Development of singleplex and multiplex real-time (Taqman®) RT-PCR assays for the detection of viruses associated with fig mosaic disease

Alsaheli, Zeinab; Abdallah, Ali E.; Incerti, Ornella; Shalaby, A. E. M.; Youssef, Sahar A.; Digiaro, M.; Elbeaino, Toufic

doi:10.1016/j.jviromet.2021.114145

Cited by 8 publications

(4 citation statements)

References 11 publications

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“…[ 15 ], who found FMV, FLMaV-1, FLMaV-2, and FMMaV in fig leaf samples, and Ref. [ 30 ], who found FMV, FLMaV-1, FLMaV-2, FMMaV, FFKaV, FCV-1, and FLV-1 in fig leaf samples during a preliminary survey conducted on a small number in some Egyptian governorates. In comparison with our findings, all 137 tested trees from all cultivars were infected by at least one virus, with FMV and FLMaV-2 being the most prevalent viruses with incidence rates of 49% and 21.8% in all tested plants, respectively, followed by FLMaV-1 with an incidence rate of 10.9% in all tested plants.…”

Section: Discussionmentioning

confidence: 99%

“…Various molecular approaches are currently highly effective in detecting viruses or viral particles in plants [ 23 , 28 , 29 ]. RT-PCR was therefore an accurate, rapid, and dependable technology used by several authors to detect FMD viruses [ 2 , 10 , 13 , 15 , 16 , 19 , 30 ]. In recent years, the use of nucleotide fragment sequence analysis and comparison with different fig virus isolates from around the world has significantly aided in the detection of fig mosaic disease viruses and understanding the ecology of the disease risk, emergence, and dynamics of fig mosaic disease [ 10 , 19 ].…”

Section: Introductionmentioning

confidence: 99%

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Incidence, Molecular Detection, and Partial Nucleotide Sequencing of Some Viruses Causing Fig Mosaic Disease (FMD) on Fig Plants in Egypt

Toima

M.El-Banna

Sayed

et al. 2022

International Journal of Microbiology

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Fig mosaic disease (FMD) is a viral disease that poses a significant danger to Egypt’s fig-producing economy. During the two growing seasons 2017 and 2018, fig leaves and fruits displaying a variety of symptoms linked with fig mosaic disease (FMD) were collected and differentiated from the most famous fig-growing governorates in Egypt, Mersa Matruh, Ismailia, and Giza. Symptomatic samples were tested for the presence of fig mosaic virus (FMV), fig leaf mottle-associated virus 1 (FLMaV-1), fig leaf mottle-associated virus 2 (FLMaV-2), fig mild mottle-associated virus (FMMaV), fig latent virus 1 (FLV-1), fig fleck-associated virus (FFkaV), and fig cryptic virus (FCV) using reverse transcription-polymerase chain reaction (RT-PCR) with specific primers. Three viruses were detected in mixed infections and showed positive results. FMV was detected with infection rate 49% followed by FLMaV-2 with infection rate 21.8% and FLMaV-1 with infection rate 10.9%, respectively, whereas all tested samples were negative for the other viruses. According to the sequence and phylogenetic analysis, the Egyptian FMV isolate was closely related to other FMV isolates, particularly the Argentina ones (Acc. No. KP796424), with 99% identity. While FLMaV-1 showed more than 98% identity with reference isolate FLMaV-1 (Acc. No. LN873219), on the other hand, the isolate of FLMaV-2 showed 100% identity with reference FLMaV-2 isolate (Acc. No. FJ473383) based on phylogenetic analysis. Because fig output in Egypt is expanding, our findings suggest that greater attention should be paid to improving the phytosanitary condition of fig trees in Egypt.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Incidence, Molecular Detection, and Partial Nucleotide Sequencing of Some Viruses Causing Fig Mosaic Disease (FMD) on Fig Plants in Egypt

Toima

M.El-Banna

Sayed

et al. 2022

International Journal of Microbiology

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“…In real-time PCR, controls must be prepared from the RM for each gene doping test [ 25 ], either by diluting high-concentration RMs for each test or by preparing diluted controls for long-term storage. The former method is generally used for quantitative operations requiring strict dilutions because a high concentration is more stable than a low concentration [ 1 ]. The latter method is easier to perform, but the stability of PTCs with low copy numbers is a concern.…”

Section: Discussionmentioning

confidence: 99%

Design and storage stability of reference materials for microfluidic quantitative PCR-based equine gene doping tests

Tozaki

Ohnuma

Kikuchi

et al. 2021

JES

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One method of gene doping in horseracing is administering of exogenous genetic materials, known as transgenes. Several polymerase chain reaction (PCR)-based methods have been developed for detecting transgenes with high sensitivity and specificity. However, novel designs for reference materials (RMs) and/or positive template controls (PTCs) are necessary for simultaneous analysis of multiple transgene targets. In this study, we designed and developed a novel RM for simultaneously detecting multiple targets via microfluidic quantitative PCR (MFQPCR). Twelve equine genes were selected as targets in this study. A sequence region including primers and probes for quantitative PCR was designed, and a 10 bp sequence was inserted to allow the RM to be distinguished from the original transgene sequences. The sequences of individual detection sites were then connected for 12 genes and cloned into a single plasmid vector. We performed fragment size analysis to distinguish between the PCR products of the original transgene sequence and those of the RM, enabling identification of RM contamination. PTCs diluted to 10,000, 1,000, 100, and 10 copies/µl with horse genomic DNA from RM were stably stored at 4°C for 1 year. As digital PCR enabled absolute quantification, the designed substances can serve as an RM. These findings indicate that the RM design and storage conditions were suitable for gene doping tests using MFQPCR.

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“…Multiplex TaqMan real-time PCR has been applied widely for pathogen detection in humans [14,[24][25][26][27] and animals [10,[28][29][30][31][32][33][34][35]. Recently, this method has also been used to detect viruses in sweet potatoes, pome fruit trees, stone fruit trees, and fig trees [36][37][38][39][40]. However, this method has not been used for the simultaneous detection of LSV, LMoV, CMV, SYSV, and PlAMV in lily plants.…”

Section: Introductionmentioning

confidence: 99%

Application of Multiplex TaqMan Real-Time PCR Assay in Survey of Five Lily Viruses Infecting Lilium spp.

Song

Jun

2021

Agronomy

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Lily symptomless virus (LSV), Lily mottle virus (LMoV), Cucumber mosaic virus (CMV), Shallot yellow stripe virus (SYSV), and Plantago asiatica mosaic virus (PlAMV) are five of the economically important viruses infecting lilies (Lilium spp.) worldwide. In order to prevent the occurrence and spread of these viruses, it is necessary to develop a rapid, effective, and sensitive detection method for the simultaneous detection and specific quantification of these viruses. In this study, specific primers and probes for multiplex TaqMan real-time PCR assays designed from conserved regions of the coat protein sequence of each virus were used for the simultaneous detection of these viruses in lilies (Lilium spp.). The optimal concentration of primers and probes and reaction annealing temperature were 20 µM and 55.9 °C, respectively. The detection limits of the assay were 1.33 × 102, 1.27 × 101, 1.28 × 101, 2.33 × 102, and 2.01 × 102 copies·μL−1 for LSV, LMoV, CMV, SYSV, and PlAMV, respectively. Specificity was determined using seven viral pathogens of lilies. Variability tests of intra- and inter-assays showed high reproducibility with coefficients of variation <2%. The multiplex TaqMan real-time PCR assay was used to detect these viruses from lily samples in China. In brief, our developed assay showed high specificity, sensitivity, and reproducibility for the simultaneous detection and differentiation of five lily-infecting viruses and can be used for certification and quarantine programs.

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Development of singleplex and multiplex real-time (Taqman®) RT-PCR assays for the detection of viruses associated with fig mosaic disease

Cited by 8 publications

References 11 publications

Incidence, Molecular Detection, and Partial Nucleotide Sequencing of Some Viruses Causing Fig Mosaic Disease (FMD) on Fig Plants in Egypt

Incidence, Molecular Detection, and Partial Nucleotide Sequencing of Some Viruses Causing Fig Mosaic Disease (FMD) on Fig Plants in Egypt

Design and storage stability of reference materials for microfluidic quantitative PCR-based equine gene doping tests

Application of Multiplex TaqMan Real-Time PCR Assay in Survey of Five Lily Viruses Infecting Lilium spp.

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