Details

Autor(en) / Beteiligte

• Munawar, Mustafa Ahmad
• Martin, Frank
• Toljamo, Anna
• Kokko, Harri
• Oksanen, Elina

Titel

RPA-PCR couple: an approach to expedite plant diagnostics and overcome PCR inhibitors

Ist Teil von

• BioTechniques, 2020-10, Vol.69 (4), p.270-280

Ort / Verlag

Future Science Ltd

Erscheinungsjahr

2020

Quelle

Free E-Journal (出版社公開部分のみ）

Beschreibungen/Notizen

• DNA extraction can be lengthy and sometimes ends up with amplification inhibitors. We present the potential of recombinase polymerase amplification (RPA) to replace plant DNA extraction. In our rapid ‘RPA-PCR couple’ concept, RPA is tuned to slower reaction kinetics to promote amplification of long targets. RPA primers amplify target and some flanking regions directly from simple plant macerates. Then PCR primers exponentially amplify the target directly from the RPA reaction. We present the coupling of RPA with conventional, TaqMan and SYBR Green PCR assays. We applied the concept to strawberry pathogens and the identification marker . We found RPA-PCR couple specific, sensitive and reliable. The approach may also benefit other difficult samples such as food, feces and ancient samples. Crudely macerate plant tissue in water for a few minutes and transfer supernatant in a recombinase polymerase amplification (RPA) reaction. Incubate RPA reaction for 20 min at 39°C and thereafter directly utilize RPA reaction to exponentially amplify target through standard PCR.