TABLE 1.
Real-time PCR methodology used by NRC and participating laboratories
| Laboratory | Extraction kit | Target genes | Real-time apparatus type | No. of cycles | Probe type | Origin of reaction mixture | Reaction vol (μl) | Eq. sample used for PCRd | No. of separate areas | UNG usee | Extraction controls | Amplification controls | Primer/probe supplier | Primer/probe origin or reference
|
|||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| IS481 | IS1001 | ptxA-Pr | Porin | ||||||||||||||
| 1 (NRC) | High Purea | IS481, ptxA-Pr, IS1001 | LC 1.0b | 45 | FRET | FastStart Master+c | 20 | 5 | 4 | Yes | Yes | Yes | TibMolBiol | 18 | 18 | 1 | NAh |
| 2 | High Pure | IS481 | LC 2.0 | 45 | FRET | FastStart Master+ | 20 | 10 | 3 | Yes | Yes | Yes | TibMolBiol | 18 | NA | NA | NA |
| 3 | High Pure | IS481, IS1001 | Rotorgene | 40 | TaqMan | Platinum Taq DNA Polj | 25 | 10 | 4 | No | Yes | Yes | Eurogentec | In-house | In-house | NA | NA |
| 4 | Genomic KF (Promega) | IS481, IS1001, porin | LC 1.0, SmartCycler 2.0f | 45 | TaqMan | Multiplex PCR Qiagen | 25 | 10 | 4 | Yes | Yes | Yes | Sigma | 11 | 11 | NA | In-house |
| 5 | MagNA Pureg | IS481 | SmartCycler 2.0 | 40 | TaqMan | Premix Ex TaqTM | 25 | 5 | 3 | No | Yes | Yes | Eurogentec | In-house | NA | NA | NA |
| 6 | QiAamp DNA mini kit | IS481 | LC 2.0 | 50 | FRET | FastStart Master+ | 20 | 2.5 | 3 | Yes | No | Yes | GenProbe | 18 | NA | NA | NA |
| 7 | EZ1 robot (Qiagen) | IS481 | iQ cycler | 48 | Molecular Beacon | iQ Supermix (Bio-Rad) | 50 | 5 | 3 | No | Yes | Yes | Sigma | 21 | 21 | NA | NA |
| 8 | MagNAPure | IS481 | Applied 7300 | 45 | TaqMan | Applied | 50 | 5 | 4 | Yes | Yes | Yes | Genset Oligos | In-house | NA | NA | NA |
| 9 | QIAamp DNA mini kit | IS481 | SmartCycler 2.0 | 40 | TaqMan | Eurogentec | 25 | 5 | 3 | Yes | Yes | Yes | Eurogentec | 18i | NA | NA | NA |
a
High Pure PCR template preparation kit (Roche).
b
LC, LightCycler.
c
FastStart Master +, LightCycler FastStart DNA master plus hybridization probes (Roche).
d
Equivalent sample used, determined as the starting volume (if fluidified, dilution factor included) used for concentration and extraction × fraction of extracted volume added to the amplification mix.
e
UNG, uracyl-N-glycosylase.
f
For QC8 and QC9, SmartCycler 2.0 apparatus used with qPCR mastermix noRox probes (Eurogentec).
g
MagNA Pure Compact nucleic acid isolation kit (Roche) with use of the robot.
h
NA, not applicable.
i
Only for the primers; in-house probe design.
j
Pol, polymerase.