Development and application of a sensitive droplet digital PCR for the detection of red mark syndrome infection in rainbow trout (Oncorhynchus mykiss)

Orioles, Massimo; Bulfoni, Michela; Saccà, Elena; Cesselli, Daniela; Schmidt, J. F.; Galeotti, Marco

doi:10.1016/j.aquaculture.2022.737910

Cited by 10 publications

(13 citation statements)

References 21 publications

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“…Our results indicated that ddPCR was 60 times more sensitive than qPCR; the detection limit of ddPCR and qPCR were 0.07 and 4.63 copies/μl, respectively. In comparison to the results of previous studies, the detection limit of ddPCR in this study was found to be higher than infectious spleen and kidney necrosis virus (1.5 copies/μl; Lin et al, 2020), carp oedema virus (2.2 copies/μl) (Wang et al, 2021), and red mark syndrome (RMS) (2.25 copies/μl; Orioles et al, 2022). Relative to ddPCR, qPCR is more popular and has been widely used for several decades, plus there are numerous commercial kits to choose from.…”

Section: Detection Of Tipv Using the Ddpcr And Qpcr Methodscontrasting

confidence: 69%

Development and application of a sensitive droplet digital PCR‐based method to detect tilapia parvovirus

Zhao

Zhou

Fan

et al. 2023

Journal of Fish Diseases

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Tilapia parvovirus (TiPV) causes severe mortality rates in cultured tilapia, resulting in substantial losses to the fish industry. Droplet digital PCR (ddPCR) is a sensitive, accurate, and absolute quantitation method, plus it does not require a standard curve. Herein we report the development and application of a sensitive ddPCR‐based method to rapidly detect and quantify TiPV. Optimal annealing temperature was determined to be 59.3°C, and optimal primer and probe concentrations were 900 nmol/L and 250 nmol/L, respectively. Our ddPCR method was highly specific to TiPV and showed no cross‐reactivity with other viruses. Further, the detection limit of ddPCR was 0.07 copies/μl, being lower than that of real‐time PCR (qPCR, 4.63 copies/μl). We also investigated the ability of ddPCR to detect TiPV in 50 samples and compared the outcome with qPCR data in terms of sensitivity and accuracy. The results showed that the positive detection rate of ddPCR (32%) was higher than that of qPCR (18%). To conclude, our ddPCR method was effective at detecting TiPV in samples with low viral loads. We believe that its application can facilitate the surveillance of sources and transmission routes of TiPV.

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Section: Detection Of Tipv Using the Ddpcr And Qpcr Methodscontrasting

confidence: 69%

Development and application of a sensitive droplet digital PCR‐based method to detect tilapia parvovirus

Zhao

Zhou

Fan

et al. 2023

Journal of Fish Diseases

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“…The ddPCR method was performed on the DNA extracted in accordance with recent literature (Orioles, Bulfoni, et al, 2022 ) and manufacturer's instructions (Bio‐Rad, USA).…”

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confidence: 99%

Observations on Red Mark Syndrome in juvenile rainbow trout farmed in RAS system

Orioles

Saccà

Metselaar

et al. 2022

Journal of Fish Diseases

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“…It can threaten land-based rearing sites in Southern Europe during cooler months [ 36 ], and the outbreaks described here exemplify a typical scenario. Various PCR methods have been developed in recent years to detect RMS-causing Rickettsia-like organisms (RLO) [ 19 , 24 , 37 , 38 ]. PCR and nested PCR are highly sensitive and fast and can simultaneously detect pathogens [ 39 , 40 ].…”

Section: Discussionmentioning

confidence: 99%

Understanding the Pathogenesis of Red Mark Syndrome in Rainbow Trout (Oncorhynchus mykiss) through an Integrated Morphological and Molecular Approach

Galeotti

Orioles

Saccà

et al. 2023

Animals

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Red mark syndrome (RMS) is a widespread skin disorder of rainbow trout in freshwater aquaculture, believed to be caused by a Midichloria-like organism (MLO). Here, we aimed to study the pathologic mechanisms at the origin of RMS by analyzing field samples from a recent outbreak through gene expression, MLO PCR, quantitative PCR, and a histopathological scoring system proposed for RMS lesions. Statistical analyses included a One-Way Analysis of Variance (ANOVA) with a Dunnett’s multiple comparisons test to assess differences among gene expression groups and a nonparametric Spearman correlation between various categories of skin lesions and PCR results. In short, the results confirmed the presence of a high quantity of 16S gene copy numbers of Midichloria-like organisms in diseased skin tissues. However, the number of Midichloria-like organisms detected was not correlated to the degree of severity of skin disease. Midichloria-like organism DNA was found in the spleen and head kidney. The spleen showed pathologic changes mainly of hyperplastic type, reflecting its direct involvement during infection. The most severe skin lesions were characterized by a high level of inflammatory cytokines sustaining and modulating the severe inflammatory process. IL-1 β, IL-6, IL-10, MHC-II, and TCR were upregulated in severe skin lesions, while IL-10 was highly expressed in moderate to severe ones. In the moderate form, the response was driven to produce immunoglobulins, which appeared crucial in controlling the skin disease’s severity. Altogether our results illustrated a complex immune interaction between the host and Midichloria-like organism.

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Development and application of a sensitive droplet digital PCR for the detection of red mark syndrome infection in rainbow trout (Oncorhynchus mykiss)

Cited by 10 publications

References 21 publications

Development and application of a sensitive droplet digital PCR‐based method to detect tilapia parvovirus

Development and application of a sensitive droplet digital PCR‐based method to detect tilapia parvovirus

Observations on Red Mark Syndrome in juvenile rainbow trout farmed in RAS system

Understanding the Pathogenesis of Red Mark Syndrome in Rainbow Trout (Oncorhynchus mykiss) through an Integrated Morphological and Molecular Approach

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