Table 4.
Technical or methodological details of the microsatellite typing technique as retrieved by questionnaire and further personal communications from laboratories participating in a ring trial on microsatellite typing of Toxoplasma gondii
| Details | Laboratory, operator, or software | |||||
|---|---|---|---|---|---|---|
| A-1a | A-2a | B | C | D | E-1 or E-2b | |
| Real-time PCR protocol used to quantify DNA | Adapted from [25] | Adapted from [25] | [26, 27] | [28] | [29] | Adapted from [30] |
| Target of real-time PCR used to quantify DNA | REP529, GenBank accession no. AF146527 | REP529, GenBank accession no. AF146527 | REP529, GenBank accession no. AF146527 | REP529, GenBank accession no. AF146527 | REP529, GenBank accession no. AF146527 | REP529, GenBank accession no. AF146527 |
| Template volumes used in real-time PCR | 5 μL | 5 μL | 5 μL | 5 μL | 3 μL | 10 μL |
| PCR MS typing (Part I) | 15 MS multiplex | 15 MS multiplex | 15 MS multiplex + singleplexes | 15 MS multiplex | 8 MS multiplex (typing markers) + 7 MS multiplex (fingerprinting) | 15 MS multiplex |
| Supplier/provider of typing primers | IN | IN | AB | AB | EU | ME (EU in comparative experiments) |
| Template volume used for MS multiplex PCR | 5 μL | 5 μL | 5 μL | 5 μL | 5 μL | 5 μL |
| Volume of amplification product used for fragment length analysis | 1 μL | 1 μL | 1 μL | 1 μL | 1 μL | 1 μL |
| Multiplex PCR kit used for typing | QIAGEN Multiplex PCR Kit (cat. no. 206143) | QIAGEN Multiplex PCR Kit (cat. no. 206143) | QIAGEN Multiplex PCR Kit (cat. no. 206143) | QIAGEN Multiplex PCR Kit (cat. no. 206143) | QIAGEN Multiplex PCR Kit (cat. no. 206143) | QIAGEN Multiplex PCR Kit (cat. no. 206143) |
| Capillary sequencer | 3730 DNA Analyzer (ABI) | 3730 DNA Analyzer (ABI) | 3130xl Genetic Analyzer (ABI) | SeqStudio Genetic Analyzer (ABI) | 3730 Genetic Analyzer (ABI) | Hitachi 3500 Genetic Analyzer (ABI) |
| Size standard | LIZ 500 (TF) | LIZ 500 (TF) | ROX 500 (ABI) | ROX 500 (ABI) | LIZ 500 (ABI) | ROX 500 (ABI) |
| MS typing software | Peak Scanner 2.0 (no bins established for allele identification) | Osiris (first part of ring trial only, no bins established for allele identification) | Gene-Mapper (bins established for allele identification) | Peak Scanner online (semi-automatic MS EXCEL template to ease allele identification) | Geneious Prime (bins established for allele identification) | Geneious Prime (bins established for allele identification) |
aIn laboratory A, two different software tools were used, i.e., Peak Scanner 2.0 (A-1) or Osiris (A-2). bIn laboratory E, two operators assessed raw data, i.e., electrophoresis profiles