Development and Application of a Quantitative Real‐time Polymerase Chain Reaction Assay for the Detection of <i>Aeromonas salmonicida</i>

Du, Yishuai; Liu, Ying; Peng, Xiao; Meng, Lingjie; Liu, Pengfei

doi:10.1111/jwas.12395

Cited by 8 publications

(9 citation statements)

References 33 publications

(73 reference statements)

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“…For example, Hiney et al (1992) reported a polymerase chain reaction (PCR) that was capable of detecting ~ 2 cells of A. salmonicida, whereas Høie et al (1997) detected 10 3 and 10 4 colony forming units (CFUs) of A. salmonicida in 100 ml of kidney suspension with 16S rRNA sequencing and plasmid primers, respectively. Subsequent developments included nested PCR (Taylor and Winton 2002), terminal-restriction fragment length polymorphism (RFLP) (Nilsson and Strom 2002), PCR-RFLP (Puah et al 2018), multiplex PCR (Chapela et al 2018), real-time PCR (Keeling et al 2013), quantitative real-time-PCR (Du et al 2017), real-time recombinase polymerase amplification (Pang et al 2019) and reverse transcription-multiplex PCR (Rattanachaikunsopon and Phumkhachorn 2012). All these techniques reported extremely high levels of sensitivity, detecting numbers of cells well below the level associated with occurrences of clinical disease (Austin and Austin 2016).…”

Section: Culture-independent Methods-molecular Biologymentioning

confidence: 99%

Methods for the diagnosis of bacterial fish diseases

Austin

2019

Mar Life Sci Technol

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The diagnosis of bacterial fish diseases has progressed from traditional culture-dependent methods involving the recovery of pathogens on agar-containing media and identification by examination of phenotypic traits. Newer approaches centre on culture-independent approaches. A problem with culturing is that it lacks sensitivity, tends to be slow, and its success depends on the composition of the media and incubation conditions employed. In contrast, culture-independent methods, now centring on molecular methods, are highly specific and sensitive. This raises an important issue that detection of very low numbers of bacterial cells does not necessarily imply the presence of clinical disease. Positivity could reflect background populations of the pathogen that may be present in the aquatic environment.

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Section: Culture-independent Methods-molecular Biologymentioning

confidence: 99%

Methods for the diagnosis of bacterial fish diseases

Austin

2019

Mar Life Sci Technol

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“…The positive results on type V and type VI spiked water samples support the potential use of the molecular assays for monitoring purposes in hatchery sites. In general, Aeromonas salmonicida isolation from environmental samples has been attempted on solid medium cultivation methods in the past with not encouraging results (Du et al., 2017; Teska & Cipriano, 1993). The bacterium is difficult to recover from these samples where mixed bacterial communities co‐exist due to the lack of selective media and overgrowth of concomitant bacteria.…”

Section: Discussionmentioning

confidence: 99%

Development of diagnostic assays for differentiation of atypical Aeromonas salmonicidavapA type V and type VI in ballan wrasse (Labrus bergylta, Ascanius)

Papadopoulou

Davie

Monaghan

et al. 2021

Journal of Fish Diseases

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Aeromonas salmonicida (As) is a highly heterogeneous bacterial species, and strains’ host specificity has been reported. Ballan wrasse (Labrus bergylta Ascanius, 1767) is susceptible to atypical As (aAs) vapA type V and type VI in Scotland and Norway. Identification of the bacterium is achieved by culture and molecular techniques; however, the available methods used to distinguish the As types are costly and time‐consuming. This paper describes the development of a PCR and a restriction enzyme assay for the detection of aAs vapA type V and type VI in ballan wrasse, respectively. Type V‐specific primers were designed on conserved regions of the vapA gene, and the restriction enzyme assay was performed on the PCR products of the hypervariable region of vapA gene for the detection of type VI isolates. Amplification product was produced for type V (254 bp) and restriction bands (368 and 254 bp) for type VI isolates only. In addition, the assays detected type V and type VI isolates in spiked water samples and type V in diagnostic tissue samples. The assays are fast, specific and cost‐effective and can be used as specific diagnostic tools for cleaner fish, to detect infectious divergence strains, and to manage and mitigate aAs disease outbreaks through vaccine development.

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“…In many quantitative analysis of pathogens, the detection of pathogens relied upon plate cultivation (Farto et al., ; Kashulin & Sorum, ; Liu et al., ). Compared with this traditional method, qPCR used here was rapid, economical, specific and sensitive for the quantitative detection of AS‐C4, with just a detection limit of 5.6 copies of one PCR reaction (Du, Liu, et al., ). AS‐C4 was detected in almost all tissue samples of fish from the bath test.…”

Section: Discussionmentioning

confidence: 99%

“…| 1827 (N = 4) were also sampled according the above protocols and all samples were kept at À80°C for genomic DNA extraction. A new qPCR assay was designed to detect the copy numbers of A. salmonicida in different tissues of Atlantic salmon in our laboratory (Du, Liu, et al, 2016). The design of primers in this new qPCR system was based on the virulence array protein (vapA) gene of A. salmonicida (GenBank accession no.…”

Section: Bath Challenge and Samplingmentioning

confidence: 99%

“…So the confirmation of colonization of ASM strains in tissues of Atlantic salmon during infection will provide important information to the infection route of ASM strains in the host. In our previous study, a new qPCR assay was designed to detect the copy numbers of A. salmonicida in different tissues of Atlantic salmon (Du, Liu, Xiao, Meng, & Liu, ). In the present study, the numbers of ASM copies in different tissues of Atlantic salmon after the bath challenge were determined to speculate the colonization of ASM strains during infection.…”

Section: Introductionmentioning

confidence: 99%

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Colonization ofAeromonas salmonicidasubsp.masoucidastrains in Atlantic salmon (Salmo salarL.) during infection

Liu

Meng

et al. 2018

Aquac Res

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Aeromonas salmonicida subsp. masoucida (ASM) is classified as atypical A. salmonicida and brought huge economic damages to the local salmonid aquaculture in China. An ASM strain named AS‐C4 was used to investigate the colonization of ASM in Atlantic salmon (Salmo salar L.) by an immersion challenge with the control group (T0, no AS‐C4), group T1 (2.67 × 104 CFU/ml AS‐C4) and group T2 (2.67 × 107 CFU/ml AS‐C4). The numbers of AS‐C4 copies in different fish tissues (gill, intestine, skin, blood, muscle, spleen, liver and kidney) were determined at different time points post challenge using the quantitative real‐time PCR (qRT‐PCR). AS‐C4 were detected in the gill and intestine as early as 0 hr after the challenge both in T1 and T2 groups, suggesting that the gill and intestine were probably the portals of entry of AS‐C4 into salmon. Although AS‐C4 could not be detected in the skin until 24 hr after the challenge in T1 group, it could be detected in the skin as early as 0 hr after the challenge in T2 group, indicating that the skin may also be a portal of entry of AS‐C4 into salmon. AS‐C4 was immediately detected in the blood within 3 hr after it entered the host, suggesting that AS‐C4 successfully invaded the bloodstream of fish. After AS‐C4 colonized the host, it colonized the internal tissues, such as the spleen, liver, kidney and muscle. The results of this study will contribute to the understanding of the pathogenesis of the ASM strains and give a broader understanding of the infection route of ASM in it's host, providing more information for the development of new therapeutic strategies to protect against this pathogen in aquaculture.

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Development and Application of a Quantitative Real‐time Polymerase Chain Reaction Assay for the Detection of Aeromonas salmonicida

Cited by 8 publications

References 33 publications

Methods for the diagnosis of bacterial fish diseases

Methods for the diagnosis of bacterial fish diseases

Development of diagnostic assays for differentiation of atypical Aeromonas salmonicidavapA type V and type VI in ballan wrasse (Labrus bergylta, Ascanius)

Colonization ofAeromonas salmonicidasubsp.masoucidastrains in Atlantic salmon (Salmo salarL.) during infection

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