A novel rolling circle amplification assay to detect members of the family Anelloviridae in pigs and humans

2011 ◽  
Vol 160 (1-2) ◽  
pp. 424-427 ◽  
Author(s):  
Lisa Macera ◽  
Martí Cortey ◽  
Fabrizio Maggi ◽  
Joaquim Segalés ◽  
Tuija Kekarainen
2014 ◽  
Vol 97 (5) ◽  
pp. 1410-1415
Author(s):  
Yu-ran Zhao ◽  
Wei-li Yin ◽  
Zhi-qin Yue ◽  
Ba-fang Li

Abstract White spot syndrome virus (WSSV) is a global threat to the prawn industry, and there is no simple method for field-based testing of this virus. We designed a padlock probe and primers to the capsid protein gene VP28 of WSSV, and established a hyperbranched rolling circle amplification (HRCA) assay and a corresponding strip-based test. The assay and the test strip both had similar high accuracy and specificity, and their sensitivity was about 10 copies/μL, which is 100 times higher than conventional PCR. In this study, 68 batches of prawns were tested for WSSV with the HRCA assay and test strip, and the results were compared with the PCR assay. The results indicated that both the assay and test strip had accuracy similar to each other and to the PCR results. However, the assay and strip were more sensitive and user-friendly than PCR. Establishment of this method will provide a rapid detection of WSSV and also a basis for field-based detection of animal disease.


2016 ◽  
Vol 148 (2-3) ◽  
pp. 237-240 ◽  
Author(s):  
Alfredo Diaz-Lara ◽  
David H. Gent ◽  
Robert R. Martin

During a survey for new viruses affecting hop plants, a circular DNA molecule was identified via rolling circle amplification (RCA) and later characterized. A small region of the 5.7-kb long molecule aligned with a microsatellite region in the Humulus lupulus genome, and no coding sequence was identified. Sequence analysis and literature review suggest that the small DNA molecule is an extranuclear DNA element, specifically, an extrachromosomal circular DNA (eccDNA), and its presence was confirmed by electron microscopy. This work is the first report of eccDNAs in the family Cannabaceae. Additionally, this work highlights the advantages of using RCA to study extrachromosomal DNA in higher plants.


2018 ◽  
Vol 108 (7) ◽  
pp. 902-909 ◽  
Author(s):  
Luz Marcela Yepes ◽  
Elizabeth Cieniewicz ◽  
Björn Krenz ◽  
Heather McLane ◽  
Jeremy R. Thompson ◽  
...  

Grapevine red blotch virus (GRBV) has a monopartite single-stranded DNA genome and is the type species of the genus Grablovirus in the family Geminiviridae. To address the etiological role of GRBV in the recently recognized red blotch disease of grapevine, infectious GRBV clones were engineered from the genome of each of the two previously identified phylogenetic clades for Agrobacterium tumefaciens-mediated inoculations of tissue culture-grown Vitis spp. plants. Following agroinoculation and one or two dormancy cycles, systemic GRBV infection was detected by multiplex polymerase chain reaction (PCR) in Vitis vinifera exhibiting foliar disease symptoms but not in asymptomatic vines. Infected rootstock genotype SO4 (V. berlandieri × V. riparia) exhibited leaf chlorosis and cupping, while infection was asymptomatic in agroinoculated 110R (V. berlandieri × V. rupestris), 3309C (V. riparia × V. rupestris), and V. rupestris. Spliced GRBV transcripts of the replicase-associated protein coding region accumulated in leaves of agroinfected vines, as shown by reverse-transcription PCR; this was consistent with systemic infection resulting from virus replication. Additionally, a virus progeny identical in nucleotide sequence to the infectious GRBV clones was recovered from agroinfected vines by rolling circle amplification, cloning, and sequencing. Concomitantly, subjecting naturally infected grapevines to microshoot tip culture resulted in an asymptomatic plant progeny that tested negative for GRBV in multiplex PCR. Altogether, our agroinoculation and therapeutic experiments fulfilled Koch’s postulates and revealed the causative role of GRBV in red blotch disease.


Plant Disease ◽  
2016 ◽  
Vol 100 (11) ◽  
pp. 2211-2214 ◽  
Author(s):  
Alfredo Diaz-Lara ◽  
Robert R. Martin

This study describes the nucleotide sequence and genome organization of a new DNA virus isolated from ‘Bluecrop’ blueberry plants exhibiting fruit-drop symptoms and named Blueberry fruit drop-associated virus (BFDaV). Blueberry fruit drop disease was first detected in blueberry plants in British Columbia, Canada in the late 1990s, and in a single field in northern Washington state in the United States in 2012. Infected bushes abort nearly 100% of their fruit about three weeks prior to harvest, when the berries are about 3 to 5 mm in diameter. At harvest, the affected plants appear taller than healthy ones as there is no fruit weighing down the branches. The virus was amplified from diseased material using rolling circle amplification, followed by enzyme digestion, cloning, and sequencing. The full genome of BFDaV is 9,850 bp in length and contains a single open reading frame, encoding for a polyprotein, and a large noncoding region. Based on the genome size and organization and phylogenetics, BFDaV is proposed as a new and the largest member of family Caulimoviridae. Finally, in mapping part of a field with fruit-drop symptoms, there was a nearly perfect correlation between the presence of the virus and fruit-drop symptoms.


2006 ◽  
Vol 87 (12) ◽  
pp. 3551-3557 ◽  
Author(s):  
Kurt Tobler ◽  
Claude Favrot ◽  
Gilles Nespeca ◽  
Mathias Ackermann

Epidermodysplasia verruciformis (EV) is a rare human genetic predisposition to develop flat warts, some of which subsequently undergo cancer transformation. Some human papillomaviruses (HPVs), i.e. HPV 5 and 8, have been associated with cancer development as a sequela of EV. As similar diseases have been observed in dogs, it was hypothesized that unknown canine papillomaviruses (CPVs) may exist and that they may be present in cases of canine EV. Consequently, DNA was extracted from a malignant lesion of a dog with EV and circular DNA was amplified by multiple-primed rolling-circle amplification (RCA). Indeed, sequence determination and analysis of the RCA-amplified and cloned DNA from a malignant canine EV lesion resulted in the detection and primary description of a third CPV (CPV3). Typical papillomavirus genes were identified, with deduced amino acid similarities ranging from 20 to 57 % for E1, E2, E6, E7, L1 and L2, respectively. According to the sequence of the L1 gene, which is used for papillomavirus classification, the new isolate meets the majority of criteria needed to declare detection of a novel genus among the papillomaviruses. Thus, CPV3 may represent the prototype of this novel genus. As the novel virus was found in a dog in association with lesions reminiscent of human EV, it should be interesting to test in the future whether this condition can be reproduced in experimental animals. If such were the case, a new model for EV could be established.


LWT ◽  
2021 ◽  
pp. 111807
Author(s):  
Arun Prince Milton ◽  
Kasanchi M. Momin ◽  
G. Bhuvana Priya ◽  
Samir Das ◽  
M. Angappan ◽  
...  

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 50
Author(s):  
Joanna Kaczorowska ◽  
Mila Sparreboom ◽  
Martin Deijs ◽  
Maarten F. Jebbink ◽  
Lia van der Hoek

Torque teno viruses (TTVs), the most well-known members of the family Anelloviridae, are not associated with any disease. They are one of the most abundant and divergent entities in the viral world; however, the cause of their variability is not currently known. In this study, a set of longitudinally collected serum samples from two HIV-1 infected and two non-infected persons was analyzed for the presence of TTVs and other Anelloviridae using a genera-specific quantitative PCR. The samples positive for TTVs were selected for the quantitative heteroduplex tracking assay (QHTA), which showed repeating patterns of TTV genotypes. Sanger sequencing of the partial viral sequences revealed that the same strains of TTVs are most probably disappearing and returning in different stages of life, with scarcely any new introductions. The partial sequences were grouped into phylogenetic genogroups, and samples representing each of these genogroups were selected for full-length genome sequencing using an in-house optimized rolling circle amplification (RCA)–Illumina protocol.


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