An alkaline solution simplifies nucleic acid preparation for RT-PCR and infectivity assays of viroids from crude sap and spotted membrane.

Formats of a simple protocol for the preparation of nucleic acids for infectivity and RT-PCR detection of viroids from minute amounts of plant material are described. The method consists of preparing crude extracts in a NaOH-EDTA solution and then testing the supernatant. The NaOH-EDTA extract can be used at four distinct stages of preparation depending upon the accuracy desired, namely: (1) incubation of extract for 15 min at room temperature and the use of the supernatant for RT-PCR; (2) the supernatant can be spotted onto a nitrocellulose membrane (NCM) without vacuum, and the water-eluted liquid is used for RT-PCR; (3) centrifugation of the extract and use of supernatant in RT-PCR; (4) for quantitative accuracy, spotting the centrifuged supernatant on NCM using a vacuum device and then using the water-eluted liquid for RT-PCR.

Evaluation of a simple membrane-based nucleic acid preparation protocol for RT-PCR detection of potato viruses from aphid and plant tissues.

A rapid and simple protocol for preparing viral RNA from aphids (Myzus persicae) and potato (Solanum tuberosum L.) tissue (leaves, sprouts, and tubers) for reverse transcription-polymerase chain reaction (RT-PCR) was developed. The four-step method involves: (1) preparing plant crude sap or aphid macerates in a buffered detergent (Triton XL-80N) solution; (2) immobilizing clarified sap on a nitrocellulose membrane; (3) performing reverse transcription using eluted water extract from cut-out spot from membrane; and (4) amplifying cDNA through PCR.