PCR Detection of Microbial Pathogens PCR Detection of Microbial ...

PCR Detection of Trichinella 303
Table 2
Multiplex-PCR Amplicon Sizes
of Primer Sets of 10 Trichinella Genotypes
Primer
pair Ts Tna Tb Tps-Ne Tps-Pa Tps-Au Tm T6 Tne Tpa T11
I 173 127 127 310 340 360 127 127 155 240 264
II 253
III 210
IV 316
V 404
Abbreviations: T. spiralis (Ts); T. nativa (Tna); T. britovi (Tb); T. pseudospiralis (Tps) of
Nearctic (Ne), Palearctic (Pa), and Australian (Au) regions; T. murrelli (Tm); Trichinella T6
(T6); T. nelsoni (Tne); T. papuae (Tpa); and Trichinella T11 (T11).
2.3. Primer Sets for Multiplex-PCR and for PCR-RFLP
2.3.1. Multiplex PCR (for amplicon sizes see Table 2):
Primer pair I: 5'-GTTCCATGTGAACAGCAGT-3';5'-CGAAAACATA
CGACAACTGC-3'
Primer pair II: 5'-GCTACATCCTTTTGATCTGTT-3';5'-AGACACAAT
ATCAACCACAGTACA-3'
Primer pair III: 5'-GCGGAAGGATCATTATCGTGT-3';5'-ATGGATTA
CAAAGAAAACCATCACT-3'
Primer pair IV: 5'-GTGAGCGTAATAAAGGTGCAG-3';5'-TTCATCAC
ACATCTTCCACTA-3'
Primer pair V: 5'-CAATTGAAAACCGCTTAGCGTGTTT-3';5'-TGATC
TGAGGTCGACATTTCC-3'
Each primer is diluted at 100 pmol/µL in sterile H2O. Multiplex primer set concentration: combine the same vol of each primer;
final concentration: 10 pmol/µL of each primer (see Note 3).
2.3.2. Primer Sets for PCR-RFLP, 43 kDa (E/S)
Ts43CAF: 5'- ATGCGAATATACATTTTTCTTA-3'
Ts43CAR: 5'- TTAGCTGTATGGGCAAGG-3'
Each primer is diluted at 100 pmol/µL in sterile H 2O.
2.4. Preparation and Amplification of Larva DNA
1. PBS washing buffer: 137 mM NaCl (8 g/L), 7 mM K 2HPO 4 (1.21 g/L), KH 2PO 4
(0.34 g/L).
2. Sterile Tris-HCl buffer: 1 mM Tris-HCl, pH 7.6.