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Additional file 1
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MIQE guidelines
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Minimum Information for Publication of Quantitative Real-Time PCR Experiments
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Essential information (E) and Desirable information (D)
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1. Experimental design
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Definition of experimental and control groups (E) and number within each group (E)
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i) Twenty-three previously characterized Campylobacter strains were used for experimental
10
analyses (Table 1).
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ii) Twenty-two previously characterized bacterial strains other than Campylobacter were used
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for specificity screening (Table 1).
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iii) 30 bacterial strains (genome sequences respectively) were used for in silico analyses
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(Table 3).
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Assay carried out by the core or investigator's lab? (D)
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Assay was carried out by investigator's lab
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2. Sample
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Description (E)
1
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Pure cultures of abovementioned Campylobacter strains (Table 1) were included in an
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experimental design of de novo protocol. Culture conditions – Park and Sanders broth
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(HiMedia, India); 24-48 h; steadily; at 42°C; under microaerobic atmosphere (5% O2, 10%
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CO2 and 85% N2; O2/CO2 incubator, MCO-18, Sanyo, USA). The strains C. jejuni CCM
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6212, C. coli CCM 6211 and C. lari CCM 4897 were further used also for food sample
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spiking.
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Pure cultures of abovementioned non-campylobacter bacterial strains (Table 1) were included
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for specificity screening. Culture conditions – BHI broth (brain heart infusion; Merck,
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Germany); 24 h; steadily; at 37°C; aerobically.
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Three chicken wings (local butchery, Prague, Czech Republic), whole frozen chicken without
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giblets (local hypermarket, Prague, Czech Republic) and fried chicken strips (fast food
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restaurant, Prague, Czech Republic) were used for validation of designed protocol when
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applied on different real food samples.
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Microdissection or Macrodissection (E)
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Not applied
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Processing procedure (E)
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Chicken rinse preparation:
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Each wing was hand massaged in plastic bag containing 65 ml of physiological saline for 3
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minutes. Afterwards, the wing was removed and 50 ml of the rinse centrifuged for 15 min at
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16000 × g. The supernatant was discarded and the pellet was resuspended in 50 ml of the
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second rinse which was conducted under the same conditions. In order to remove larger
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particles from rinse fluid low-speed centrifugation was applied (5 min at 1880 × g).
2
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Supernatant was transferred to another tube and centrifuged for 15 min at 16000 × g. The
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supernatant was discarded and the pellet was resuspended in 50 ml of physiological saline.
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Chicken juice preparation:
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Commercially frozen chicken was placed into a container and left to thaw overnight at
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ambient temperature. About 20 ml of chicken juice was collected and centrifuged for 15
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minutes at 16000 × g. The supernatant was discarded and the pellet resuspended in a 40 ml of
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physiological saline. In order to remove larger particles from the sample low-speed
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centrifugation was applied (5 min at 1880 × g). The supernatant was transferred to another
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tube and centrifuged for 15 min at 16000 × g. The supernatant was discarded and the pellet
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was resuspended in 50 ml of physiological saline.
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Preparation of fried chicken strips homogenate:
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Homogenate sample was made by adding 100 g of fried chicken strips to 200 ml of
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physiological saline into a plastic bag with a filter and mixing in a stomacher for 3 minutes.
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Afterwards, 50 ml of the homogenate were centrifuged for 15 min at 16000 × g. The
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supernatant was discarded and the pellet was resuspended in 50 ml of a physiological saline.
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In order to remove larger particles from the sample low-speed centrifugation was applied (5
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min at 1880 × g). The supernatant was transferred to another tube and centrifuged for 15 min
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at 16000 × g. The supernatant was discarded and the pellet was resuspended in 50 ml of
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physiological saline.
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3
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If frozen, how and how quickly? (E)
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Not applied
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If fixed, with what and how quickly? (E)
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Not applied
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Sample storage conditions and duration (E)
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Food samples were processed immediately after the purchase.
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3. Nucleic acid extraction
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Procedure and/or instrumentation (E)
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i) Thermal lysis (protocol designed and adjusted in investigator's laboratory):
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From pure bacterial cultures DNA was extracted from the volume of 1 ml. Suspension was
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centrifuged for 10 min at 10000 × g. The supernatant was discarded and the pellet was
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resuspended in 1 ml of physiological saline and then centrifuged for the second time under the
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same conditions. The supernatant was discarded and the pellet was resuspended in 100 µl of
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nuclease-free water (Promega, USA). Lysis was performed at 95°C for 20 min. Cell lysate
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was immediately cooled on ice, shortly vortexed and then centrifuged for 3 min at 10000 × g.
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Extracted DNA was present in the supernatant (if needed for further experiments, DNA was
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stored at -20°C)
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ii) DNA extraction kit:
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DNA from all food samples was isolated using commercial PrepSEQ® Spin Sample
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Preparation Kit with Protocol (Applied Biosystems, USA) in accordance with manufacturer's
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recommendations.
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Name of kit and details of any modifications (E)
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PrepSEQ® Spin Sample Preparation Kit with Protocol (Applied Biosystems, USA)
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Details of DNase or RNase treatment (E)
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Not applied
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Contamination assessment (DNA or RNA), (E)
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Not applied
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Nucleic acid quantification (E)
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DNA concentration (ng/µl) was determined spectrophotometrically using NanoPhotometerTM
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(Implen, Germany)
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Instrument and method (E)
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NanoPhotometerTM (Implen, Germany)
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Performed in accordance with manufacturer's recommendations
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Purity (A260/A280), (D)
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DNA purity was determined spectrophotometrically using NanoPhotometerTM (Implen,
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Germany)
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Only samples whose A260/A280 ratio ranged from 1.7 to 2.1 were used for further analyses
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(indication of good DNA purity)
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RNA integrity: method/instrument (E)
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Not applied
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RIN/RQI or Cq of 3' and 5' transcripts (E)
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Not applied
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Inhibition testing (Cq dilutions, spike, or other), (E)
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Serial dilutions followed by qPCR and Cq determination were performed (details in section 7
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– qPCR protocol; Complete reaction conditions)
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No inhibition was observed when DNA isolated from pure cultures was used.
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Inhibition occurred only when DNA from food samples (chicken rinses) was isolated by
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thermal lysis which was obviously because of a high level of inhibiting components.
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Therefore only DNA isolated by commercial PrepSEQ® Spin Sample Preparation Kit with
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Protocol (Applied Biosystems, USA) was further used when food samples processed.
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Inhibition testing during food sample analyses:
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Food samples (chicken juice and homogenate prepared from fried chicken strips) were spiked
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with mixed suspension of the C. jejuni CCM 6212, C. coli CCM 6211 and C. lari CCM 4897
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(final concentration of each strain was 101 CFU/ml). Simultaneously mixed pure suspension
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of the same final cell concentration was prepared in physiological saline. DNA was isolated
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from 750 µl using commercial PrepSEQ® Spin Sample Preparation Kit with Protocol
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(Applied Biosystems, USA). Multiplex qPCR was performed as described in section 7. qPCR
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protocol. Quantification cycles were compared and no inhibition during PCR reaction was
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observed (Table 6).
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Table 6: Results of inhibition testing during food sample analyses
Strain
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*
physiological saline
SD
Cq mean
chicken juice
SD
fried strips homogenate
SD
*
*
C. jejuni
32.66
0.24
-
-
32.30
0.08
C. coli
32.44
1.15
33.70
0.15
32.77
0.17
31.21
0.27
31.47
0.19
32.06
C. lari
chicken juice was naturally contaminated with therefore inhibition testing for this specie was not possible
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4. Reverse transcription
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Not applied
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5. qPCR target information
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Gene symbol (E)
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hipO, glyA, pepT
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Sequence accession number (E)
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hipO NC_002163.1, glyA AF136494.1, pepT NC_012039.1
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Location of amplicon (within target), (D)
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hipO 809-932, glyA 271-404, pepT 519-604
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Amplicon length (E)
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hipO 124 bp, glyA 133 bp, pepT 86 bp
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In silico specificity screen (BLAST, and so on), (E)
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0.09
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NCBI standard nucleotide BLAST (nBLAST) – specificity check of primers and probes
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against 30 bacterial genomes (Table 3; control group iii) in section 1 – Definition of
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experimental and control groups)
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Additional more comprehensive in silico analysis for primer pair specificity checking was
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conducted using Primer-BLAST tool at NCBI. As a database query “Genome (chromosome
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of all organisms)” was selected and as an organism query was selected “bacteria (taxid:2)”.
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Secondary structure analysis of amplicon (D)
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FastPCR© molecular biology software [1] – prediction of amplicons' sizes, melting
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temperatures, GC content and secondary structure formations
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Location of each primer by exon or intron (if applicable), (E)
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Not applied – prokaryote
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What splice variants are targeted? (E)
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Not applied – prokaryote
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6. qPCR oligonucleotides
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Primer sequences, 5'→ 3', (E)
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hipO [2]
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F: TGCACCAGTGACTATGAATAACGA, R: TCCAAAATCCTCACTTGCCATT
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glyA [3] with alteration: 3' end modification in forward primer (this section below – Location
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and identity of any modifications)
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F: CATATTGTAAAACCAAAGCTTATCGTG, R: AGTCCAGCAATGTGTGCAATG
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pepT [2]
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F: TTAGATTGTTGTGAAATAGGCGAGTT, R: TGAGCTGATTTGCCTATAAATTCG
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Probe sequence, 5'→ 3', (D)
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hipO [2]
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JOE-TTGCAACCTCACTAGCAAAATCCACAGCT-Eclipse
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glyA [3]
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FAM-TAAGCTCCAACTTCATCCGCAATCTCTCTAAATTT- Eclipse
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pepT [2]; with alteration (this section below – Location and identity of any modifications)
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CY5-TGAAAATTGGAAdCGdCAGGTG-BHQ
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Location and identity of any modifications (E)
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forward primer glyA altered from reference publication [3] – two bases at 3' end
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original forward primer: CATATTGTAAAACCAAAGCTTATCGG
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altered forward primer: CATATTGTAAAACCAAAGCTTATCGTG
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pepT probe altered from reference publication [2] – two internal modifications: propynyl dC
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original probe: TGAAAATTGGAACGCAGGTG
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altered probe: TGAAAATTGGAAdCGdCAGGTG
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Manufacturer of oligonucleotides (D)
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East Port, Prague, Czech Republic
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Purification method (D)
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Primers – desalted; Probes – HPLC
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7. qPCR protocol
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Complete reaction conditions (E)
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a) SYBR Green melt curve analysis:
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For primers' specificity and secondary structures formation check
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DNA samples: C. jejuni subsp. jejuni CCM 6212, C. coli CCM 6211, C. lari CCM
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4897, C. upsaliensis ATCC 43954 and C. fetus subsp. fetus CCM 6213
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Cycling parameters: from 60°C to 95°C
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Reaction mixture consisted of 12.5 µl 2 × Power SYBR Green PCR Master Mix
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(Applied Biosystems, USA), 1 µl of each 10 µM primer (0.4 µM final), 5.5 of µl
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nuclease-free water (Promega, USA) and 5 µl of DNA; final volume 25 µl
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b) Singleplex qPCR:
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DNA samples: Genomic DNA isolated from all bacteria listed in Table 1.
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Cycling parameters: 95°C for 10 min, followed by 40 cycles consisting of 95°C for 20
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s and 60°C for 60 s
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Each reaction mixture consisted of 12.5 µl 2 × TaqMan® Universal PCR Master Mix,
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No AmpErase® UNG (Applied Biosystems, USA), 1 µl of each 10 µM primer (0.4
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µM final), 0.5 µl of 10 µM hydrolysis probe (0.2 µM final), 5 µl of nuclease-free
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water (Promega, USA) and 5 µl of DNA; final volume 25 µl
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c) Optimized multiplex qPCR:
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Cycling parameters: 95°C for 10 min, followed by 40 cycles consisting of 95°C for 20
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s and 60°C for 60 s
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Reaction mixture consisted of 15 µl 2 × TaqMan® Universal PCR Master Mix, No
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AmpErase® UNG (Applied Biosystems, USA), 0.24 µl of 100 µM C. jejuni primers
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(0.8 µM final) , 0.12 µl of 100 µM C. coli primers ( 0.4 µM final), 0.15 µl of 10 µM
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C. lari primers (0.05 µM final), 0.6 µl of each 10 µM probe (0.2 µM final), 7.18 µl of
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nuclease-free water (Promega, USA) and 5 µl of DNA for standard curve construction.
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For unknown samples (food samples), the same volume and concentrations of all
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components were used except for nuclease-free water (2.18 µl) and DNA (10 µl).
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Final volume: 30 µl
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Reaction volume and amount of cDNA/DNA (E)
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Reaction volume: SYBR Green melt curve assay 25 µl; singleplex qPCR assay 25 µl;
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multiplex qPCR assay 30 µl
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DNA volume for standard curves: 5 µl; real copy number of genomes in a well was
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determined using the formula [2]:
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Genome copies/µl = (C x NA x 10-9)/(genome length (bp) x Mw)
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Where C is a measured concentration of extracted DNA (ng/µl), NA is Avogadro's number
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(6.02 x 1023 molecule/mole) and Mw is molecular weight of 1 bp (660 Da).
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Genome length (bp):
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C. jejuni 1641481 (NCBI NC_002163); C. coli 1714000 [4]; C. lari 1525460 (NCBI
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NC_012039)
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DNA volume for unknown samples: 10 µl; quantification in accordance with standard curves
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Primer, probe, Mg2+, and dNTP concentrations (E)
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SYBR Green assay: 0.4 µM primers
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Singleplex qPCR: 0.4 µM primers and 0.2 µM probe
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Optimized multiplex qPCR: 0.8 µM C. jejuni primers, 0.4 µM C. coli primers, 0.05 µM C.
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lari primers, 0.2 µM probes
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Mg2+ and dNTPs were components of commercial 2 × TaqMan® Universal PCR Master Mix,
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No AmpErase® UNG (Applied Biosystems, USA) and 2 × Power SYBR Green PCR Master
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Mix (Applied Biosystems, USA), concentrations unknown
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Polymerase identity and concentration (E)
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AmpliTaq Gold® DNA Polymerase - a component of commercial 2 × TaqMan® Universal
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PCR Master Mix, No AmpErase® UNG (Applied Biosystems, USA) and 2 × Power SYBR
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Green PCR Master Mix (Applied Biosystems, USA), concentrations unknown
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Buffer/Kit identity and manufacturer (E)
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2 × TaqMan® Universal PCR Master Mix, No AmpErase® UNG (Applied Biosystems,
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USA)
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2 × Power SYBR Green PCR Master Mix (Applied Biosystems, USA)
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Additives (SYBR Green I, DMSO, and so forth), (E)
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Not applied
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Manufacturer of plates/tubes and catalogue number (D)
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MicroAmp® Optical 96-Well Reaction Plate (catalogue number 4306737, Applied
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Biosystems, USA)
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Complete thermocycling parameters (E)
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Melt curve analysis for primers' specificity and secondary structures formation check: from
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60°C to 95°C
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Singleplex and multiplex qPCR: 95°C for 10 min, followed by 40 cycles consisting of 95°C
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for 20 s and 60°C for 60 s
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Reaction setup (manual/robotic), (D)
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Manual
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Manufacturer of qPCR (E)
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7500 Real-Time PCR System (Applied Biosystems, USA)
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8. qPCR validation
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Specificity (gel, sequence, melt or digest), (E)
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Horizontal agarose-gel electrophoresis
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Melt curve analysis (from 60°C to 95°C)
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For SYBR Green I, Cq of the NTC (E)
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No signal detected in NTCs, Cq of the NTCs undetermined or higher than 38
13
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Calibration curves with slope and y intercept (E)
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Singleplex:
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hipO (slope = -3.757; y-intercept = 41.308); glyA (slope = -3.61; y-intercept = 37.393);
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pepT (slope = -3.996; y-intercept = 40.234)
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Multiplex with DNA sample from each strain individually:
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hipO (slope = -3.549; y-intercept = 43.818); glyA (slope = -3.443; y-intercept =
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41.653); pepT (slope = -3.604; y-intercept = 41.966)
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Multiplex with mixed DNA sample:
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hipO (slope = -3.565; y-intercept = 42.227); glyA (slope = -3.399; y-intercept =
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40.040); pepT (slope = -3.506; y-intercept = 39.548)
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PCR efficiency calculated from slope (%), (E)
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Singleplex:
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275
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hipO (E = 84.56); glyA (E = 89.24); pepT (E = 77.93)
Multiplex with DNA sample from each strain individually:
hipO (E = 91.347); glyA (E = 95.229); pepT (E = 92.117)
Multiplex with mixed DNA sample:
hipO (E = 90.848); glyA (E = 96.973); pepT (E = 92.888)
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R2 of calibration curve (E)
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Singleplex:
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hipO (R2 = 0.999); glyA (R2 = 1.0); pepT (R2 = 0.993)
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282
283
284
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Multiplex with DNA sample from each strain individually:
hipO (R2 = 0.999); glyA (R2 = 0.998); pepT (R2 = 0.998)
Multiplex with mixed DNA sample:
hipO (R2 = 0.998); glyA (R2 = 0.998); pepT (R2 = 0.998)
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Linear dynamic range (E)
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Eight points of ten-fold serial dilutions in the range from approximately 100 to 107 genome
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copies/well were tested for multiplex qPCR
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In the tested range, reactions were linear from 101 to 107 genome copies/well, and had
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potential to cover wider range at higher orders of magnitude
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Cq variation at LOD (E)
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0.4 – 0.7 cycle
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Evidence for LOD (E)
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No amplification after 38 cycles
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If multiplex, efficiency (%) and LOD of each assay (E)
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Multiplex with DNA sample from each strain individually:
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hipO (E = 91.347); glyA (E = 95.229); pepT (E = 92.117)
Multiplex with mixed DNA sample:
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hipO (E = 90.848); glyA (E = 96.973); pepT (E = 92.888)
300
(this section above – PCR efficiency calculated from slope)
15
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LOD (CFU equivalent/well) for optimized multiplex qPCR with mixed DNA:
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Cut off cycle 38 at 95% confidence level
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C. jejuni: 6.62 < LOD < 16.10
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C. coli: 5.13 < LOD < 6.30
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C. lari: 4.87 < LOD < 5.23
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9. Data analysis
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Six independent experiments were conducted and data analysed
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qPCR analysis program (source, version), (E)
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Instrument compliant 7500 Software (Applied Biosystems, USA, version 2.0.5)
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Microsoft Office Excel 2007 (Microsoft, USA, version 2007)
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GenEx software (MultiD Analyses AB, Sweden, version GenEx 5 Enterprise)
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Method of Cq determination (E)
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Second derivative maximum
315
Outlier identification and disposition (E)
316
Grubbs test (MultiD Analyses AB, Sweden, version GenEx 5 Enterprise)
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Results for NTCs (E)
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No signal detected in NTCs
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Cq of the NTCs undetermined or higher than 38
16
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Justification of number and choice of reference genes (E)
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Not applied
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Description of normalization method (E)
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Not applied
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Number and stage (reverse transcription or qPCR) of technical replicates (E)
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All samples were run in duplicates; non-template (NTC) and positive controls were included
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Repeatability (intraassay variation), (E)
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Cq deviation from mean Cq expressed as SD
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C. jejuni – singleplex SD mean 0.039; optimized multiplex SD mean 0.118
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C. coli – singleplex SD mean 0.318; optimized multiplex SD mean 0.104
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C. lari – singleplex SD mean 0.091; optimized multiplex SD mean 0.091
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Statistical methods for results significance (E)
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Not applied
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Software (source, version), (E)
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GenEx software (MultiD Analyses AB, Sweden, version GenEx 5 Enterprise)
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Microsoft Office Excel 2007 (Microsoft, USA, version 2007)
336
337
338
References
17
339
1.
Kalendar R, Lee D, Schulman AH: FastPCR software for PCR primer and probe
340
design and repeat search. Genes, Genomes and Genomics 2009, 3:1-14.
341
[www.biocenter.helsinki.fi/bi/programs/fastpcr.htm]
342
2.
He YP, Yao XM, Gunther NW, Xie YP, Tu SI, Shi XM: Simultaneous detection and
343
differentiation of Campylobacter jejuni, C. coli, and C. lari in chickens using a
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multiplex real-time PCR assay. Food Anal Methods 2010, 3:321-329.
345
3.
LaGier MJ, Joseph LA, Passaretti TV, Musser KA, Cirino NA: A real-time
346
multiplexed PCR assay for rapid detection and differentiation of Campylobacter
347
jejuni and Campylobacter coli. Mol Cell Probes 2004, 18:275-282.
348
4.
Chang N, Taylor DE: Use of pulsed-field agarose-gel electrophoresis to size
349
genomes of Campylobacter species and to construct a SalI map of Campylobacter
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jejuni UA580. J Bacteriol 1990, 172:5211-5217.
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