Testing Lab

RNA Extraction Kit

RT-PCR
Kit

Probe source

Thermocycler

# Cycles

CT cut-off

Instrument

Software

OIE Ref Lab, A

QIAamp viral RNA extraction kit Qiagen

LightCycler 480 RNA Master Hydrolysis Probes

IDT

LightCycler 480

LightCycler software release 1.5.0

45

No CT is negative

Lab B

High Pure RNA Tissue kit Roche

LightCycler 480 RNA Master Hydrolysis Probes

IDT or Applied Biosystems

LightCycler 480

LightCycler 480 software release 1.5.0

45

No CT is negative

Lab C

High Pure RNA Tissue kit Roche

LightCycler 480 RNA Master Hydrolysis Probes

Roche

LightCycler 480

LightCycler second derivative max

45

No CT is negative

Lab D

Total RNA kit I (E.Z.N.A)

Express One-Step qPCR SuperScript Mix

Applied Biosystems

StepOne (Applied Biosystems)

StepOne Software v2.0 FAST mode

45

No CT is negative

Lab E

Total RNA kit I (E.Z.N.A)

Invitrogen

 

Stratagene MX 3000P

MxPro

 

>38 is inconclusive
No CT is negative

Lab F

Total RNA kit I (E.Z.N.A)

Invitrogen

 

Stratagene MX 3000P

MxPro

 

>38 is inconclusive
No CT is negative

Lab G

Total RNA kit I (E.Z.N.A)

TaqMan® RNA-to-CtTM 1-Step Kit

Applied Biosystems

StepOnepPlus Real-Time PCR System (Applied Biosystems)

StepOne Software v2.0

45

>37.81 or no CT is negative

Lab H

No report

No report

No report

No report

No report

No report

No report

Lab I

High Pure RNA Tissue kit Roche

LightCycler 480 RNA Master Hydrolysis Probes

Roche

LightCycler 480

LightCycler 480 second derivative max

45

No CT is negative

Lab J

High Pure Viral Nucleic Acid kit Roche

LightCycler RNA Master Hydrolysis Probes

 

LightCycler 1.5

LightCycler 4.0

45

No CT is negative

Lab K

RNeasy Mini Kit Qiagen

One-step real-time RT-PCR Quantitech Probe Kit Qiagen

 

Stratagene MX 3000P

MxPro

 

No CT is negative

Lab L

Total RNA kit I (E.Z.N.A)

SuperScript III Platinum One-Step Quantitative RT-PCR system (Invitrogen)

 

Stratagene MX 3000P

MxPro

40

>38 or no CT is negative

Lab M

QIAamp viral RNA extraction kit Qiagen

SuperScript III Platinum One-Step Quantitative RT-PCR system (Invitrogen)

IDT

Stratagene MX 3000P

MxPro 3005 V 4.01 build 369

45

Seg 8 >35 & ELF1α <33 is negative

Lab N

Total RNA kit I (E.Z.N.A)

Stratagene Brilliant®II qRT-PCR Master Mix Kit

Applied Biosystems

Stratagene MX 3000P

MxPro QPCR default settings

45

No CT is negative

Lab O

MagMAX-96 Viral RNA Isolation Kit (MagMAXTM Express-96 Magnetic Particle Processors)

VetMAX Multiplex RT-PCR Reagents

confidential

Applied Biosystems 7500 Fast System

7500 System SDS Software Version 1.4.0.25

40

≥38 is negative

Lab V

Total RNA kit I (E.Z.N.A)

SuperScript III Platinum One-Step Quantitative RT-PCR system (Invitrogen)

 

Stratagene MX 3000P

MxPro

45

>40 is negative

Table 2: Real-time RT-PCR protocols used by each laboratory for amplification of ISAV segment 8.