Amplification of different nucleic acid targets in the same reaction (multiplex polymerase chain reaction) is challenging but an extremely useful tool especially for viroid diagnosis. In the amplification mixtures, several pairs of primers work together in the same conditions to detect different targets. Here, we describe the development and use of a multiplex reverse transcription polymerase chain reaction protocol highlighting the most crucial factors that can significantly affect the quality of the method. First, particular attention must be paid to primer design. Then, the amplification mixture and temperature conditions must be calibrated precisely to avoid cross reactivity or loss in sensitivity. Finally, the detection system of the amplification results must allow a specific identification of the amplified target(s).
Keywords: Agarose gel; Amplicon size; One-step mRT-PCR; Primer design; Two-step mRT-PCR; Viroids.
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