Low-Cost and Rapid Method of DNA Extraction from Scaled Fish Blood and Skin Mucus

Liu, Gui; Li, Xinyu; Lin, Shentao; Zhao, Yun; Lin, Pei-Yao; Wang, Bingqi; Tang, Rongkang; Guo, Jing; Zu, Yao; Zhou, Yan; Li, Mingyou

doi:10.3390/v14040840

Cited by 8 publications

(8 citation statements)

References 25 publications

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“…The results demonstrated that swabs were as effective as organ samples. Mucus swabs were previously used in a Chinese study on crucian carp [45]. A positive result indicated Carassius auratus herpes virus DNA could be detected using the mucus swab sampling method.…”

Section: Discussionmentioning

confidence: 99%

Molecular epidemiology of Megalocytivirus in freshwater angelfish (Pterophyllum scalare) from Johor, Malaysia

Johan,

Abdullah,

Emilia

et al. 2023

Vet World

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Background and Aim: Malaysia has more than 630 culturists who are involved in the ornamental fish industry and culture 250 species, including local and exotic species. Among these viruses, megalocytiviruses have been associated with severe systemic diseases and economic losses in ornamental fish. The intensity of Megalocytivirus infection in Pterophyllum scalare in Malaysia remains unknown. Thus, this study aimed to investigate the occurrence of Megalocytivirus while discovering its associated risk factors and the genotypes of its causative agents in an ornamental fish farm in Malaysia. Materials and Methods: Seven broodstock pairs of P. scalare were used in this study to follow the life stages of fish, from egg to market size. Water samples and other samples, such as mucus swabs, gill swabs, P. scalare eggs, fries, juveniles, snails, snail eggs, live feed (Tubifex worms and Moina spp.), sediment samples, and wild fish, were collected periodically for initial environmental sampling from day 0 to day 60. Nested polymerase chain reaction amplifications were performed for megalocytivirus-related sequences. The phylogenetic tree, including the sampled causative agents of megalocytiviruses, was inferred from the major capsid protein genes of all known Iridoviridae species. Pearson’s correlation coefficients were calculated to determine the strength of the correlation between the presence of megalocytiviruses in P. scalare samples and the associated risk factors. Results: A total of 312 out of 935 pooled and individual samples tested positive for the presence of Megalocytivirus-related sequences, except snail eggs and wild fish (Poecilia reticulata). No clinical symptoms were observed in any fish samples. Megalocytivirus-associated viruses detected in water samples indicate horizontal transmission of the virus. All the nucleotide sequences found in this study had high nucleotide identities of 95%–99 % and were closely related to Megalocytivirus genotype I infectious spleen and kidney necrosis virus. Risk factors associated with Megalocytivirus include water temperature, dissolved oxygen (DO), pH, ammonia, nitrate, nitrite, and the life stages of P. scalare. High Megalocytivirus infection was detected when the water temperature, DO, and pH were high in P. scalare, high water temperature and nitrate in the water samples, and the same rate of Megalocytivirus infection in P. scalare fry and juveniles. Conclusion: This is the first study to confirm the existence of different possible routes of megalocytivirus distribution in ornamental fish farms in Malaysia. Nevertheless, the connection between the mode of transmission and the risk factors for this virus needs to be explored further to recognize the evolution and potential new host species. Keywords: epidemiology, genotype I, infectious spleen and kidney necrosis virus, Megalocytivirus, ornamental fish, risk factors.

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Section: Discussionmentioning

confidence: 99%

Molecular epidemiology of Megalocytivirus in freshwater angelfish (Pterophyllum scalare) from Johor, Malaysia

Johan,

Abdullah,

Emilia

et al. 2023

Vet World

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“…CaHV DNA was extracted from the crucian carp infected with CaHV, by using skin mucus swabbing and the disc method as we described before ( Gui et al., 2022 ). Briefly, healthy crucian carp were injected intraperitoneally with diseased fish tissue filtrate (viral suspension) kindly provided by Dr. Qiya Zhang (Institute of Hydrobiology, Chinese Academy of Sciences).…”

Section: Methodsmentioning

confidence: 99%

“…Based on our previous study of the extraction of DNA from fish skin mucus within 30 seconds with low cost (USD $0.02) ( Gui et al., 2022 ), we established a rapid, sensitive and cheap method to detect CaHV within 35min from sample collection to result interpretation. Our study could provide a simple, rapid, and low-cost detecting application for POCT in aquaculture industry.…”

Section: Introductionmentioning

confidence: 99%

Quick detection of Carassius auratus herpesvirus (CaHV) by recombinase-aid amplification lateral flow dipstick (RAA-LFD) method

Liu

Zhao²,

Xu³

et al. 2022

Front. Cell. Infect. Microbiol.

Self Cite

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Crucian carp (Carassius auratus) is one of the major freshwater species and is also a common food fish in China. Recently, Carassius auratus herpesvirus (CaHV) could induce fatal viral disease with high mortality of crucian carp, which had caused huge economic losses. In this study, we described a rapid and simple recombinase-aid amplification (RAA) assay coupled with lateral flow dipstick (LFD), which could achieve sensitive diagnosis of tumor necrosis factor receptor (TNFR) of CaHV within 35 min at 40°C. Our RAA-LFD method had a satisfactory detection limit of 100 gene copies per reaction, which was 100-fold more sensitive than traditional PCR. In addition, no cross-reaction was observed with other viral pathogens, including koi herpesvirus (KHV), cyprinid herpesvirus 2 (CyHV-2), infectious hematopoietic necrosis virus (IHNV), spring viremia of carp virus (SVCV) and grass carp reovirus (GCRV). Furthermore, the overall cost of the method was cut in half compared to previous studies. In conclusion, RAA-LFD assay is therefore, a promising alternative for point-of-care testing (POCT) of CaHV, which is feasible and of certain value in application of aquatic disease control.

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“…However, limited advances have been made toward non-lethal pathogen detection in fish, particularly in small-sized ones. The successful detection and isolation of fish viruses obtained from the swabbing of epithelial cells [ 31 , 32 ], as well as the consistent results obtained in amphibians [ 33 ], suggests that this approach may be a reliable method for molecular detection of viral DNA [ 34 ], and an alternative for sampling a threatened species.…”

Section: Introductionmentioning

confidence: 99%

Non-Lethal Detection of Ranavirus in Fish

Coutinho

Ford

Trafford

et al. 2023

Viruses

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Emergent infectious diseases have an increasing impact on both farmed animals and wildlife. The ability to screen for pathogens is critical for understanding host–pathogen dynamics and informing better management. Ranavirus is a pathogen of concern, associated with disease outbreaks worldwide, affecting a broad range of fish, amphibian, and reptile hosts, but research has been limited. The traditional screening of internal tissues, such as the liver, has been regarded as the most effective for detecting and quantifying Ranavirus. However, such methodology imposes several limitations from ethical and conservation standpoints. Non-lethal sampling methods of viral detection were explored by comparing the efficacy of both buccal swabbing and fin clipping. The study was conducted on two Iberian, threatened freshwater fish (Iberochondrostoma lusitanicum and Cobitis paludica), and all samples were screened using qPCR. While for C. paludica both methods were reliable in detecting Ranavirus, on I. lusitanicum, there was a significantly higher detection rate in buccal swabs than in fin tissue. This study, therefore, reports that fin clipping may yield false Ranavirus negatives when in small-bodied freshwater fish. Overall, buccal swabbing is found to be good as an alternative to more invasive procedures, which is of extreme relevance, particularly when dealing with a threatened species.

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Low-Cost and Rapid Method of DNA Extraction from Scaled Fish Blood and Skin Mucus

Cited by 8 publications

References 25 publications

Molecular epidemiology of Megalocytivirus in freshwater angelfish (Pterophyllum scalare) from Johor, Malaysia

Molecular epidemiology of Megalocytivirus in freshwater angelfish (Pterophyllum scalare) from Johor, Malaysia

Quick detection of Carassius auratus herpesvirus (CaHV) by recombinase-aid amplification lateral flow dipstick (RAA-LFD) method

Non-Lethal Detection of Ranavirus in Fish

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