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Table 3 Overview of DNA extraction and M. bovis PCR methods used by laboratories participating in the PCR ring trial

From: A European interlaboratory trial to evaluate the performance of different PCR methods for Mycoplasma bovis diagnosis

  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]