eISSN: 2353-9461
ISSN: 0860-7796
BioTechnologia
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2/2018
vol. 99
 
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abstract:
RESEARCH PAPERS

The detection of avocado sunblotch viroid in avocado using a real-time reverse transcriptase polymerase chain reaction

Gabriel Morey-León
,
Eddy Ortega-Ramirez
,
Carmen Julca-Chunga
,
Cesar Santos-Chanta
,
Lissette Graterol-Caldera
,
Eric Mialhe

BioTechnologia vol. 99(2) C pp. 99–107 C 2018
Online publish date: 2018/06/26
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The sunblotch disease is one of the diseases that affects avocado (Persea americana Mill) crops in particular, causing serious crop losses. The causal agent is the avocado sunblotch viroid (ASBVd), a circular RNA-based pathogen built of 247 nucleotides, which causes stem discoloration, bleaching, variegation, and distortion on the leaves and sunken yellow or red blotches on the fruits. In this study, a novel SYBR green-based real-time RT-PCR (RT-qPCR) assay specific for ASB viroid was developed. The assay was followed by a standard curve and a melting curve analysis for the detection, identification, and quantification of the full-length ASB viroid. The assay’s lower limit of detection in the samples of avocado leaves was 8.8 copies/μl. The RT-qPCR was 100 times more sensitive to ASBVd than the conventional RT-PCR. The melting curve analysis showed a variation in the specific melting temperature (Tm) between positive samples, probably due to the alteration in the nucleotide sequences of viroids. In conclusion, the use of RT-qPCR assays established in this study, as a routine diagnostic method in breeding programs, could decrease the viroid incidence in avocado crops and its further accumulation in plants.
keywords:

Persea americana, avocado sunblotch viroid, RNA-based pathogen, real-time RT-PCR, sensitivity, specificity

 
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