Multiplex PCR assay for detection of four major bacterial pathogens causing rainbow trout disease

Altınok, İlhan

doi:10.3354/dao02300

Cited by 25 publications

(16 citation statements)

References 26 publications

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“…We can speculate that a possible cause can be due to the primer length used and the nucleotide content. Our results are in agreement with Altinok (2011) who noted that the high amounts of DNA from one bacterial species had a significant effect on the amplification sensitivity of the other bacterial species when these are present in lower concentration in the multiplex reaction.…”

Section: Resultssupporting

confidence: 93%

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Multiplex PCR for the detection of Piscirickettsia salmonis, Vibrio anguillarum, Aeromonas salmonicida and Streptococcus phocae in Chilean marine farms

Tapia‐Cammas¹,

Yáñez²,

Arancibia³

et al. 2011

Dis. Aquat. Org.

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A multiplex (m-)PCR-based protocol was designed for the simultaneous detection of the main marine bacterial pathogens in Chilean salmon farms: Streptococcus phocae, Aeromonas salmonicida, Vibrio anguillarum and Piscirickettsia salmonis. Each of the 4 oligonucleotide primer pairs exclusively amplified the target gene of the specific bacterial pathogen. The detection limit of the m-PCR using purified total bacterial DNA was 50 pg µl −1 for V. anguillarum, 500 fg µl −1 for P. salmonis, and 5 pg µl −1 for S. phocae and A. salmonicida. This corresponded to average limits in the m-PCR sensitivity of 3.69 × 10 5 CFU ml −1 of V. anguillarum, 1.26 × 10 4 CFU ml −1 of S. phocae, and 5.33 × 10 4 CFU ml −1 of A. salmonicida, while the detection limits for the spiked fish tissues, regardless of the sample (spleen, kidney, liver or muscle) were 2.64 ± 0.54 × 10 7 CFU g −1 for V. anguillarum, 9.03 ± 1.84 × 10 5 CFU g −1 for S. phocae, 3.8 ± 0.78 × 10 3 CFU mg −1 for A. salmonicida and 100 P. salmonis cells. However, high amounts of DNA from 3 bacterial species had a reduction of ~1 log-unit on the amplification sensitivity of S. phocae or A. salmonicida when these were present in lower concentration in the multiplex reaction. The assay described in this study is a rapid, sensitive and efficient tool to detect the presence of S. phocae, A. salmonicida, V. anguillarum and P. salmonis simul taneously from pure cultures and tissues from clinically diseased fish. Therefore, it may be a useful alternative to culture-based methods for the diagnosis of infections in fish obtained from Chilean salmon farms. KEY WORDS: Multiplex PCR · Fish pathogens · Atlantic salmon · Analytic sensitivity test · Specificity test Resale or republication not permitted without written consent of the publisherDis Aquat Org 97: [135][136][137][138][139][140][141][142] 2011 marine Chilean farming every year with annual economic losses of US $100 million http://aqua. merck-animal-health.com/diseases/piscirickettsiosis/ product additional_ 127_113333.aspx. Affected fish are dark in colouration, show inappetence, are lethargic and swim near the surface or in edges of the cages. Internally enlarged spleen, discoloured kidney and ana emia, the principal characteristic of the disease, can be observed. In addition, some fish show distinct circular nodules in the liver (see Fryer & Hedrick 2003 for review).In the last decade, incidence of other pathogens such as Streptococcus phocae , Vibrio anguillarum (Silva-Rubio et al. 2008) and atypical Aeromonas salmonicida has increased in Chilean salmon culture, provoking significant mortalities also in rainbow trout Onchorhynchus mykiss and Pacific salmon Oncorhynchus kisutch (Bravo 2000, Godoy et al. 2010.Each disease is diagnosed presumptively based on the clinical signs of the affected fish. However, Streptococcus phocae, Aeromonas salmonicida, Vibrio anguillarum and Piscirickettsia salmonis often appear in mixed infections, increasing the possibility of misdiagnosis commonly attributed to P. salmonis perhap...

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Section: Resultssupporting

confidence: 93%

“…An m-PCR approach has been successfully applied to detect multiple bacterial pathogens of marine (Kulkarni et al 2009) and/or freshwater fish (del Cerro et al 2002, Mata et al 2004, Altinok et al 2008, Altinok 2011.…”

Section: Resultsmentioning

confidence: 99%

Multiplex PCR for the detection of Piscirickettsia salmonis, Vibrio anguillarum, Aeromonas salmonicida and Streptococcus phocae in Chilean marine farms

Tapia‐Cammas¹,

Yáñez²,

Arancibia³

et al. 2011

Dis. Aquat. Org.

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“…At first, all L. garvieae isolates were confirmed by PCR assay as described by Altinok (2). Briefly, a forward primer LgF (5'-CCA ACT TCC GTG GTG TGA CG-3') and a reverse primer LgR (5'-AGT GGC TCA ACC ATT GTG TGC-3') for PCR amplification of a less conserved region of the small subunit (16S) ribosomal RNA (rRNA) gene sequence of L. garvieae were synthesised by Life Technologies (Grand Island, USA).…”

Section: Bacterialmentioning

confidence: 99%

Phenotypic and Genotypic Antimicrobial Resistance of Lactococcus Sp. Strains Isolated from Rainbow Trout (Oncorhynchus Mykiss)

Türe¹,

Boran

2015

Bulletin of the Veterinary Institute in Pulawy

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A current profile of antimicrobial resistance and plasmid of 29 Lactococcus garvieae and one Lactococcus lactis strains isolated from rainbow trouts (Oncorhynchus mykiss) from farms throughout Turkey were investigated. All isolates were sensitive to penicillin G (90%), ampicillin (86.7%), florfenicol (83.3%), amoxicillin (80.1%), and tetracycline (73.4%), and resistant to trimethoprim+sulfamethoxazole (86.6%) and gentamycin (46.6%) by disc diffusion method. Twenty-eight (93%) isolates had two to seven antibiotic resistance genes (ARGs) determined by PCR. The most prevalent ARGs were tetracycline (tetB), erythromycin (ereB), and β-lactam (blaTEM). Bacterial strains were also screened for plasmid DNA by agarose gel electrophoresis and two strains harboured plasmids, with sizes ranging from 3 to 9 kb.

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“…Biochemical tests, DNA homology, and protease variability techniques have also been extensively used, but these techniques have major disadvantages, such as the need for initial isolation of the pathogen and insufficient sensitivity to detect low levels of pathogen. Molecular techniques have slowly established a place in the diagnosis of disease in aquaculture (Chatterjee and Haldar, 2012), by overcoming the problems related to the sensitivity and specificity of pathogen detection and identification (Altinok, 2011).…”

Section: Introductionmentioning

confidence: 99%

Development of a new multiplex-PCR tool for the simultaneous detection of the fish pathogens Vibrio alginolyticus, Vibrio anguillarum, Vibrio harveyi and Edwardsiella tarda

Pinto

Baptista

Afonso

2017

Aquat. Living Resour.

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Disease assessment and management in cultured aquatic animals is a major concern in commercial aquaculture. Disease outbreaks have direct effects on fish production, causing serious economic losses in this industry. This can be overcome by early detection through molecular high sensitivity tools such as PCR (polymerase chain reaction). One of the most critical steps in the study of bacterial fish diseases is the precise identification of the infectious agent. Considering the damage that some bacteria can cause to fish and humans, the development of a rapid detection method for the four target species that demonstrates to be simple, accurate and low cost is an essential step for the prevention and early treatment of these diseases. Edwardsiella tarda (ACC 36.1), Vibrio harveyi (DSM 19023), Vibrio anguillarum (AQV 55.1) and Vibrio alginolyticus (CECT 521) were selected as targets of a multiplex PCR tool. The multiplex PCR reactions were performed in various reaction conditions, including different annealing temperatures (between 49°C and 55°C) and changes in the MgCl 2 concentration from 2 mM to 8 mM. Best results were obtained at 51°C and MgCl 2 concentration from 4 mM to 6 mM. Primers were tested using purified DNA from the respective bacterial strains, yielding bands at expected sizes, which can be identified on agarose gels with clearness and without overlapping sizes. Results indicate that this multiplex PCR tool is suitable for the detection of target pathogens and may, in the future, have extended practical application in commercial aquaculture.

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Multiplex PCR assay for detection of four major bacterial pathogens causing rainbow trout disease

Cited by 25 publications

References 26 publications

Multiplex PCR for the detection of Piscirickettsia salmonis, Vibrio anguillarum, Aeromonas salmonicida and Streptococcus phocae in Chilean marine farms

Multiplex PCR for the detection of Piscirickettsia salmonis, Vibrio anguillarum, Aeromonas salmonicida and Streptococcus phocae in Chilean marine farms

Phenotypic and Genotypic Antimicrobial Resistance of Lactococcus Sp. Strains Isolated from Rainbow Trout (Oncorhynchus Mykiss)

Development of a new multiplex-PCR tool for the simultaneous detection of the fish pathogens Vibrio alginolyticus, Vibrio anguillarum, Vibrio harveyi and Edwardsiella tarda

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