Table 3 Overview of DNA extraction and M. bovis PCR methods used by laboratories participating in the PCR ring trial
Lab 1 | Lab 2 | Lab 3 | Lab 4 | Lab 5 | Lab 6 | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
DNA extraction methods | |||||||||||
Method (kit) | QIAamp DNA Mini Kit (Qiagen) | Promega Wizard | QIAamp DNA Mini Kit (Qiagen) | QIAamp DNA Mini Kit (Qiagen) | EZ1 DNA Tissue Kit, (Qiagen) | MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche) | |||||
Extraction equipment | QIAamp DNA Mini Kit spin column | Promega Maxwell automated DNA extraction | QIAamp DNA Mini Kit spin column | QIAamp DNA Mini Kit spin column | EZ1 robot, (Qiagen) | MagNA Pure LC Instrument (Roche) | |||||
Sample lysis buffer | Buffer AL (Qiagen) | Buffer AL (Qiagen) | Buffer AL (Qiagen) | Buffer AL (Qiagen) | Kit based | “Total NA External lysis” protocol | |||||
Sample volume (μl) | 200 | 300 | 200 | 200 | 200 | 200 | |||||
Elution volume (μl) | 200 | 300 | 200 | 200 | 100 | 200 | |||||
PCR methods | |||||||||||
PCR assay | Real-time | End-point | Real-time | End-point | PCR-DGGE | Real-time | Real-time | Real-time | Real-time | ||
Targeted genome region | polC | polC | polC | uvrC | 16S rRNA | oppD | polC | oppD | uvrC | oppD | 16S rRNA (V3-V4) |
Method (kit) | MPBO50 (Thermo Fisher Scientific) | MPBO50 (Thermo Fisher Scientific) | NA | Jumpstart ready mix (Sigma) | MPBO50 (Thermo Fisher Scientific) | NA | NA | QuantiFast triplex Kit Real Time-PCR kit (Qiagen) | |||
DNA volume (μl) | 5 | 5 | 1 | 1 | 1 | 1 | 5 | 5 | 2 | 5 | |
Total PCR volume (μl) | 25 | 25 | 25 | 50 | 50 | 25 | 25 | 22 | 25 | 15 | 20 |
DNA polymerase | Provided in the kit | GoTaq (Promega) | Provided in the kit | Taq Gold (Applied Biosystems) | Jumpstart ready mix (Sigma) | Provided in the kit | iTaq universal | PerfeCTa qPCR ToughMix (Quanta Bio) | Provided in the kit | ||
Thermocycler | LightCycler 480 | Bio-Rad | Stratagene MX3000 | Bio-Rad C1000 Touch and iCycler | Rotor-Gene (Qiagen) | Bio-Rad CFX96 | ABI 7500 Fast | ABI 7500 | |||
Number of cycles | 45 | 45 | 45 | 30 | 45 | 45 | 45 | 45 | 40 | ||
Cut-off (Ct) | 37 | NA | 37 | NA | 36 | 37 | 37 | Doubtful 35 < Ct < 40; Positive Ct > 40 | |||
Electrophoresis methods | |||||||||||
Method | NA | Qiaxcel | NA | E-Gel™ EX Agarose Gels, 2% (Thermo Fisher Scientific) | NA | NA | NA | NA | NA | ||
Volume per lane (μl) | NA | 0.2 | NA | 20 | NA | NA | NA | NA | NA | ||
Reference (if the method has been published) | |||||||||||
NA | [25] | NA | [26] | [35] | [23] | NA | [23] | [24] | [23] | [22] | |