Quantitative detection of Citrus tristeza virus in plant tissues and single aphids by real-time RT-PCR

Abstract

TaqMan real-time reverse transcriptase (RT)-polymerase chain reaction (PCR) using purified RNA targets or coupled to tissue-print and squash procedures was developed to detect and quantify Citrus tristeza virus (CTV) RNA-targets in plant tissues and in single aphids. With this method all CTV isolates tested from different hosts and origins were detected. The sensitivity of conventional real-time RT-PCR was 1,000 times higher than immunocapture (IC)-RT-nested PCR and 10(6) times higher than enzyme linked immunosorbent assay (ELISA). The quantitation limit ranged from 1.7x10(2) to 1.7x10(9) transcript copies. The estimated number of CTV RNA-targets detected in different organs of a CTV-infected tree ranged from 4.5x10(5) to 6.5x10(8) copies when purified RNA was used as template and from 1.9x10(4) to 3.7x10(6) when tissue-printed material was used. In single squashed aphids the number of copies ranged from 4.73x10(3) to 1.23x10(5). Reliable quantitation of CTV targets present in infected plant material or acquired by single aphid species, was achieved with tissue-print and squash procedures combined with real-time RT-PCR, both of which do not require extraction procedures or nucleic acid purification.