Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for In-Field Detection of American Plum Line Pattern Virus

• Published in: Viruses Vol. 16; no. 10; p. 1572
• Main Authors: Matić, Slavica, Myrta, Arben
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 01.10.2024; MDPI
• Subjects: American plum line pattern virus; Analysis; Capsids; Coat protein; Diseases and pests; Enzyme-linked immunosorbent assay; Flowering; Fruit trees; Gene amplification; Genetic aspects; Genetic diversity; Growth; Health aspects; Ilarvirus; Ilarvirus - classification; Ilarvirus - genetics; Ilarvirus - isolation & purification; isothermal amplification; Italy; Leaves; Maximum likelihood method; Molecular Diagnostic Techniques - methods; Movement protein; Nucleic Acid Amplification Techniques - methods; Ornamental plants; Pathogens; phylogenetic analyses; Plant Diseases - virology; Plant extracts; Plant virus diseases; Plant viruses; Plum; point-of-care detection; Polymerase chain reaction; Proteins; Prunus - virology; Prunus avium - virology; Prunus serrulata; Prunus serrulata Lindl; Quarantine; RNA, Viral - genetics; Sensitivity and Specificity; sequencing; Viruses; Italy; Lebanon; United States > US; Germany; Albania; point-of-care detection; quarantine pathogen; Prunus serrulata Lindl; isothermal amplification; American plum line pattern virus; phylogenetic analyses; sequencing
• ISSN: 1999-4915; 1999-4915
• DOI: 10.3390/v16101572

American plum line pattern virus (APLPV) is the most infrequently reported Ilarvirus infecting stone fruit trees and is of sufficient severity to be classified as an EPPO quarantine A1 pathogen. In late spring, yellow line pattern symptoms were observed on leaves in a few flowering cherries (Prunus serrulata Lindl.) grown in a public garden in Northwest Italy. RNA extracts from twenty flowering cherries were submitted to Ilarvirus multiplex and APLPV-specific RT-PCR assays already reported or developed in this study. One flowering cherry (T22) with mixed prunus necrotic ringspot virus (PNRSV) and prune dwarf virus (PDV) infection also showed infection with APLPV. Blastn analysis of PCR products of the full coat protein (CP) and movement protein (MP) genes obtained from flowering cherry T22 showed 98.23% and 98.34% nucleotide identity with reference APLPV isolate NC_003453.1 from the USA. Then, a LAMP-specific assay was designed to facilitate the fast and low-cost identification of this virus either in the laboratory or directly in the field. The developed assay allowed not only the confirmation of APLPV (PSer22IT isolate) infection in the T22 flowering cherry but also the identification of APLPV in an asymptomatic flowering cherry tree (TL1). The LAMP assay successfully worked with crude flowering cherry extracts, obtained after manually shaking a single plant extract in the ELISA extraction buffer for 3–5 min. The developed rapid, specific and economic LAMP assay was able to detect APLPV using crude plant extracts rather that RNA preparation in less than 20 min, making it suitable for in-field detection. Moreover, the LAMP assay proved to be more sensitive in APLPV detection in flowering cherry compared to the specific one-step RT-PCR assay. The new LAMP assay will permit the estimation of APLPV geographic spread in the territory, paying particular attention to surrounding gardens and propagated flowering cherries in ornamental nurseries.