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Sensitive detection and discrimination method for studying multiple infections of five major plant viruses infecting ornamental plants in nursery environments
Author(s) -
Dobhal S.,
Arif M.,
Olson J.,
MendozaYerbafría A.,
AguilarMoreno S.,
PerezGarcia M.,
OchoaCorona F. M.
Publication year - 2015
Publication title -
annals of applied biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.677
H-Index - 80
eISSN - 1744-7348
pISSN - 0003-4746
DOI - 10.1111/aab.12182
Subject(s) - biology , cucumber mosaic virus , virology , ornamental plant , multiplex , tospovirus , virus , primer (cosmetics) , tobacco mosaic virus , plant virus , multiplex polymerase chain reaction , polymerase chain reaction , tomato spotted wilt virus , gene , genetics , botany , chemistry , organic chemistry
Tobacco mosaic virus ( TMV ), Hosta virus X ( HVX ), Cucumber mosaic virus ( CMV ), Tomato spotted wilt virus ( TSWV ) and Impatiens necrotic spot virus ( INSV ) are a few of the major viruses that infect ornamental and nursery plants. These viruses cause significant losses that impact growers and the ornamental industry. Often, a single ornamental plant is co‐infected by more than one virus, which makes identification and discrimination of these viruses a difficult task, thus creating delays and limiting regulatory measures for effective quarantine. The aim of this study is to develop a sensitive, rapid, economic, and reliable multiplex Reverse Transcription PCR ( RT‐PCR ) for simultaneous detection and discrimination of these five viruses. Specific PCR primers were designed using the consensus sequences of corresponding capsid protein ( CP ) genes of HVX and CMV , the nucleocapsid protein ( NP ) genes of TSWV and INSV , and the movement protein ( MP ) and CP genes of TMV . The primers were validated in vitro using single and multiplex RT‐PCR assays. The detection limit of each primer set in multiplex RT‐PCR was 100 fg ( TMV ), 1 fg ( HVX ), 10 fg ( CMV ), 10 pg ( TSWV ) and 10 pg ( INSV ). Forty‐six infected nursery samples collected from different locations in the USA were screened for virus infections using this multiplex RT‐PCR . The multiplex RT‐PCR has a potential for its application in routine diagnostics, quarantine, and epidemiological studies. The developed method is reliable, sensitive, and economic for testing a wide range of ornamental and nursery plants for detection of these viruses.