Abstract：
:A molecular diagnostic assay for the rapid, sensitive and specific detection of Apple stem pitting virus (ASPV) in infected samples, utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 42 °C and the designed target-specific primers, was developed. The RT-RPA assay was able to be used in ASPV-infected leaves, rootstocks and fruits. Sensitivity tests, using ASPV transcripts, showed that the RT-RPA with the ASPV-specific primers was more sensitive than the conventional RT-PCR, with a detection limit of 1 fg/μL of RNA. In addition, the reaction time for the amplification of ASPV was shortened to as little as 1 min. The assay was highly specific and did not give a positive reaction to other viruses infecting pears. Moreover, the amplified genomic fragment of ASPV produced by the assay could be determined within 4 min using a portable capillary gel electrophoresis system. The entire process, excluding the extraction of total RNA, could be completed in 5 min using portable equipment in the field. This is the first report of utilizing an RT-RPA assay to detect a pear tree virus and the assay could be used both in the laboratory and in the field for ASPV detection.
journal_name
J Virol Methodsjournal_title
Journal of virological methodsauthors
Kim NY,Lee HJ,Jeong RDdoi
10.1016/j.jviromet.2019.113747subject
Has Abstractpub_date
2019-12-01 00:00:00pages
113747eissn
0166-0934issn
1879-0984pii
S0166-0934(19)30137-5journal_volume
274pub_type
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