A rapid method based on the one‐step reverse transcriptase‐polymerase chain reaction (RT‐PCR) technique for detection of different strains of citrus tristeza virus

Abstract

A rapid and sensitive assay based on the one‐step reverse transcriptase‐polymerase chain reaction (RT‐PCR) technique was developed for the detection of citrus tristeza virus (CTV) in citrus plants. This method combines reverse transcription (RT) and polymerase chain reaction (PCR) in one tube using an optimized buffer condition and efficient enzyme mix. In comparison with the two‐step RT‐PCR and enzyme‐linked immunosorbent assay (ELISA) tests, the one‐step RT‐PCR method has a higher sensitivity in CTV detection especially when the CTV concentration is low in its host. After graft‐inoculation, CTV in the infected plant was first monitored by the one‐step RT‐PCR method at the fourth week which is one week earlier than by the two‐step RT‐PCR method and two weeks earlier than by the ELISA test. Three major strains of CTV in Taiwan: seedling yellows (SY), sweet orange stem‐pitting (SO/SP), and pummelo stem‐pitting (Pum/SP) strains, could be accurately detected by this method with a CTV‐specific primer pair. These CTV strains have their different pathogenicities in citrus cultivars grown in Taiwan such as Ponkan mandarin (Citrus poonensis Hort.), Luchen sweet orange (C. sinensis Osb.), and Wentan pummelo (C. grandis f. buntan Hay.). CTV is usually latent in these hosts, and the apparent symptoms appear only in the Luchen host infected by SO/SP strain and the Wentan host infected by Pum/SP strain. RT‐PCR detection of CTV demonstrated that both SY and SO/SP strains could infect Ponkan and Luchen whereas Pum/SP could infect Luchen and Wentan. The one‐step RT‐PCR assay could detect CTV in both fresh and dried tissues. Detection in dried tissues offers a safe way for international cooperation in CTV‐strain research without the possible spreading of exotic CTV strains.