Polymerase chain reaction detection of greening bacterium (Candidatus Liberibacter asiaticus) and Citrus mosaic virus in citrus tissues, by means of a simplified template-preparation protocol

Application of a simplified protocol for nucleic acid preparation for polymerase chain reaction (PCR) detection of greening bacterium (Candidatus Liberibacter asiaticus; Cla) and Citrus mosaic virus (CMBV) associated with citrus is described. Crude extracts of citrus tissues in NaOH - ethylenediaminetetraacetic acid solution, prepared without the use of liquid nitrogen, were spotted on a nitrocellulose membrane (NCM) and then eluted in water at 80 °C for 10 min. Both the greening bacterium and CMBV were detected by PCR from the eluted liquid from the spotted NCMs. The detection efficacy for the templates obtained in the NCM-eluted liquid was comparable with that of templates obtained with a multistep laboratory method or a commercial kit for nucleic acid preparation. The protocol is simple, inexpensive, rapid, and applicable to large-scale surveys of citrus trees. The spotted-membrane methodology can also be used for short-term sample storage for future testing or for long-distance sample transport to a d...