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Loop-mediated isothermal amplification as an emerging technology for detection of Yersinia ruckeri the causative agent of enteric red mouth disease in fish.

Saleh M, Soliman H, El-Matbouli M - BMC Vet. Res. (2008)

Bottom Line: These diagnostic methods are laborious, time consuming and need well trained personnel.Amplification products were detected by visual inspection, agarose gel electrophoresis and by real-time monitoring of turbidity resulted by formation of LAMP amplicons.Digestion with HphI restriction enzyme demonstrated that the amplified product was unique.

View Article: PubMed Central - HTML - PubMed

Affiliation: Clinic for Fish and Reptiles, Faculty of Veterinary Medicine, University of Munich, Munich, Germany. saleh@zoofish.vetmed.uni-muenchen.de

ABSTRACT

Background: Enteric Redmouth (ERM) disease also known as Yersiniosis is a contagious disease affecting salmonids, mainly rainbow trout. The causative agent is the gram-negative bacterium Yersinia ruckeri. The disease can be diagnosed by isolation and identification of the causative agent, or detection of the Pathogen using fluorescent antibody tests, ELISA and PCR assays. These diagnostic methods are laborious, time consuming and need well trained personnel.

Results: A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for detection of Y. ruckeri the etiological agent of enteric red mouth (ERM) disease in salmonids. The assay was optimised to amplify the yruI/yruR gene, which encodes Y. ruckeri quorum sensing system, in the presence of a specific primer set and Bst DNA polymerase at an isothermal temperature of 63 degrees C for one hour. Amplification products were detected by visual inspection, agarose gel electrophoresis and by real-time monitoring of turbidity resulted by formation of LAMP amplicons. Digestion with HphI restriction enzyme demonstrated that the amplified product was unique. The specificity of the assay was verified by the absence of amplification products when tested against related bacteria. The assay had 10-fold higher sensitivity compared with conventional PCR and successfully detected Y. ruckeri not only in pure bacterial culture but also in tissue homogenates of infected fish.

Conclusion: The ERM-LAMP assay represents a practical alternative to the microbiological approach for rapid, sensitive and specific detection of Y. ruckeri in fish farms. The assay is carried out in one hour and needs only a heating block or water bath as laboratory furniture. The advantages of the ERM-LAMP assay make it a promising tool for molecular detection of enteric red mouth disease in fish farms.

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Visual detection of ERM-LAMP product. Using different naked eye detection methods: 1 = Negative control of ERM-LAMP reaction using Rox- labelled probe, there is neither pellet nor red fluorescence; 2 = Positive ERM-LAMP reaction using Rox- labelled probe, the pellet emitted red fluorescence; 3 = positive sample by using FDR, emitted strong green fluorescence when exposed to UV light; 4 = negative sample by using FDR, did not emitted strong green fluorescence under UV light; 5 = positive sample with green colour by using SYBR green I stain; 6 = negative sample with orange colour by using SYBR green I stain.
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Figure 2: Visual detection of ERM-LAMP product. Using different naked eye detection methods: 1 = Negative control of ERM-LAMP reaction using Rox- labelled probe, there is neither pellet nor red fluorescence; 2 = Positive ERM-LAMP reaction using Rox- labelled probe, the pellet emitted red fluorescence; 3 = positive sample by using FDR, emitted strong green fluorescence when exposed to UV light; 4 = negative sample by using FDR, did not emitted strong green fluorescence under UV light; 5 = positive sample with green colour by using SYBR green I stain; 6 = negative sample with orange colour by using SYBR green I stain.

Mentions: Optimal amplification of the Y. ruckeri yruI/yruR gene by ERM-LAMP assay was obtained at 63°C for 60 min, as shown by both agarose gel electrophoresis and real time turbidity measurements. Amplified products exhibited a ladder-like pattern on the gel (Fig. 1). Specificity of the amplification was confirmed by digestion of the LAMP products using HphI restriction enzyme (Fig. 1), the sizes of the resultant digestion products were as predicted (87 bp and 108 bp). Results obtained with the visual detection methods correlated with agarose gel electrophoresis results. When FDR used, a strong green fluorescence was emitted by LAMP positive reactions (F ig. 2, Tube No.3) when exposed to UV light and no fluorescence was evident for a negative reaction (Fig. 2, Tube No. 4). Likewise, after addition of SYBR Green I dye, the ERM-LAMP products appeared green (Fig. 2, Tube No. 5), while in the negative control tube the original orange colour of SYBR Green I did not change (Fig. 2, Tube No. 6). With Rox-labelled probe, a pellet formed emitted a red fluorescence for a positive reaction (Fig. 2, Tube No. 2), but there was neither pellet nor fluorescence observed in the negative control tube (Fig. 2, Tube No. 1).


Loop-mediated isothermal amplification as an emerging technology for detection of Yersinia ruckeri the causative agent of enteric red mouth disease in fish.

Saleh M, Soliman H, El-Matbouli M - BMC Vet. Res. (2008)

Visual detection of ERM-LAMP product. Using different naked eye detection methods: 1 = Negative control of ERM-LAMP reaction using Rox- labelled probe, there is neither pellet nor red fluorescence; 2 = Positive ERM-LAMP reaction using Rox- labelled probe, the pellet emitted red fluorescence; 3 = positive sample by using FDR, emitted strong green fluorescence when exposed to UV light; 4 = negative sample by using FDR, did not emitted strong green fluorescence under UV light; 5 = positive sample with green colour by using SYBR green I stain; 6 = negative sample with orange colour by using SYBR green I stain.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2531098&req=5

Figure 2: Visual detection of ERM-LAMP product. Using different naked eye detection methods: 1 = Negative control of ERM-LAMP reaction using Rox- labelled probe, there is neither pellet nor red fluorescence; 2 = Positive ERM-LAMP reaction using Rox- labelled probe, the pellet emitted red fluorescence; 3 = positive sample by using FDR, emitted strong green fluorescence when exposed to UV light; 4 = negative sample by using FDR, did not emitted strong green fluorescence under UV light; 5 = positive sample with green colour by using SYBR green I stain; 6 = negative sample with orange colour by using SYBR green I stain.
Mentions: Optimal amplification of the Y. ruckeri yruI/yruR gene by ERM-LAMP assay was obtained at 63°C for 60 min, as shown by both agarose gel electrophoresis and real time turbidity measurements. Amplified products exhibited a ladder-like pattern on the gel (Fig. 1). Specificity of the amplification was confirmed by digestion of the LAMP products using HphI restriction enzyme (Fig. 1), the sizes of the resultant digestion products were as predicted (87 bp and 108 bp). Results obtained with the visual detection methods correlated with agarose gel electrophoresis results. When FDR used, a strong green fluorescence was emitted by LAMP positive reactions (F ig. 2, Tube No.3) when exposed to UV light and no fluorescence was evident for a negative reaction (Fig. 2, Tube No. 4). Likewise, after addition of SYBR Green I dye, the ERM-LAMP products appeared green (Fig. 2, Tube No. 5), while in the negative control tube the original orange colour of SYBR Green I did not change (Fig. 2, Tube No. 6). With Rox-labelled probe, a pellet formed emitted a red fluorescence for a positive reaction (Fig. 2, Tube No. 2), but there was neither pellet nor fluorescence observed in the negative control tube (Fig. 2, Tube No. 1).

Bottom Line: These diagnostic methods are laborious, time consuming and need well trained personnel.Amplification products were detected by visual inspection, agarose gel electrophoresis and by real-time monitoring of turbidity resulted by formation of LAMP amplicons.Digestion with HphI restriction enzyme demonstrated that the amplified product was unique.

View Article: PubMed Central - HTML - PubMed

Affiliation: Clinic for Fish and Reptiles, Faculty of Veterinary Medicine, University of Munich, Munich, Germany. saleh@zoofish.vetmed.uni-muenchen.de

ABSTRACT

Background: Enteric Redmouth (ERM) disease also known as Yersiniosis is a contagious disease affecting salmonids, mainly rainbow trout. The causative agent is the gram-negative bacterium Yersinia ruckeri. The disease can be diagnosed by isolation and identification of the causative agent, or detection of the Pathogen using fluorescent antibody tests, ELISA and PCR assays. These diagnostic methods are laborious, time consuming and need well trained personnel.

Results: A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for detection of Y. ruckeri the etiological agent of enteric red mouth (ERM) disease in salmonids. The assay was optimised to amplify the yruI/yruR gene, which encodes Y. ruckeri quorum sensing system, in the presence of a specific primer set and Bst DNA polymerase at an isothermal temperature of 63 degrees C for one hour. Amplification products were detected by visual inspection, agarose gel electrophoresis and by real-time monitoring of turbidity resulted by formation of LAMP amplicons. Digestion with HphI restriction enzyme demonstrated that the amplified product was unique. The specificity of the assay was verified by the absence of amplification products when tested against related bacteria. The assay had 10-fold higher sensitivity compared with conventional PCR and successfully detected Y. ruckeri not only in pure bacterial culture but also in tissue homogenates of infected fish.

Conclusion: The ERM-LAMP assay represents a practical alternative to the microbiological approach for rapid, sensitive and specific detection of Y. ruckeri in fish farms. The assay is carried out in one hour and needs only a heating block or water bath as laboratory furniture. The advantages of the ERM-LAMP assay make it a promising tool for molecular detection of enteric red mouth disease in fish farms.

Show MeSH
Related in: MedlinePlus