Sensitive and Rapid Detection of Edwardsiellosis in Fish by a Loop-Mediated Isothermal Amplification Method

Savan, Ram; Igarashi, Arisa; Matsuoka, Satoru; Sakai, Masahiro

doi:10.1128/aem.70.1.621-624.2004

Cited by 90 publications

(68 citation statements)

References 11 publications

(10 reference statements)

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“…A method of amplifying nucleic acid sequences with high specificity, sensitivity and rapidity under isothermal conditions (60 to 65°C), loop-mediated isothermal amplification (LAMP) has been developed more recently (Notomi et al 2000) and applied to fish pathogen detection (Caipang et al 2004, Savan et al 2004, Yeh et al 2005, Itano et al 2006. The assay uses a set of 4 or 6 primers that create a targetspecific stem loop DNA structure during initial amplification steps; subsequent LAMP auto-cycling is performed by the Bst DNA polymerase large fragment (Notomi et al 2000).…”

Section: Introductionmentioning

confidence: 99%

Development of a sensitive and specific LAMP PCR assay for detection of fish pathogen Lactococcus garvieae

Tsai¹,

Wang²,

Yoshida³

et al. 2013

Dis. Aquat. Org.

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Based on use of a loop-mediated isothermal amplification (LAMP) identification protocol, this study attempted to detect Lactococcus garvieae in fish by using primer sets designed from an L. garvieae alpha/beta fold family hydrolase gene. Reaction time and temperatures were optimized for 60 min at 60°C with the resulting amplicons visualized by adding SYBR Green I to the reaction tube. The assay specificity was assessed using 45 different bacterial strains. Positive results were observed in all 30 L. garvieae isolates from various aquatic animals. No false-positive results were observed in 15 non-L. garvieae strains. The detection limit of the LAMP assay was 10-fold more sensitive than the conventional polymerase chain reaction (PCR) targeting 16S rDNA when using purified L. garvieae DNA. The detection limit of the LAMP assay was approximately 300 colony-forming units (CFU) using crude bacterial lysates, 100-fold more sensitive than PCR. Furthermore, L. garvieae in spleen, kidney and brain of experimentally challenged tilapia and grey mullet were detected using this optimized LAMP assay. Results of this study demonstrate the effectiveness of LAMP in providing a rapid yet simple test for detecting L. garvieae in fish.KEY WORDS: Lactococcosis · Alpha/beta fold family · Hydrolase gene · Loop-mediated isothermal amplification · Polymerase chain reaction · SYBR Green I Resale or republication not permitted without written consent of the publisherDis Aquat Org 102: [225][226][227][228][229][230][231][232][233][234][235] 2013 swimming. However, similar clinical signs are also found in Streptococcus sp. infections (Eldar & Ghittino 1999).Molecular detection methods of Lactococcus garvieae have been developed, including polymerase chain reaction (PCR) (Zlotkin et al. 1998, Aoki et al. 2000, Pu et al. 2002 and 16S rRNA and sodA sequence analysis (Fihman et al. 2006). However, although considered rapid and specific methods for detection, these require expensive equipment and other costly materials (Gunimaladevi et al. 2005).A method of amplifying nucleic acid sequences with high specificity, sensitivity and rapidity under isothermal conditions (60 to 65°C), loop-mediated isothermal amplification (LAMP) has been developed more recently (Notomi et al. 2000) and applied to fish pathogen detection (Caipang et al. 2004, Savan et al. 2004, Yeh et al. 2005, Itano et al. 2006. The assay uses a set of 4 or 6 primers that create a targetspecific stem loop DNA structure during initial amplification steps; subsequent LAMP auto-cycling is performed by the Bst DNA polymerase large fragment (Notomi et al. 2000). Since it does not require complicated equipment, the LAMP method provides a cost-effective, simple, and rapid (producing a result within 60 min) DNA amplification method.Recently, a fosmid library of Lactococcus garvieae from grey mullet has been constructed and many new genes of L. garvieae were cloned (M. A. Tsai et al. unpubl. data). The clone flg08-6 contained an alpha/beta fold family hydrolase gene that is also...

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Section: Introductionmentioning

confidence: 99%

Development of a sensitive and specific LAMP PCR assay for detection of fish pathogen Lactococcus garvieae

Tsai¹,

Wang²,

Yoshida³

et al. 2013

Dis. Aquat. Org.

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“…Fish diseases are a major problem in the aqua culture industry because of their financial impact (Savan et al 2004). SVC is a transmissible disease of farmed fish that causes considerable economic losses (Koutná et al 2003).…”

Section: Discussionmentioning

confidence: 99%

Direct detection of unamplified spring viraemia of carp virus RNA using unmodified gold nanoparticles

Saleh¹,

Soliman²,

Schachner³

et al. 2012

Dis. Aquat. Org.

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Spring viraemia of carp (SVC) is a viral disease that mainly affects carp Cyprinus carpio and other cyprinid fish, causing severe economic losses. Rapid detection and identification of spring viraemia of carp virus (SVCV) is crucial for effective disease management. Recent advances in nanoscience are having a significant impact on many scientific fields, especially biodiagnostics, where a number of nanoparticle-based assays have been introduced for biomolecular detection. Single-and double-stranded oligonucleotides can be adsorbed on gold nanoparticles (AuNPs) in colloidal solution under certain conditions. We exploited this phenomenon to develop a specific hybridization assay for direct detection of SVCV-RNA without prior amplification. The result of the hybridization process could be detected visually within 1 min when the colour of the reaction mixture changed from red to blue (positive reaction) or remains red (negative). The lower detection limit of the assay was estimated to be 10 −3 TCID 50 ml −1 SVCV-RNA, and it has the feasibility to detect the target virus-RNA in clinical specimens without previous amplification. In order to obtain an indication of the assay's performance on clinical samples we compared the optimized assay with nested RT-PCR in detection of SVCV-RNA in infected fish samples. The concordance of the 2 methods was defined as 100% when compared to nested RT-PCR positive and negative samples. The SVC-AuNPs assay requires only 15 min, eliminates the need for thermal cycling or detection instruments and is a specific and rapid tool for detection of SVCV-RNA directly from clinical samples. KEY WORDS: SVCV · AuNPs · Colorimetric detection · Diagnosis Resale or republication not permitted without written consent of the publisherDis Aquat Org 100: [3][4][5][6][7][8][9][10] 2012 rescence, immunoperoxidase, and ELISA (Amos 1985, OIE 2009). Rapid ad vances in molecular biology techniques have led to the development of several molecular methods for SVCV detection, including hybridization assays, RT-PCR, nested RT-PCR (nRT-PCR) and real-time PCR (Oreshkova et al. 1999, Koutná et al. 2003, Yue et al. 2008. Although these assays are specific and sen sitive, they are time consuming and require sophis ticated apparatus and complex post-run manipu lations.Recently, noble metal nanoparticles, particularly gold nanoparticles (AuNPs), have been introduced as a promising approach to the development of the next generation of diagnostic assays (Mirkin et al. 1996, Storhoff et al. 2000. AuNPs have become an important alternative as imaging agents due to their noncytotoxicity, facile immunotargeting and nonsusceptibility to photobleaching or chemical/thermal de naturation, a problem commonly associated with dyes (Jain et al. 2006). Advances in functionalizing particles with oligonucleotides and tailoring their surface properties have paved the way to design a series of new and practical systems for nucleic acid detection (Mirkin et al. 1996, Elghanian et al. 1997, Storhoff et al. 2000, Daniel & Astru...

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“…Outbreaks of edwardsiellosis appear first in the form of infections affecting only a few fish, and these diseased fish are able to survive only for a few days. When early detection and proper measurement are not undertaken and the diseased fish not treated as soon as possible, the infection spreads to the surrounding areas to create an edwardsiellosis epidemic [6]. Since early detection is critical for controlling this disease, rapid and accurate methods for edwardsiellosis diagnosis would be very valuable.…”

Section: Detection Of E Tarda In Artificially Contaminated Fish Tissuementioning

confidence: 99%

“…During the past decade, a number of immunoassay methods, including enzyme-linked immunosorbent assay (ELISA) and dot blot immunoassay, have been used to detect E. tarda in infected organs like the liver, spleen, and kidney of the diseased fish [2,3]. Polymerase chain reaction (PCR) and modified PCR methods such as loop-mediated isothermal amplification (LAMP) were also used in the diagnoses of E. tarda [1,4,5], and the LAMP method was proved to be successful for E. tarda detection with a limit of detection (LOD) of 10 colony-forming units (CFU) [6]. Although the above methods are effective for E. tarda diagnosis, they are time-consuming (2 h to 7 days) and depend on expensive instruments and highly skilled personnel.…”

Section: Introductionmentioning

confidence: 99%

An immunochromatographic test strip for rapid detection of fish pathogen Edwardsiella tarda

Liu

Wang

Xiao

et al. 2015

Bioresour. Bioprocess.

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Background: Edwardsiella tarda, the etiologic agent of edwardsiellosis, is a devastating fish pathogen prevailing in worldwide aquaculture industries and accounting for severe economic losses. There is a raising concern about E. tarda being a significant zoonotic pathogen, and it is urgent to develop a rapid detection of this pathogen. This is the first study to develop a test strip for rapid detection of E. tarda in turbot. Results: Mouse monoclonal antibodies (MAbs) and rabbit polyclonal antibody (PAb) against E. tarda were generated from immunization of mice and rabbits with a virulent isolate of E. tarda EIB202. Two MAbs specific to isolates of E. tarda were obtained, and one of them (25C1) was selected to conjugate with colloidal gold as the detector antibody. Rabbit PAb was used as the capture antibody. It was found the strip had no cross-reactivity with non-E. tarda bacterial microbes and the limit of detection (LOD) was 1 × 10 5 colony-forming units (CFU)/ml. The detection could be visually observed by the naked eye within 5 min. This test strip was verified with a similar detection limit and much less analysis time compared with a dot blot immunoassay (1 × 10 5 CFU/ml for LOD and 120 min for reaction time). When the samples were mixed with turbot tissue homogenates, strong immunoreactivity was observed over 10 5 CFU/ml, which suggested that the turbot tissue homogenates did not affect the detection of the strip. Pre-enrichment with homogenized turbot tissue for 12 h could increase the detection limit of the E. tarda present in the sample up to 1 to 10 CFU/ml. In practice, in detecting 20 turbot ascite samples infected by E. tarda, the immunochromatographic test strip showed a high accuracy (100% positive). Conclusions: The immunochromatographic test strip offers great promise for a rapid, simple, and economical method of E. tarda on-site detection, and with different antibodies, it might be used to detect other aquatic pathogens.

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Sensitive and Rapid Detection of Edwardsiellosis in Fish by a Loop-Mediated Isothermal Amplification Method

Cited by 90 publications

References 11 publications

Development of a sensitive and specific LAMP PCR assay for detection of fish pathogen Lactococcus garvieae

Development of a sensitive and specific LAMP PCR assay for detection of fish pathogen Lactococcus garvieae

Direct detection of unamplified spring viraemia of carp virus RNA using unmodified gold nanoparticles

An immunochromatographic test strip for rapid detection of fish pathogen Edwardsiella tarda

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