Duplex realtime RT-PCR assay for the detection of Potato spindle tuber viroid (PSTVd) along with ef 1-α gene of potato

Abstract

SYBR green based realtime RT-PCR assay coupled with melt curve analysis was developed for the detection of Potato spindle tuber viroid (PSTVd) along with and without internal control from potato. The amplification of the specific targets was identified by their melting points viz., 85.93±0.22, 85.62±0.34 and 82.07±0.23 for primer pairs PSTVd-1F/PSTVd-1R, PSTVd-NFP/PSTVd-NRP and PSTVd-QFP1/PSTVd-QRP1, respectively. The realtime RT-PCR assay was 1×104 times more sensitive than RT-PCR assay and the assay could detect up to 20 copies of the target using serially diluted plasmid and up to 0.025 fg of total RNA from infected tissues diluted in healthy plant RNA. Duplex realtime RT-PCR assay was also standardized to detect PSTVd along with elongation factor 1- α (ef-1α) gene, a stable housekeeping gene of potato. Duplex realtime RT-PCR assay showed two melt peaks indicating the successful amplification both the PSTVd RNA and internal control RNA. The developed assays could consistently detect PSTVd and proved to be highly sensitive and rapid for the detection of PSTVd in post entry quarantine testing.