Supplemental information

Preparation of DNA

DNA was purified from the following organisms: Pseudomonas aeruginosa (DSM50071), Staphylococcus aureus: MSSA (DSM6148), Staphylococcus aureus: MRSA (ATCC BAA 42), Candida albicans (DSM6569) and Candida glabrata (DSM11226) as follows. For each organism, a 50 mL Falcon tube was prepared with 10 mL of Tryptone Soya Broth (TSB) and 0.5 mL of the suspension strain, from -80°C stock. Tubes were incubated at 37°C for 24±2 h. Cell density was checked for all organisms following incubation using DAPI microscopy. For each of the organisms, a dilution series was made from 108 to 101 cells/mL using Peptone Salt Solution (SPO) as dilution medium. Colony forming units (CFU) were determined for each cell suspension in a dilution series by transferring 1 mL (in duplicate) to petri dishes. Tryptone Soya Agar (TSA) (15-20 mL) was added to each of the bacterial strains and Yeast Extract Agar (15-20 mL) was added to each of the yeast strains. The plates were incubated at 37°C for 24±2 h and colony forming units were determined. DNA extractions were performed on 5 mL of cell suspension from each dilution of 108 to 101 cells/mL, using the FastDNA SPIN Kit for Soil (MP Biomedicals) according to the manufacturer’s instructions. Elutions were performed in 100 μL elution buffer and stored at -20°C until further processing.

Targets

The following genes were targeted: oprL (P. aeruginosa), femB (S. aureus: MRSA and MSSA), mecA (S. aureus: MRSA), D1D2 regions of 26S rRNA specific for C. albicans or C. glabrata. Primer sets have previously been published or were designed using LAMP Designer (Premier Biosoft International) or PrimerExplorer v4 (Eiken), details of which are presented in Supplemental Data Fig. 1 and Supplemental Data Table 1.

Amplification and detection

The purified DNA sample eluates were amplified and detected using single step LAMP, two-step assays comprising a first-stage PCR reaction followed by a second-stage qPCR or LAMP and finally the isoPCR method. The volumes of eluates used corresponded to specific copy numbers in each reaction and were referred to as copies per reaction or simply copies. Reactions were performed on 1000, 100, 10, 5, 2, 1 and 0.1 copies.

Conventional PCR reactions were used as first-stage PCR preamplification. PCR reagent mixture (20 mL) consisted of: 2 mL purified DNA or H2O, 10 µl of Paq5000 Hotstart PCR mastermix (Agilent Technologies) and 0.4 mM of each primer, FP+BP, F3+B3 or FIP+BIP. Using a thermocycler (2720 Thermal Cycler from Applied Biosystems), the PCR reagent mixture was subjected to an initial hotstart of 2 min at 95°C, followed by 18 thermocycles of 30 s at 95°C, 20 s at 60°C, and 20 s at 72°C. Final elongation was performed for 5 min at 72°C. First-stage PCR products were stored at -20°C if not processed directly in second-stage reactions.

LAMP reactions were performed as follows. LAMP reaction mixture (10 mL) consisted of: 6 mL of Isothermal Mastermix (Optigene); 1-2 mL of purified DNA or first-step PCR amplified product or H2O; and primers as specified in the Results section in final concentrations of 0.2 mM F3 and B3; 1.6 mM FIP and BIP; 0.8 mM LF and LB. Reactions were performed at 63°C for at least 45 min using the Genie II device (Optigene). The device measured the development in fluorescent signal in real-time. The time for detection was determined by setting a threshold value of 2000 fluorescent units, corresponding to 5-10% of the maximum obtainable fluorescent level.

Nested qPCR assays consisted of a first-stage PCR as previously described, followed by a second-stage qPCR. The second-stage qPCR reactions (25 mL) were performed using 1x iQ Supermix (Bio-Rad), 1x Eva Green, primers were 0.4 mM F3 and B3 and template was 2.5 µL of first-stage PCR product or H2O for no template control (NTC). Using a thermocycler (DNA Engine Opticon System from Bio-Rad) the qPCR reagent mixture was subjected to an initial 2 min at 95°C, followed by 40 thermocycles of 30 s at 95°C, 20 s at 60°C, and 20 s at 72°C. For comparative reasons, the resulting CT values were converted to a detection time by taking into account the hotstart time, thermocycle times, and heating and cooling times. A melting point analysis was performed to confirm amplification of the correct product.

The isoPCR method was performed using a first-stage PCR reaction with FIP/BIP primer sets in concentrations of 0,4 mM. First-stage PCR reactions (20 mL) were performed as singleplex reactions targeting C.gla (sequence specific for C.glabrata) or as 4-plex reactions targeting oprL-, femB-, mecA-genes and C.alb (sequence specific for C.albicans). Subsamples of the product (1-2 mL) were used as template in one or more second-stage isothermal amplification/detection reactions, each with a single target. Second-stage reactions (10 mL), were performed similar to LAMP, as previously described, with primers of 1.6 mM FIP and BIP, and 0.8 mM LF and LB. Reactions were performed at 63°C using the Genie II device for at least 30 min and time for detection was determined as previously described for LAMP reactions.

A melting temperature analysis was performed following each individual isothermal or LAMP reaction by the Genie II device. Amplification products with melting temperatures that differed significantly (more than 0.5 degrees) from the mean of all true positive samples were marked as false positive samples.

Supplemental Data Table 1

Primer sets for oprL-, femB-, mecA-, 26S rRNA (C.albicans)-, 26S rRNA (C.glabrata)- genes. Reference shows if primer sequences have previously been published or have been designed for this study. Sequence letter K indicates G or T.

Primers / Sequences (5' to 3') / Reference
oprL
FP / AGAAGTCGTTATGCCCAAGC / This study
BP / CTGGTAGCGTTCCAGGCTTT / This study
F3 / GCGTTGCCGCCAACAATG / 1
B3 / GGATCTGGTTCTGCTGCT / This study
FIP / GTTGTCACCCCACCTCCGGGCGGCAACGTTCCTCC / 1
BIP / CTCCGTGCAGGGCGAACTGCAGGCGAGCCAACTC / 1
LF / ACCTGCCGTGCCATACC / 1
LB / GTTCATGCAGCTCCAGCAG / 1
femB
FP / GGTCGCGAGAAAAATGATGC / This study
BP / AGCACGCTCTTCAGTTTCAC / This study
F3 / TGTTTAAATCACATGGTTACGAG / 2
B3 / TCACGTTCAAGGAATCTGA / 2
FIP / TACCTTCAAGGTTTAATACGCCCATCATCATGGCTTTACAACTGAG / 2
BIP / ACACCCGAAACATTGAAAAAGACACTTTAACACCATAGTTTATCGCTT / 2
LF / CCATCGTACTTGGCTCGATG / This study
LB / TGATAGTCAACGTAAACGTAAT / This study
mecA
FP / ACAGAAAGTCGTAACTATCCTC / This study
BP / GCAGTACCTGAGCCATAATC / This study
F3 / GCGACTTCACATCTATTAGGT / 2
B3 / GCCATCTTTTTTCTTTTTCTCT / 2
FIP / TCCCTTTTTACCAATAACTGCATCATATGTTGGTCCCATTAACTCT / 2
BIP / AAGCTCCAACATGAAGATGGCCGATTGTATTGCTATTATCGTCAA / 2
LF / TAGCCTTTATATTCTTTTTGTT / This study
LB / TATCGTGTCACAATCG / This study
C.alb: sequence specific for D1D2 region of 26S rRNA in Candida albicans
FP / TAGGACTACCCGCTGAACTT / This study
BP / CGAGAGAGCAGCATGCAAAA / This study
F3 / GCATATCAATAAGCGGAGGAAAAG / 3
B3 / CCTTCCCTTTCAACAATTTCAC / 3
FIP / CTGCATTCCCAAACAACTCGACTCACAGAGGGTGAGAATCCCG / 3
BIP / TATTGGCGAGAGACCGATAGCGTTTCACTCTCTTTTCAAAGTTC / 3
LF / TCKTCGAAGGAACTTTACA / This study
LB / CAAGTACAGTGATGGAAAGATG / This study
C.gla: sequence specific for D1D2 region of 26S rRNA in Candida glabrata / 3
FP / TAGGGTTACCCGCTGAACTT / This study
BP / GCGCAAAACACCATGTCTGA / This study
F3 / GGAGAGTACCACTTTGGGACTG / This study
B3 / CCTTCCCTTTCAACAATTTCAC / 3
FIP / CTGCATTCCCAAACAACTCGACTCATGGAGGGTGAGAATCCCG / This study
BIP / TACAGGCGAGAGACCGATAGCGTTTCACTCTCTTTTCAAAGTTC / This study
LF / CAAAGAACTGACACCCTCGC / This study
LB / CAAGTACAGTGATGGAAAGATG / This study

Supplemental Data Table 2

Number of positive detections per triplicate experiments is shown for four-plex isoPCR targeting sepsis relevant organisms.

Copies per reaction
Sample / Primer set / 1000 / 100 / 10 / 5 / 2 / 1 / 0,1 / 0 (NTC)
P. aeruginosa / oprL / 3 / 3 / 3 / 1 / 0 / 0 / 0 / 0
S. aureus: MRSA / femB / 3 / 3 / 3 / 3 / 2 / 2 / 0 / 0
S. aureus: MRSA / mecA / 3 / 3 / 3 / 2 / 3 / 2 / 0 / 0
S. aureus: MSSA / femB / 3 / 3 / 3 / 3 / 3 / 0 / 0 / 0
C. albicans / C.alb / 3 / 3 / 3 / 1 / 1 / 1 / 0 / 0


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Supplemental Data Table 3

Colony forming units per cell suspension (cells / mL).

Organism / >10000 / 1000 / 100 / 10
Pseudomonas aeruginosa / >300 / >300 / 194 / 23
Staphylococcus aureus: MRSA / >300 / >300 / 127 / 15
Staphylococcus aureus: MSSA / >300 / >300 / 87 / 10
Candida albicans / >300 / >300 / 98 / 8
Candida glabrata / >300 / 255 / 21 / 3

Supplemental Data Table 4

Specificity of detection is shown for four-plex isoPCR targeting sepsis relevant organisms. Samples contain one or more organisms with 1000 copies per reaction. Human DNA was purified from whole blood.

Primer set
Sample / oprL / femB / mecA / C.alb
P. aeruginosa / + / - / - / -
S. aureus: MRSA / - / + / + / -
S. aureus: MSSA / - / + / - / -
Candida albicans / - / - / - / +
P. aeruginosa + S. aureus: MRSA / + / + / + / -
P. aeruginosa + S. aureus: MSSA / + / + / - / -
P. aeruginosa + C. albicans / + / - / - / +
S. aureus: MRSA + S. aureus: MSSA / - / + / + / -
S. aureus: MRSA + C. albicans / - / + / + / +
S. aureus: MSSA + C. albicans / - / + / - / +
Human DNA / - / - / - / -
NTC / - / - / - / -


References

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2.  Hanaki K.-I.; Sekiguchi, J.-I.; Shimada, K.; Sato, A.; Watari, H. et al. Loop-mediated isothermal amplification assays for identification of antiseptic- and methicillin-resistant Staphylococcus aureus. J. Microbiol. Meth. 2011; 84: 251-4.

3.  Inácio, J.; Flores, O.; Spencer-Martins, I. Efficient Identification of Clinically Relevant Candida Yeast Species by Use of an Assay Combining Panfungal Loop-Mediated Isothermal DNA Amplification with Hybridization to Species-Specific Oligonucleotide Probes. J. Clin. Microbiol. 2008; 46: 713-20.