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Supporting information
2
Table S1. PCR Results from mussel hemolymph spiking experiments showing number of
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samples positive for T. gondii / number of replicates for (A) screening assays using primers
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targeting multi-copy genes including ITS1, B1, and a 529 bp repetitive element (RE); and (B)
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genotyping assays using primers targeting single-copy genes including SAG1, SAG3, and
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GRA6. The RE primer set yielded results that were identical to ITS1, and thus was not further
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included in multiplexed assays or for testing field-collected mussels for T. gondii. The B1 primer
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sets were retained for testing field-collected mussels due to the dual application of this locus for
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further genotyping and molecular characterization.
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(A)
Assay type
Spiking
Multiplexed
Simplex
concentration
oocysts / mL
ITS1
B1
ITS1
B1
RE
0
0/3
0/3
0/3
0/3
0/3
1
0/3
0/3
0/3
0/3
0/3
5
1/3
0/3
1/3
1/3
1/3
10
1/3
0/3
1/3
0/3
1/3
50
3/3
2/3
3/3
3/3
3/3
100
3/3
2/3
3/3
3/3
3/3
1000
3/3
3/3
3/3
3/3
3/3
hemolymph
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1
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(B)
Assay type
Spiking
Multiplexed
Simplex
concentration
oocysts / mL
SAG1
SAG3
GRA6
SAG1
SAG3
GRA6
0
0/3
0/3
0/3
0/3
0/3
0/3
1
0/3
0/3
0/3
0/3
0/3
0/3
5
0/3
0/3
0/3
0/3
0/3
0/3
10
0/3
0/3
0/3
0/3
0/3
0/3
50
1/3
1/3
0/3
0/3
1/3
0/3
100
1/3
2/3
1/3
1/3
1/3
1/3
1000
3/3
3/3
3/3
3/3
3/3
3/3
hemolymph
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2
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Table S2. Primer sequences and thermocycler conditions used in polymerase chain reaction assays for detection of T. gondii DNA in
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mussel hemolymph. Loci used for screening assays (ITS1, B1, and the 529 bp repetitive element (RE)) were tested individually as
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simplex assays as well as multiplexed, and loci used only for genotyping (SAG1, SAG3, and GRA6) were tested individually or
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multiplexed together. Note that the B1 locus can be used both for screening and genotyping, as it is a multi-copy gene containing
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single nucleotide polymorphisms. In multiplexed assays, primers for all loci were combined in the external reaction, and then primers
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for each locus were used separately for the internal reaction, following the approach described by Su et al. (2010).
Locus
Purpose
Thermocycler conditions
Primer sequences a
Restriction
Reference
Enzyme b
ITS1
Screening
Denaturation 3 min at 94C; 35
External forward:
cycles: 40 sec at 95C, 40 sec
TACCGATTGAGTGTTCCGGTG
at 58C (external) or 59C
External reverse:
(internal), 90 sec at 72C; Post
PCR extension 4 min at 72C,
hold at 4C
GCAATTCACATTGCGTTTCGC
Internal forward:
CGTAACAAGGTTTCCGTAGG
Internal reverse:
TTCATCGTTGCGCGAGCCAAG
3
NA
(Rejmanek et
al., 2009)
B1
Screening &
External forward:
genotyping
TGTTCTGTCCTATCGCAACG
PmlI,
XhoI
External reverse:
(Grigg
and
Boothroyd,
2001)
ACGGATGCAGTTCCTTTCTG
Internal forward:
TCTTCCCAGACGTGGATTTC
Internal reverse:
CTCGACAATACGCTGCTTGA
RE
Screening
Denaturation: 7 min at 94C;
External forward:
35 cycles: 60 sec at 94C, 60
CGCTGCAGGGAGGAAGACGAAA
sec at 55C, 60 sec at 72C;
GTTG
Post PCR extension: 10 min at
External reverse:
72C
NA
(Homan
al., 2000)
(Shapiro
CGCTGCAGACACAGTGCATCTGG
ATT
Internal forward:
AGAAGGGACAGAAGTCGAAG
4
et
al., 2010)
et
Internal reverse:
CTCCACTCTTCAATTCTCTCC
SAG1
Genotyping
Denaturation 3 min at 94C
External forward:
(internal) or 95C (external);
GTTCTAACCACGCACCCTGAG
35 cycles: 40 sec at 95C
External Reverse:
(external) or 30 sec at 94C
(internal), 40 sec at 58C
(external) or 30 sec at 60C
Hae II,
Sau96 I
(Grigg et al.,
2001)
GTGGTTCTCCGTCGGTGTGAG
Internal forward:
CAATGTGCACCTGTAGGAAGC
Internal reverse:
(internal), 90 sec at 72C
TTATCTGGGCAGGTGACAAC
(external) or 45 sec at 72C
SAG3
Genotyping
(internal); Post PCR extension
External forward:
AlwNI,
(Grigg et al.,
CAACTCTCACCATTCCACCC
NciI
2001)
4 min (external) or 10 min
External Reverse:
(internal) at 72C, hold at 4C
GCGCGTTGTTAGACAAGACA
Internal forward:
TCTTGTCGGGTGTTCACTCA
5
Internal reverse:
CACAAGGAGACCGAGAAGGA
GRA6 Genotyping
External forward:
MseI
ATTTGTGTTTCCGAGCAGGT
(Fazaeli
et
al., 2000)
External Reverse:
GCACCTTCGCTTGTGGTT
Internal forward:
TTTCCGAGCAGGTGACCT
Internal reverse:
TCGCCGAAGAGTTGACATAG
All primer sequences are listed in 5’to 3’ orientation. All PCR reactions were carried out in 50 l reaction volumes with 5 l of DNA
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a
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template used in external reactions and 2 l DNA amplicon from the external reaction used in internal reactions.
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b
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SAG1, SAG3, and GRA6).
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NA = Not applicable
Restriction enzymes used for genotyping with restriction fragment length polymorphism (RFLP) assays for genotyping loci (B1,
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27
6
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References
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30
31
Fazaeli, A., Carter, P.E., Darde, M.L., and Pennington, T.H. (2000) Molecular typing of Toxoplasma gondii strains by GRA6 gene
sequence analysis. Int J Parasit 30: 637-642.
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Grigg, M.E., and Boothroyd, J.C. (2001) Rapid identification of virulent type I strains of the protozoan pathogen Toxoplasma
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gondii by PCR-restriction fragment length polymorphism analysis at the B1 gene. J Clin Microbiol 39: 398-400.
34
35
Grigg, M.E., Ganatra, J., Boothroyd, J.C., and Margolis, T.P. (2001) Unusual abundance of atypical strains associated with human
ocular toxoplasmosis. J Infect Dis 184: 633-639.
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Homan, W.L., Vercammen, M., De Braekeleer, J., and Verschueren, H. (2000) Identification of a 200- to 300-fold repetitive 529
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bp DNA fragment in Toxoplasma gondii, and its use for diagnostic and quantitative PCR. Int J Parasitol 30: 69-75.
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Rejmanek, D., Vanwormer, E., Miller, M.A., Mazet, J.A.K., Nichelason, A.E., Melli, A.C. et al. (2009) Prevalence and risk factors
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associated with Sarcocystis neurona infections in opossums (Didelphis virginiana) from central California. Vet Parasitol
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166: 8-14.
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Shapiro, K., Mazet, J.A.K., Schriewer, A., Wuertz, S., Fritz, H., Miller, W.A. et al. (2010) Detection of Toxoplasma gondii oocysts
and surrogate microspheres in water using ultrafiltration and capsule filtration. Water Res 44: 893-903.
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