A novel HRM assay for the simultaneous detection and differentiation of eight poxviruses of medical and veterinary importance

Gelaye, Esayas; Mach, Lukas; Kolodziejek, Jolanta; Grabherr, Reingard; Loitsch, Angelika; Achenbach, Jenna E.; Nowotny, Norbert; Diallo, Adama; Lamien, Charles Euloge

doi:10.1038/srep42892

Cited by 48 publications

(59 citation statements)

References 43 publications

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“…The ability to discriminate between both Photobacterium subspecies was validated on a total of 19 Phdp and 11 Phdd strains isolated from Europe, USA and Japan, proving the assay is able to overcome the documented limitation due to the genetic variability of isolates from diverse hosts and geographical origins (Costas et al., ; Magarinos, Toranzo, Barja, & Romalde, ). The observed difference of at least 0.3°C between the two subspecies has been already proved sufficient/suitable for differentiating virus and bacterial species of medical and veterinary importance (Gelaye et al., ; Miller, Zorn, & Brielmeier, ) as well as for the monitoring of bacterial communities in food (Sardaro et al., ). In fact, both classical melting curve analysis and High Resolution Melting (HRM) analysis have been described as powerful techniques for variant scanning and genotyping, enabling to analyse even single genetic variants in nucleic acid sequences (Wittwer, ).…”

Section: Discussionmentioning

confidence: 99%

Development of a real‐time PCR assay for rapid detection and quantification of Photobacterium damselae subsp. piscicida in fish tissues

Carraro

Rovere

Ferraresso

et al. 2017

Journal of Fish Diseases

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The availability of a rapid and accurate method for the diagnosis of Photobacterium damselae subsp. piscicida (Phdp), able to discriminate its strictly correlated subsp. damselae (Phdd), formally known as Vibrio damsela, is essential for managing fish pasteurellosis outbreaks in farmed fish. A single-step, high-sensitivity real-time PCR assay for simultaneous detection and quantification of P. damselae was designed targeting partial of the sequence of the bamB gene and tested for specificity and sensitivity on laboratory-generated samples as well as on experimentally infected seabream tissue samples. With a limit of detection (LOD) of one copy in pure bacterial DNA, the sensitivity was higher than all methods previously reported. Validation in target and non-target bacterial species proved the assay was able to discriminate Phdd-Phdp subspecies from diverse hosts/geographical origins and between non-target species. In addition, two SNPs in the target amplicon region determine two distinctive qPCR dissociation curves distinguishing between Phdp-Phdd. This is the first time that a molecular method for P. damselae diagnosis combines detection, quantification and subspecies identification in one step. The assay holds the potential to improve the knowledge of infection dynamics and the development of better strategies to control an important fish disease.

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Section: Discussionmentioning

confidence: 99%

Development of a real‐time PCR assay for rapid detection and quantification of Photobacterium damselae subsp. piscicida in fish tissues

Carraro

Rovere

Ferraresso

et al. 2017

Journal of Fish Diseases

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“…The samples tested negative by real-time PCR for LSD. The extracted DNA was stored and subsequently analyzed using a recently developed high resolution melting (HRM) assay for the simultaneous detection and differentiation of eight poxviruses of medical and veterinary importance [12]. A follow-up trip was undertaken on the farm in April 2018 to examine the animals clinically and obtain additional history and samples.…”

Section: Clinical and Epidemiological Investigationsmentioning

confidence: 99%

“…We tested the extracted DNA using the HRM assay for the simultaneous detection and differentiation of eight poxviruses. [12]. The reaction contained 200 nM of each primer (Table 2), 1 X SsoFast™ EvaGreen® Supermix, and 2 µl of the template.…”

Section: Hrm Assaymentioning

confidence: 99%

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First Detection and Molecular Characterisation of Pseudocowpox Virus in a Cattle Herd in Zambia

Ziba

Chitala

Settypalli³

et al. 2020

Preprint

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Background: Pseudocowpox virus (PCPV) of the genus Parapoxvirus in the family Poxviridae causes pseudocowpox in cattle worldwide and presents a zoonotic concern. Most poxviruses produce diseases of similar clinical signs in affected animals, which are impossible to differentiate clinically or by serology. It is, therefore, vital to use molecular assays to identify the causative agents of poxvirus infections rapidly. This study aimed to detect, diagnose, and characterize the causative agent of pox-like skin lesions in a cattle herd in Zambia, initially suspected to be infected with the Lumpy Skin Disease virus.Methods: We used a high resolution melting (HRM) analysis assay to detect the PCPV genome and sequenced the major envelope protein (B2L) gene for comparative sequence and phylogenetic analysis. Results: Our field investigations showed cattle presenting atypical skin lesions and high morbidity within the herd. The laboratory diagnosis based on an HRM assay revealed the PCPV genome in the samples. Phylogenetic and comparative sequence analyses confirmed PCPV in the samples. They revealed genomic differences between samples collected in 2017 and 2018 from the same farm.Conclusion: Our work is the first documented report of PCPV in Zambia. It shows the strength of molecular methods to diagnose pox-like infections in cattle and discriminate between diseases causing similar clinical signs for better veterinary interventions.

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“…High-resolution melting (HRM) curve analysis is a newly established PCR-based technique that has been used to differentiate related strains of the same animal or avian virus [16][17][18][19]. This technique was performed on double-stranded DNA resulting in rapid genotyping of genetic polymorphisms in diagnostic and routine [20].…”

Section: Introductionmentioning

confidence: 99%

High-resolution melting curve analysis for infectious bronchitis virus strain differentiation

Ababneh

Al‐Zghoul

2020

Vet World

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Background and Aim: Belonging to the Coronaviridae family, avian infectious bronchitis virus (IBV) causes respiratory, reproductive, and renal diseases in poultry. Preventative measures lie mainly in vaccination, while the gold standard for IBV classification and differentiation is based on the sequence analysis of the spike 1 (S1) gene. In this study, we tested a new assay for IBV strain classification that is less expensive and requires reduced time and effort to perform. We carried out a quantitative real-time polymerase chain reaction followed by high-resolution melting (qRT-PCR/HRM) curve analysis. Materials and Methods: In this study, qRT-PCR was conducted on a partial fragment S1 gene followed by a high resolution melting curve analysis (qRT-PCR/HRM) on 23 IBV-positive samples in Jordan. For this assay, we utilized the most common IBV vaccine strains (Mass and 4/91) as a reference in the HRM assay. To evaluate the discrimination power of the qRT-PCR/ HRM, we did the sequencing of the partial S1 gene. Results: It was shown that HRM was able to classify IBV samples into four clusters based on the degree of similarity between their melting points: The first cluster exhibited the highest similarity to the 4/91 strain, while the second was similar to the Mass-related IBV strain. Although the third cluster contained the highest number of samples, it displayed no similarity to any of the reference vaccine strains, and, after comparing them with the sequencing results, we found that the samples in the third cluster were similar to the variant II-like (IS-1494-06) IBV field strain. Finally, the fourth cluster comprised one unique sample that was found to belong to the Q1 IBV strain. Conclusion: Our developed qRT-PCR/HRM curve analysis was able to detect and rapidly identify novel and vaccine-related IBV strains as confirmed by S1 gene nucleotide sequences, making it a rapid and cost-effective tool.

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A novel HRM assay for the simultaneous detection and differentiation of eight poxviruses of medical and veterinary importance

Cited by 48 publications

References 43 publications

Development of a real‐time PCR assay for rapid detection and quantification of Photobacterium damselae subsp. piscicida in fish tissues

Development of a real‐time PCR assay for rapid detection and quantification of Photobacterium damselae subsp. piscicida in fish tissues

First Detection and Molecular Characterisation of Pseudocowpox Virus in a Cattle Herd in Zambia

High-resolution melting curve analysis for infectious bronchitis virus strain differentiation

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