RT-PCR

Real-Time PCR (RT-PCR)

This is main method for fast COVID19 testing, recommended by the CDC, etc.

Both of the following two processes are required for this to be done:

1. Reverse Transcription. This is the conversion of all ribonucleic acid (RNA) contained in the sample into deoxyribonucleic acid (DNA - also known as double standed DNA (dsDNA) or complimentary DNA (cDNA).

2. DNA amplification - the amplification of a specific sequence of target DNA.

This can be achieved from either method:

1. Using a Commercial RT-PCR Master Mix (e.g. TaqPath 1-Step)

2. Using a self-assembled/custom RT-PCR mix

Commercial RT-PCR Master Mixes (Confirmed for COVID19)

USED IN COVID19 Testing

Thermo Fisher Scientific (Applied Biosystems) TaqPath™ 1-Step RT-qPCR Master Mix, CG

• **CURRENTLY RECOMMENDED BY THE CDC**

• This is a RT-PCR master-mix including:

  • MMLV reverse transcriptase, dNTPs [deoxynucleotide trip phosphtase]

  • UNG [Uracil-N-glycosylase]

  • ROX dye [for reference]

  • Thermostable Fast DNA polymerase.

  • This is compatible with TaqMan probes.

Thermo Fisher Scientific (Invitrogen) SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase (Link)

• **Used in the paper "Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR" (Corman, VM, et al, 2020)

• REACTION BUFFER from SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase:

  • • **Proprietary buffer** - containing 3.2 mM magnesium sulphate, 0.4mM dNTPs, and stabilizers

  • SuperScript III / Taq mixture

  • Magnesium sulphate solution

Other RT-PCR Master Mixes (Not confirmed for COVID19)

Thermo Fisher Scientific SuperScript IV One-Step RT-PCR System (Link)

Probes

TaqMan probes are fluorescent DNA probes that are used in increase specificity of quantitative PCR.

Real-Time PCR Instruments

Listing of Manufacturers

Comparison of instruments, SKUs