A validated semi-nested PCR for rapid detection of scale drop disease virus (SDDV) in Asian sea bass (Lates calcarifer)

Charoenwai, Onanong; Meemetta, Watcharachai; Sonthi, Molrudee; Dong, Ha Thanh; Senapin, Saengchan

doi:10.1016/j.jviromet.2019.03.007

Cited by 20 publications

(31 citation statements)

References 11 publications

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“…The newly established qPCR assay described in this study is highly specific to SDDV and could detect down to 2 copies per reaction which is 100 times more sensitive than our previous sem-inested PCR and LAMP protocols (Charoenwai et al, 2019; Dangtip et al, 2019), and 25 times more sensitive than a previously probe-based qPCR detection (de Groof et al, 2015). When applied to detection of clinically healthy fish from unaffected farms, it was revealed that ~57.7g% (30/52) samples previously tested negative by semi-nested PCR were positive by qPCR, indicating that the newly established qPCR is suitable for detection of SDDV from subclinically infected fish populations.…”

Section: Discussionmentioning

confidence: 78%

“…DNA extracted from 10 bacteria ( Vibrio harveyi, V. parahaemolyticus, V. vulnificus, V. tubiashi, V. alginolyticus, V. cholera, Streptococcus iniae, Tenacibaculum litopenaei, Pleisiomonas shigelloides , and Nocardia seriolae ) and DNA extracted from fish infected with 2 viruses (nervous necrosis virus (NNV) and infectious spleen and kidney necrosis virus (ISKNV)) were used as template. Details of the pathogens used in the specificity assay were described previously (Charoenwai et al, 2019). DNA extracted from clinically healthy Asian sea bass were also included in the assays.…”

Section: Methodsmentioning

confidence: 99%

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A highly sensitive and specific SYBR Green quantitative polymerase chain reaction (qPCR) method for rapid detection of scale drop disease virus in Asian sea bass, Lates calcarifer

Sriisan

Boonchird

Thitamadee

et al. 2019

Preprint

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Scale drop disease virus (SDDV) is a novel Megalocytivirus causing scale drop disease (SDD) in Asian sea bass in Southeast Asia. In order to support disease diagnosis and surveillance, the present study developed a highly sensitive and specific SYBR Green qPCR assay for rapid detection of SDDV. Specific primers targeting a 135-bp fragment of ATPase coding gene of the SDDV genome were newly designed and subsequent gradient PCR assays were conducted to investigate their optimal annealing temperature. The optimized qPCR assay could detect as low as 2 viral copies per reaction and showed no cross amplification with DNA extracted from 12 viruses and bacteria commonly found in aquatic animals. The SDDV ATPase qPCR method was subsequently validated with field samples (n= 86). The results revealed that all clinically sick fish (n=34) from 5 affected farms gave positive results. Interestingly, 30/52 samples of apparently healthy fish from 8 unaffected farms which previously tested negative for SDDV by semi-nested PCR assay were positive by the newly developed qPCR method. This suggested that qPCR method is highly sensitive and suitable for early screening of SDDV from clinically healthy fish and for disease confirmation of sick fish. Investigation of tissue tropism and viral load of SDDV revealed systemic viral infection with relatively high viral load (8 × 102 to 6.8 × 104 copies per 200 ng of DNA template) in all 9 tested organs including eyes, brain, fin, gills, kidney, liver, kidney, spleen, and muscle. The newly developed qPCR method in this study delivered an accurate and reliable method for rapid detection of SDDV that may facilitate active surveillance and prevent widespread of the virus.HighlightsThis study developed a SYBR Green qPCR assay for rapid detection of SDDVThe developed qPCR assay is specific for SDDV with limit of detection of 2 viral copies per reactionThe assay could detect the virus from subclinically infected fish with low viral loadWe recommend this qPCR assay for active surveillance and early screening of SDDV

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Section: Discussionmentioning

confidence: 78%

Section: Methodsmentioning

confidence: 99%

A highly sensitive and specific SYBR Green quantitative polymerase chain reaction (qPCR) method for rapid detection of scale drop disease virus in Asian sea bass, Lates calcarifer

Sriisan

Boonchird

Thitamadee

et al. 2019

Preprint

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“…Majority of tilapia farming countries are located in low and middle-income countries. In those countries, conventional PCR remains the preferable technique for many laboratories (in terms of costs and ease of use) compared to less accessible and more expensive quantitative PCR machine (Charoenwai et al 2019). The semi-nested PCR method in this study therefore was designed based on conserved regions of genome segment 1 that was able to amplify representatives from both Thai, Israeli-2011 and Israeli-2012 clades.…”

Section: Discussionmentioning

confidence: 99%

Genetic diversity of tilapia lake virus genome segment 1 from 2011 to 2019 and a newly validated semi-nested RT-PCR method

Taengphu

Sangsuriya

Phiwsaiya

et al. 2019

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AbstractThe gene of RNA viruses, encoding RNA-directed RNA polymerase (RdRp) is relatively conserved due to its crucial function in viral genome replication and transcription making it a useful target for genetic diversity study and PCR detection. In this study, we investigated the genetic diversity of 21 tilapia lake virus (TiLV) genome segment 1 sequences predictively coding for RdRp subunit P1. Those sequences were obtained from infected fish samples collected in Ecuador, Israel, Peru, and Thailand between 2011 and 2019 (nine sequences from this study and 12 sequences from GenBank). Primers were then designed from the highly conserved regions among all 21 TiLV segment 1 sequences and used in semi-nested RT-PCR condition optimization. The result revealed that all 21 TiLV segment 1 sequences showed 95.00-99.94 and 99.00-100% nucleotide and amino acid sequence identity, respectively. These isolates were phylogenically clustered into three separate genetic clades, called i) Israeli-2011 clade (containing of TiLV isolates from Israel collected in 2011, Ecuador, and Peru isolates), ii) monophyletic Israel-2012 clade (containing only TiLV isolates collected from Israel in 2012), and iii) Thai clade (containing only sequences obtained from Thailand isolates). The newly established PCR protocol was 100 times more sensitive than our previous segment 3-based protocol when comparatively assayed with RNA extracted from infected fish. The assay was also shown to be specific when tested against negative control samples, i.e. RNA extracted from clinical healthy tilapia and from bacterial and viral pathogens (other than TiLV) commonly found in aquatic animals. Validation experiment with RNA extracted from naturally infected fish specimens collected in 2013-2019 yielded positive test results for all samples tested, confirming that our newly designed primers and detection protocol against TiLV segment 1, have a potential application for detection of all current genetic variants of TiLV.

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“…The optimized qPCR protocol was subsequently used to test for specificity against extracted genomic DNA from i) clinically healthy fish, ii) from 12 common aquatic bacterial species, and iii) from fish samples infected with either infectious spleen and kidney necrosis virus (ISKNV), nervous necrosis virus (NNV), or scale drop disease virus (SDDV). Sample sources and preparation were previously described (Charoenwai et al, 2019; Sriisan et al, 2020). DNA extracted from fin of LCHV-infected fish was used as positive control.…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, molecular detection methods are required for diagnostic and screening purposes. Several DNA-based detection methods for SDDV have been freely available such as single PCR (Senapin et al, 2019), semi-nested PCR (Charoenwai et al, 2019), loop-mediated isothermal amplification (LAMP) (Dangtip et al, 2019), probe-based qPCR (de Groof et al, 2015), and SYBR Green-based qPCR (Sriisan et al, 2020). The latter one is the most sensitive method with a detection limit of 2 copies of DNA template per reaction.…”

Section: Introductionmentioning

confidence: 99%

Development of a SYBR Green quantitative PCR assay for detection of Lates calcarifer herpesvirus (LCHV) in farmed barramundi

Meemetta

Domingos

Dong

et al. 2020

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Lates calcarifer herpes virus (LCHV) is a new virus of farmed barramundi in Southeast Asia. However, a rapid detection method is yet to be available for LCHV. This study, therefore, aimed to develop a rapid quantitative PCR (qPCR) detection method for LCHV and made it timely available to public for disease diagnostics and surveillance in barramundi farming countries. A newly designed primer set targeting a 93-bp fragment of the LCHV putative major envelope protein encoding gene (MEP) was used for developing and optimizing a SYBR Green based qPCR assay. The established protocol could detect as low as 10 viral copies per µl of DNA template in a reaction containing spiked host DNA. No cross-amplification with genomic DNA extracted from host as well as common aquatic pathogens (12 bacteria and 3 viruses) were observed. Validation test of the method with clinical samples revealed that the virus was detected in multiple organs of the clinically sick fish but not in the healthy fish. We thus recommend that barramundi farming countries should promptly initiate active surveillance for LCHV in order to understand their circulation for preventing possibly negative impact to the industry.HighlightsThis study reported a new SYBR Green qPCR method for detection of LCHVThe qPCR method had detection limit of 10 copies per µl plasmid DNA template when spiked with genomic DNA from the hostThe aforementioned method is highly specific to LCHVValidation with clinical samples revealed that LCHV could be detected from multiple organs with fin and brain the best organs for qPCR detection

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A validated semi-nested PCR for rapid detection of scale drop disease virus (SDDV) in Asian sea bass (Lates calcarifer)

Cited by 20 publications

References 11 publications

A highly sensitive and specific SYBR Green quantitative polymerase chain reaction (qPCR) method for rapid detection of scale drop disease virus in Asian sea bass, Lates calcarifer

A highly sensitive and specific SYBR Green quantitative polymerase chain reaction (qPCR) method for rapid detection of scale drop disease virus in Asian sea bass, Lates calcarifer

Genetic diversity of tilapia lake virus genome segment 1 from 2011 to 2019 and a newly validated semi-nested RT-PCR method

Development of a SYBR Green quantitative PCR assay for detection of Lates calcarifer herpesvirus (LCHV) in farmed barramundi

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