Three genes were chosen as biomarkers that demonstrate the greatest degree of segregation between CPCs and CPPs, and the corresponding CpG sites were validated for differential methylation between these tumor groups using both the initial discovery panel of 34 samples and a validation set of 22 samples. Quantitative sodium bisulfite pyrosequencing was performed for AK1 (cg14578146), PER2 (cg11903188), and PLSCR4 (cg07038342). All targeted assays were designed using the PyroMark Assay Design Software 1.0 (Qiagen). All primer sets are listed in Additional file 1 : Table S4. Sodium bisulfite-modified genomic DNA was amplified using Hot-Start Taq Master Mix (Qiagen) as previously described [16 (link)]. Regions of interest were amplified by PCR, and pyrosequencing was carried out using the PyroMark Q24 pyrosequencer (Qiagen) according to the manufacturer’s protocol (Pyro-Gold reagents). Output data were analyzed using PyroMark Q24 1.0.10 Software (Qiagen), which calculates the methylation percentage β for each CpG site, allowing quantitative comparisons.
Hotstarttaq master mix
Manufactured by Qiagen
Sourced in Germany
HotStartTaq Master Mix is a ready-to-use solution that contains HotStartTaq DNA Polymerase, buffer components, and dNTPs. The HotStartTaq DNA Polymerase is inactive at lower temperatures, preventing non-specific amplification and primer-dimer formation.
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Lab products found in correlation
Pyromark q24 pyrosequencer, by Qiagen (1 mentions)
Pyromark assay design software 1, by Qiagen (1 mentions)
T100 thermal cycler, by Bio-Rad (1 mentions)
Superscript 2 kit, by Thermo Fisher Scientific (1 mentions)
Fluorchem sp digital imaging system, by Bio-Techne (1 mentions)
Gelred, by Biotium (1 mentions)
2720 thermal cycler, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Qiamp dna mini kit, by Qiagen (1 mentions)
Lysostaphin, by Merck Group (1 mentions)
Monarch pcr dna cleanup kit, by New England Biolabs (1 mentions)
Veriti 96 well thermal cycler, by Thermo Fisher Scientific (1 mentions)
Restriction enzyme, by New England Biolabs (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Monarch plasmid miniprep kit, by New England Biolabs (1 mentions)
Ready to go pcr beads, by GE Healthcare (1 mentions)
Mastercycler gradient, by Eppendorf (1 mentions)
Bigdye terminator cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Abi prism 3100, by Thermo Fisher Scientific (1 mentions)
Pyromark q96 id, by Qiagen (1 mentions)
15 protocols using hotstarttaq master mix
1
Quantitative Methylation Analysis of Biomarker Genes
2
Antibiotic Resistance Profiling of Bacteroides and Prevotella
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Isolates belonging to the genus Bacteroides were tested for the presence of cfxA, cepA, cfiA, tetQ, ermF and nim antibiotic-resistance genes and isolates belonging to the genus Prevotella were tested for the presence of cfxA, tetQ, ermF and nim genes using targeted PCR. As a positive control, a Bacteroides strain and a Prevotella strain were used in which antibiotic-resistance genes were known to be present, as assessed by whole genome sequencing. An overview of the primers used in the PCR is shown in the Supplementary material (Table S1).
For PCR, DNA was obtained by suspending bacterial colonies in DNAse/RNAse-free water. The PCR mastermix consisted of 100 mL HotStart-Taq mastermix (100 U/mL DNA polymerase, 400 mM of each dNTP; Qiagen, Hilden, Germany), 4 mL of each of the primers (10 mM; Eurogentec, Luik, Belgium) and 84 mL DNAse/RNAse-free water. For each PCR 24 mL PCR mastermix and 1 mL DNA suspension were used, yielding an end concentration of 5 mM per primer.
The PCR reactions were run in a T100™ Thermal cycler (Bio-Rad, Hercules, CA, USA), using the conditions presented in the Supplementary material (Table S1). Strains harbouring the cfiA gene were also checked for the presence of an insertion sequence (IS) element upstream of the gene according to the method described by Soki et al. [8] .
For PCR, DNA was obtained by suspending bacterial colonies in DNAse/RNAse-free water. The PCR mastermix consisted of 100 mL HotStart-Taq mastermix (100 U/mL DNA polymerase, 400 mM of each dNTP; Qiagen, Hilden, Germany), 4 mL of each of the primers (10 mM; Eurogentec, Luik, Belgium) and 84 mL DNAse/RNAse-free water. For each PCR 24 mL PCR mastermix and 1 mL DNA suspension were used, yielding an end concentration of 5 mM per primer.
The PCR reactions were run in a T100™ Thermal cycler (Bio-Rad, Hercules, CA, USA), using the conditions presented in the Supplementary material (Table S1). Strains harbouring the cfiA gene were also checked for the presence of an insertion sequence (IS) element upstream of the gene according to the method described by Soki et al. [8] .
3
Quantitative RT-PCR Protocol for Gene Expression Analysis
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mRNA was isolated with an RNeasy Kit (Qiagen, 74104) and subjected to RT-PCR with the Superscript II kit (Invitrogen). The total volume of the PCR reaction was 25.0 µl in each tube, which contained 12.5 μl Qiagen HotStartTaq Mastermix, 2.0 µl 4.0 μM primers, the appropriate volume of cDNA, and ultra-pure water to bring the final volume to 25.0 µl. Samples were loaded into a 2720 Thermal Cycler (Applied Biosystems) and specific PCR conditions were used based on the primer composition. The PCR product was mixed with 2.0 µl 6x loading buffer (Bioline, Bio-37068) and 0.5 µl Gel Red (Biotium, 41003), then loaded into the well for agarose gel electrophoresis. The gel was photographed under UV light using the FluorChem SP Digital Imaging System (Alpha Innotech Corporation, San Leandro, California, USA). Fluorescence density of each PCR-amplified band was normalized with the corresponding value of the GAPDH cDNA band.
4
Genotyping P. larvae Strains
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The ERIC genotypes of P. larvae strains were determined by specific repetitive element PCR. The PCR reactions (20 μL) contained 2x HotStartTaq Master Mix (Qiagen, Venlo, The Netherlands), 1 μM primers ERIC1R 5′-ATGTAAGCTCCTGGGGATTCAC-3′ and ERIC2 5′-AAGTAAGTGACTGGGGTGAGCG-3′ [77 ] and 2 μL of DNA solution. The amplification conditions were the same as described Genersch et al. [10 (link)].
5
Virulence Profiling of S. suis Isolates
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All S. suis isolates were tested for three virulence-associated genes (mrp, epf, sly) by multiplex PCR using primers as described previously [22 (link)]. Additionally, a epf monoplex PCR [22 (link)] was conducted to ensure reliable detection of large variants of the epf gene designated as epf* [21 (link)]. To accurately differentiate size variants of mrp, a PCR was performed using the mrp variant primers as described previously [22 (link)]. The PCR assays were performed using HotStartTaq Master Mix (Qiagen) according to the manufacturer’s instructions. MCG sequence typing was performed as described previously [42 (link)].
6
Molecular Detection of Antibiotic Resistance
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We extracted DNA from resistant bacteria using QIAmp DNA Mini Kit (Qiagen) following manufacturer's instructions. The Check-MDR CT103XL test (Checkpoints, NL) was used for molecular detection and identification of genes encoding carbapenemase, AmpC and ESBL enzymes according to manufacturer's instructions. All suspected ESBL, AmpC and carbapenemase positives were confirmed by PCR (HotStart Taq Mastermix, Qiagen). The presence of mecA among suspected MRSA and the quinolone resistance-determining regions (QRDR) of the gyrA and parC genes from fluoroquinolone resistant E. coli or P. aeruginosa were amplified by PCR. All PCR amplicons were sent for DNA sequencing using the Sanger method at Beckman Coulter Genomics and analysed using BioNumerics (Applied Maths) software and NCBI's BLAST. All primers used in this study are listed in Table S1.
7
Construction of mCherry Expressing Plasmid
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Restriction enzymes and T4 DNA ligase were purchased from New England BioLabs (NEB). Hot Start Taq Master Mix was purchased from Qiagen (Hilden, Germany), and PCR was performed with a Veriti™ 96-Well Thermal Cycler (Applied Biosystems). Oligonucleotides, including primers and agrP3-mCherry, were synthesized by IDT DNA Technologies (Coralville, IA). The sequence of codon-optimized mCherry was obtained from NCBI under accession number LC088724 [48 (link)]. All amplicons and digested DNA were purified using Monarch® PCR & DNA Cleanup Kit (NEB). Plasmid DNA was purified using Monarch® Plasmid Miniprep Kit (NEB) after pre-treatment of S. aureus cells with 20 µg of lysostaphin (Sigma-Aldrich) for 30 min at 37 °C.
8
Genomic Island PCR Amplification
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Two regions of the genomic island were targeted for PCR amplification. A 923 bp product at the 5′ end of the island was amplified using BF1 5′-ATCTTTTCCGCGAATCACTG-3′ (Venter 70585 2049624-2049643) and BR1 5′-TTGTATTGGAGGACCAAGC-3′ (2050519-2050537) primers. Also a 1323 bp product was amplified targeting the 3′ end with EF1 5′-GCGCTGTAATATAGGCAAAGC-3′ (2054079-2054099) and ER1 5′-ATAAGCGTGTCCGCTATCGT-3′ (2055382-2055401) primers. Bacterial colonies were inoculated into each PCR reaction consisting of 12.5 μL of HotStart Taq mastermix (Qiagen), 1.25 μL of each 10 μM primer, and 10 μL of ddH2O. Reactions were incubated at 95°C for 15 minutes, followed by 30 cycles each of 94°C for 30 s, x°C for 30 s, and 72°C for 60 s, and finally 72°C for 10 min, where x equals 52.5°C for BF1/BR1 and 54.0°C for EF1/ER1 primers. Additional reactions were also carried out with Ready-To-Go PCR beads (GE Healthcare) with 0.5 μL of each 10 μM primer and 24 μL of ddH2O. Reactions were carried out with the same conditions for each respective primer pair. Any samples with discrepant or negative PCR results were repeated.
9
Profiling Microbial Diversity via T-RFLP Analysis
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The T-RFLP analysis was set-up and performed as described previously (15 (link)) (Fig. 1 a). In brief, bacterial 16S rRNA genes were amplified using the universal primers ENV 1 (5′-6-FAM-AGA GTT TGA TII TGG CTC AG-3′ (for Escherichia coli nucleotides (nt) 8-27) and ENV 2 (5′-CGG ITA CCT TGT TAC GAC TT-3′ (for E. coli nt 1511-1492) (16 (link)), with the forward primer being fluorescently labelled with 6-FAM at the 5′ -end. The PCR mixture contained 100 ng DNA, 25 µl Hot Start Taq Master Mix (2.5 U Taq DNA polymerase, 1.5 mM MgCl2, 200 µM dNTP) (Qiagen, Hilden, Germany), 0.3 µM primer, 1 mM MgCl2, and H2O to a final volume of 50 µl for each buccal swab sample. The PCR reaction was performed in an Eppendorf Mastercycler Gradient (Eppendorf, Hamburg, Germany) using the following steps: initial activation of Taq polymerase at 95°C for 15 min; 25 cycles of 94°C for 1 min; 50°C for 45 s; 72°C for 2 min; and a final extension step at 72°C for 7 min. Negative controls that contained all the PCR reagents were included throughout the analysis, to detect potential DNA contamination. Each buccal swab sample was analysed in three independent PCRs and then the triplicate PCR products were pooled.
10
CYP1A2 Gene Amplification Protocol
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The primer pairs designed to amplify the 5′ flanking regions, all exons and all introns of the CYP1A2 gene are listed in Table I . The PCR was conducted in a total volume of 10 µl consisting of 1 µl genomic DNA (20 ng/µl), 0.5 µl each primer pair (5 µM), 5 µl HotStart TaqMasterMix (Qiagen China Co., Ltd., Shanghai, China), and 3 µl deionized water. PCR amplification consisted of an initial denaturation step at 95°C for 15 min followed by 35 cycles of denaturation at 95°C for 30 sec, annealing at 55–64°C for 30 sec, extension at 72°C for 1 min. The final extension step was performed at 72°C for 3 min. The PCR products were purified and sequenced on an ABI Prism 3100 sequencer (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) using a BigDye Terminator Cycle Sequencing kit (version, 3.1; Applied Biosystems; Thermo Fisher Scientific, Inc.).
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