Table 3 Overview of primers, enzymes and conditions used for multiplex multilocus nested PCR-RFLP typing of the Toxoplasma gondii isolates in the present study.

From: Investigation of Toxoplasma gondii in wastewater and surface water in the Qinghai-Tibet Plateau, China using real-time PCR and multilocus genotyping

Marker

 

PCR primers (5ʹ–3ʹ)

Size (bp)

Restriction enzymes

Enzyme digestion conditions

SAG187

SAG1 Ex F:

GTTCTAACCACGCACCCTGAG

390

Cfr13I + BfoI, Thermo Scientific (double digest)

37 °C 1 h

SAG1 Ex R:

AAGAGTGGGAGGCTCTGTGA

SAG1 In F:

CAATGTGCACCTGTAGGAAGC

SAG1 In R:

GTGGTTCTCCGTCGGTGTGAG

SAG275

SAG2 Ex F:

GGAACGCGAACAATGAGTTT

546

HinfI + TaqI, Thermo scientific

37 °C 1 h + 65 °C 1 h

SAG2 Ex R:

GCACTGTTGTCCAGGGTTTT

SAG2 In F:

ACCCATCTGCGAAGAAAACG

SAG2 In R:

ATTTCGACCAGCGGGAGCAC

SAG387

SAG3 Ex F:

CAACTCTCACCATTCCACCC

311

BcnI, Thermo Scientific

37 °C 1 h

SAG3 Ex R:

GCGCGTTGTTAGACAAGACA

SAG3 In F:

TCTTGTCGGGTGTTCACTCA

SAG3 In R:

CACAAGGAGACCGAGAAGGA

c 22-875

C22-8 Ex F:

TGATGCATCCATGCGTTTAT

521

Alw26I + MboII, Thermo Scientific (double digest)

37 °C 1 h + 55 °C 1 h

C22-8 Ex R:

CCTCCACTTCTTCGGTCTCA

C22-8 In F:

TCTCTCTACGTGGACGCC

C22-8 In R:

AGGTGCTTGGATATTCGC

c 22-975

C29-2 Ex F:

ACCCACTGAGCGAAAAGAAA

446

TaiI + RsaI, Thermo Scientific (double digest)

37 °C 1 h

C29-2 Ex R:

AGGGTCTCTTGCGCATACAT

C29-2 In F:

AGTTCTGCAGAGTGTCGC

C29-2 In R:

TGTCTAGGAAAGAGGCGC

L35875

L358 Ex F:

TCTCTCGACTTCGCCTCTTC

418

BsuRI + Hin1II, Thermo Scientific (double digest)

37 °C 1 h

L358 Ex R:

GCAATTTCCTCGAAGACAGG

L358 In F:

AGGAGGCGTAGCGCAAGT

L358 In R:

CCCTCTGGCTGCAGTGCT

BTUB75

BTUB Ex F:

TCCAAAATGAGAGAAATCGT

411

Bsh1285I + TaqI, Thermo Scientific (double digest)

65 °C 1 h

BTUB Ex R:

AAATTGAAATGACGGAAGAA

BTUB In F:

GAGGTCATCTCGGACGAACA

BTUB In R:

TTGTAGGAACACCCGGACGC

PK175

L358 Ex F:

TCTCTCGACTTCGCCTCTTC

903

Eco88I + RsaI, Thermo Scientific (double digest)

37 °C 1 h

L358 Ex R:

GCAATTTCCTCGAAGACAGG

L358 In F:

AGGAGGCGTAGCGCAAGT

L358 In R:

CCCTCTGGCTGCAGTGCT

GRA675

GRA6 Ex F:

ATTTGTGTTTCCGAGCAGGT

344

Tru1I, Thermo Scientific

37 °C 1 h

GRAG Ex R:

GCACCTTCGCTTGTGGTT

GRA6 In F:

TTTCCGAGCAGGTGACCT

GRA 6 In R:

TCGCCGAAGAGTTGACATAG

Apico75

APICO Ex F:

TGGTTTTAACCCTAGATTGTGG

640

AflII + DdeI, Thermo Scientific (double digest)

37 °C 1 h

APICO Ex R:

AAACGGAATTAATGAGATTTGAA

APICO In F:

GCAAATTCTTGAATTCTCAGTT

APICO In R:

GGGATTCGAACCCTTGATA

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