivity tests
The limits of identification and detection were determined by serially diluting (log2-fold) cultures with known counts (CFU) of E. coli in phosphate-buffered saline (PBS) and subjecting the samples to Tm mapping identification using IMLL Q-probes. The limits of identification were determined to be the final log2 dilution of the template in which the Tm mapping result was correct, with the correct number of Tm values and a difference value of ≤0.5. The limit of detection (LOD) was determined to be the final log2 dilution of the template in which at least one of the seven Tm values was observed.
Nucleotide sequence-based analysis of bacterial genomic DNA
Amplicons from the samples used in the first PCR procedure were purified (QIAquick PCR Purification Kit; Qiagen) and then sequenced (3500 Genetic Analyzer; Applied Biosystems) using the Region 1 forward primer and the Region 5 reverse primer. An online homology search was performed for strain identification using the BLAST nucleotide database tool of the DNA Data Bank of Japan (http://www.ddbj.nig.ac.jp/index-j.html).
Culture-based biochemical identification of bacteria
The whole blood samples (one aerobic blood culture bottle and one anaerobic blood culture bottle) were collected simultaneously with the blood sample for Tm analysis from the same puncture site. The whole blood samples were then analyzed according to standard methods used by the Clinical Laboratory Center (certified ISO15189) at Toyama University Hospital. The blood culture procedures were performed using the BacT/ALERT 3D system (bioMerieux, Inc., Mercy-l’Etoile, France). Positive blood culture bottles were subcultured in the appropriate media and incubated aerobically or anaerobically until sufficient growth was present to proceed with testing (usually 18 to 24 h). The specific identification methods differed according to the organism, although they included the MicroScan WalkAway system (Siemens Healthcare Diagnostics, IL, USA), RapID ANA II (Thermo Fisher SCIENTICIC, UK), and various latex agglutination and biochemical spot tests.