Supplemental Materials

FIG. S1. Correlation between template copy number and detection by multiplex PCR-Luminex assay (cMFI) for Aeromonas hydrophila, Campylobacter jejuni, Salmonella enterica serovar Enteritidis, Shigella sonnei, Yersinia enterocolitica and extrinsic control, PhHV. Best fit lines and regression (R2) were extrapolated.

FIG. S2. Correlation of cMFI target/extrinsic control ratio with known bacterial copy number. Two thousand PhHV copies and 103 to 108 CFU of S. Enteritidis were spiked into uninfected fecal samples and analyzed with the PCR-Luminex assay. cMFI ratio was calculated as cMFI of Salmonella divided by cMFI of PhHV. Dotted line reveals 95% confidence interval of the regression line, whose equation is x=0.9128lny+5.0388.

Table S1. Species, target genes, primers, and probes.

Species / Target gene / Primer/Probe Sequence a,b / Location of 5’
(Accession No.) / Reference
Aeromonas spp. / aerolysin / F / TYCGYTACCAGTGGGACAAG / 1664 (M16495) / This study
R / CCRGCAAACTGGCTCTCG / 1835 (M16495) / This study
P / CAGTTCCAGTCCCACCACTT / 1705 (M16495) / This study
Campylobacter jejuni and coli / cadF / F / CTGCTAAACCATAGAAATAAAATTTCTCAC / 289 (AF104303) / (1)
R / CTTTGAAGGTAATTTAGATATGGATAATCG / 69 (AF104303) / (1)
P / CATTTTGACGATTTTTGGCTTGA / 127 (AF104303) / (1)
Salmonella spp. / invA / F / TCGGGCAATTCGTTATTGG / 389 (M90846) / This study
R / GATAAACTGGACCACGGTGACA / 465 (M90846) / This study
P / GAAGACAACAAAACCCACCGCC / 417 (M90846) / This study
Shigella spp. / ipaH / F / CGGAATCCGGAGGTATTGC / 1128 (M32063) / (6)
R / CCTTTTCCGCGTTCCTTGA / 1065 (M32063) / (6)
P / CGCCTTTCCGATACCGTCTCTGCA / 1085 (M32063) / (6)
Vibrio spp. / toxRc / F / GTTTGGCGWGAGCAAGGTTT / 420/334 (M21249/L11929) / This study
Rcholerae
Rparahaemolyticus / TCTCTTCTTCAACCGTTTCCA
AAGCGGGCTTAGGCGTTC / 585(M21249)
498 (L11929) / This study
This study
P / CGCAGAGTMGAAATGGCTTGG / 464/378 (M21249/L11929) / This study
Yersinia spp. / lysP / F / TGATTCACCAGCAGCAATAC / 3363 (FR718566) / (1)
R / GGCATCATGAAAGGCGG / 3208 (FR718566) / (1)
P / TGTCGGTTTCTCCTTCCAGG / 3309 (FR718566) / (1)
Extrinsic control (Phocine Herpes Virus) / gB / F / GCGGTTCCAAACGTACCAA / 355 (Z68147) / (4, 5)
(4, 5)
This study
R / GGGCGAATCACAGATTGAATC / 267 (Z68147)
P / TATGTGTCCGCCACCATCTG / 309 (Z68147)
Secondary assays
Aeromonas / 16s rRNA / F
R / GCAGCGGGAAAGTAGCTTG
CTCAGACCAGCTAGGGATCG / 62 (HQ832869)
294 (HQ832869) / This study
This study
Campylobacter / 16s rRNA / F
R / GGATGACACTTTTCGGAGC
CATTGTAGCACGTGTGTC / 409 (GQ479822)
1485 (GQ479822) / (2)
(2)
Salmonella / ompC / F
R / ATCGCTGACTTATGCAATCG
CGGGTTGCGTTATAGGTCTG / 1017 (AF039309)
1220 (AF039309) / (3)
(3)
Shigella / virA / F
R / CTGCATTCTGGCAATCTCTTCACATC
TGATGAGCTAACTTCGTAAGCCCTCC / 827 (AY206441)
1041 (AY206441) / (7)
(7)
Vibrio / hlyA / F
R / ATCGTCAGTTTGGAGCCAGT-3’
TCGATGCGTTAAACACGAAG-3’ / 977 (M36855)
1078 (M36855) / This study
This study
tlh / F
R / AAGAGCACGGTTTCGTGAAC
CTCTGCAACATAGCGGTGAG / 1160 (M36437)
1335 (M36437) / This study
This study

a Mixed bases are as follows: Y, C or T; R, A or G; B, not A; N, any.

b F, R and P represent forward primer (5’ biotinylated), reverse primer and capture oligonucleotide probe (5’ amine modified), respectively.

c Two reverse primers were designed in order to detect both Vibrio cholerae and V. parahaemolyticus.

TABLE S2. Inclusivity and exclusivity panels tested.

Inclusivity Panela / Exclusivity Panel
Aeromonas caviae (1)
Aeromonas hydrophila (5)
Aeromonas veronii biovar sobria (1)
Campylobacter coli (2)
Campylobacter jejuni (2)
Salmonella enterica* (93)
Salmonella arizonae (1)
Salmonella diarizonae (1)
Shigella boydii (2)
Shigella dysenteriae (2)
Shigella flexneri (3)
Shigella sonnei (4)
Enteroinvasive Escherichia coli (1)
Vibrio cholerae (6)
Vibrio parahaemolyticus (2)
Yersinia enterocolitica (2)
Yersinia pseudotuberculosis (1) / Adenovirus Types 40 and 41
Patient samples positive for rotavirus and norovirus GII
Campylobacter hyointestinalis
Campylobacter upsaliensis
Citrobacter freundii
Clostridium difficile
Escherichia coli (ATCC25922)
Enteroaggregative Escherichia coli (EAEC)
Enterohaemorrhagic Escherichia coli (EHEC)
Enteropathogenic Escherichia coli (EPEC)
Enterotoxigenic Escherichia coli (ETEC)
Helicobacter pylori
Cryptosporidium parvum
Cyclospora cayetanensis
Entamoeba dispar
Entamoeba histolytica
Enterocytozoon bieneusi
Giardia lamblia
Isospora belli
Ancylostoma duodenale
Ascaris lumbricoides
Necator americanus
Schistosoma mansoni
Strongyloides stercoralis
Trichuris trichiura

a, number of strains is included in the parentheses.

*, 43 different Salmonella enterica serovars were tested as follows.

S. Agona

S. Albany

S. Anatum

S. Brandenberg

S. Choleraesuis

S. Cubana

S. Derby

S. Dublin

S. Duisburg

S. Enteritidis

S. Emek

S. Gaminara

S. Hadar

S. Haifa

S. Heidelberg

S. Hvittingfoss

S. Indiana

S. Infantis

S. Miami

S. Minnesota

S. Montevideo

S. Muenchen

S. Newport

S. Panama

S. Paratyphi A

S. Paratyphi B

S. Paratyphi C

S. Pullorum

S. Reading

S. Rubislaw

S. Saintpaul

S. Schwarzengrund

S. Sendai

S. Senftenberg

S. Stanley

S. Stanleyville

S. Thompson

S. Typhi

S. Typhimurium

S. Typhisuis

S. Virchow

S. Weltevreden

S. Wien


TABLE S3. Quantification affected by mixed infection. Each PCR reaction contained 107 copies of each of the two targets. Reported here are cMFIs for Aeromonas (A), Campylobacter (C), Salmonella (Sa), Shigella (Sh), Vibrio (V), and Yersinia (Y). Data shown as mean±SD cMFI. All assays run in duplicate.

Mixed infection / Aeromonas / Campylobacter / Salmonella / Shigella / Vibrio / Yersinia
Aeromonas
Campylobacter
Salmonella
Shigella
Vibrio
Yersinia / Aeromonas alone: 29.2±4.3
-
-
-
-
- / A: 28.9±0.9
C: 103.0±14.2
Campy alone: 119.5±5.8
-
-
-
- / A: 23.4±4.3
Sa: 70.6±1.9
C: 58.5±1.2
Sa: 76.0±2.8
Salmonella alone: 77.5±0.3
-
-
- / A: 28.4±3.4
Sh: 71.2±2.4
C: 112.0±20.0
Sh: 74.0±0.6
Sa: 74.0±0.1
Sh: 68.6±4.8
Shigella alone: 73.7±0.6
-
- / A: 28.6±1.3
V: 50.2±7.3
C: 56.0±6.1
V: 54.3±5.2
Sa: 76.4±2.3
V: 37.1±7.5
Sh: 66.8±2.8
V: 47.1±6.4
Vibrio alone: 46.2±4.8
- / A: 29.0±3.1
Y: 53.0±8.3
C: 127.7±21.1
Y: 53.7±8.2
Sa: 74.2±1.1
Y: 54.4±6.9
Sh: 74.6±0.7
57.1±4.0
V: 42.9±5.1
58.4±1.6
Yersinia alone: 61.0±0.1

References

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2. Linton, D., R. J. Owen, and J. Stanley. 1996. Rapid identification by PCR of the genus Campylobacter and of five Campylobacter species enteropathogenic for man and animals. Res Microbiol 147:707-718.

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5. Taniuchi, M., J. J. Verweij, Z. Noor, S. U. Sobuz, L. Lieshout, W. A. Petri, Jr., R. Haque, and E. R. Houpt. 2011. High throughput multiplex PCR and probe-based detection with Luminex beads for seven intestinal parasites. Am J Trop Med Hyg 84:332-337.

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