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An Elegant Analysis of White Spot Syndrome Virus Using a Graphene Oxide/Methylene Blue based Electrochemical Immunosensor Platform

View Article: PubMed Central - PubMed

ABSTRACT

White spot syndrome virus (WSSV) is a major devastating virus in aquaculture industry. A sensitive and selective diagnostic method for WSSV is a pressing need for the early detection and protection of the aquaculture farms. Herein, we first report, a simple electrochemical immunosensor based on methylene blue dye (MB) immobilized graphene oxide modified glassy carbon electrode (GCE/GO@MB) for selective, quick (35 ± 5 mins) and raw sample analysis of WSSV. The immunosensor was prepared by sequential modification of primary antibody, blocking agent (bovine serum album), antigen (as vp28 protein), secondary antibody coupled with horseradish peroxidase (Ab2-HRP) on the GCE/GO@MB. The modified electrode showed a well-defined redox peak at an equilibrium potential (E1/2), −0.4 V vs Ag/AgCl and mediated H2O2 reduction reaction without any false positive result and dissolved oxygen interferences in pH 7 phosphate buffer solution. Under an optimal condition, constructed calibration plot was linear in a range of 1.36 × 10−3 to 1.36 × 107 copies μL−1 of vp28. It is about four orders higher sensitive than that of the values observed with polymerase chain reaction (PCR) and western blot based WSSV detection techniques. Direct electrochemical immunosensing of WSSV in raw tissue samples were successfully demonstrated as a real sample system.

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CV responses of different antigens modified GCE/GO@MB-Ab1-BSA-Ag-Ab2-HRP without and with 500 μM H2O2 containing pH 7 PBS at v = 10 mV s−1; (A) Enterocytozoon Hepatopenaei, (B) Infectious Hypodermal and Haematapoietic Necrosis Virus, (C) Fish intestine and (D) Fish Muscle. (E) Agarose gel showing PCR amplification of WSSV vp28 gene. M-100 bp DNA Marker, NC-Negative Control, PC-positive control-WSSV (white Spot Syndrome Virus), 2-IHHNV (Infectious Hypodermal and Haematopoietic necrosis Virus) DNA, 3-EHP (Enterocytozoon Hepatopenaei) DNA, 4-vibrio parahaemolyticus infected Fish intestine, and 5-vibrio harveyi infected fish muscle samples.
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f7: CV responses of different antigens modified GCE/GO@MB-Ab1-BSA-Ag-Ab2-HRP without and with 500 μM H2O2 containing pH 7 PBS at v = 10 mV s−1; (A) Enterocytozoon Hepatopenaei, (B) Infectious Hypodermal and Haematapoietic Necrosis Virus, (C) Fish intestine and (D) Fish Muscle. (E) Agarose gel showing PCR amplification of WSSV vp28 gene. M-100 bp DNA Marker, NC-Negative Control, PC-positive control-WSSV (white Spot Syndrome Virus), 2-IHHNV (Infectious Hypodermal and Haematopoietic necrosis Virus) DNA, 3-EHP (Enterocytozoon Hepatopenaei) DNA, 4-vibrio parahaemolyticus infected Fish intestine, and 5-vibrio harveyi infected fish muscle samples.

Mentions: Electrochemical immunosensor specificity is major concern in the real sample analysis. Figure 7(A–D) is CV response of the GCE/GO@MB for various cross-reactivity samples based on different aquatic pathogens such as EHP, IHHNV, Vibrio parahaemolyticus and Vibrio harveyi. For the preparation of different aquatic pathogen modified GCE/GO@MB electrodes, procedure mentioned in Fig. 1 and Fig. 2G was adopted. Interestingly, these immunosensor systems failed to show any signal for H2O2 reduction current, unlike to the WSSV-vp28, postulating the selectiveness of the present sensor for the WSSV. The specificity study was cross-confirmed with discreet PCR as well. Fig. 7(E) is the typical PCR analysis result of WSSV-infected positive DNA (PC, vp28) and healthy animals-negative DNA (NC) samples. The symbol “M” in the PCR photograph is the standard commercial 100 base pair DNA ladder. As can be seen, there were no target specific bands observed with the cross-reactivity samples when compared with the controls.


An Elegant Analysis of White Spot Syndrome Virus Using a Graphene Oxide/Methylene Blue based Electrochemical Immunosensor Platform
CV responses of different antigens modified GCE/GO@MB-Ab1-BSA-Ag-Ab2-HRP without and with 500 μM H2O2 containing pH 7 PBS at v = 10 mV s−1; (A) Enterocytozoon Hepatopenaei, (B) Infectious Hypodermal and Haematapoietic Necrosis Virus, (C) Fish intestine and (D) Fish Muscle. (E) Agarose gel showing PCR amplification of WSSV vp28 gene. M-100 bp DNA Marker, NC-Negative Control, PC-positive control-WSSV (white Spot Syndrome Virus), 2-IHHNV (Infectious Hypodermal and Haematopoietic necrosis Virus) DNA, 3-EHP (Enterocytozoon Hepatopenaei) DNA, 4-vibrio parahaemolyticus infected Fish intestine, and 5-vibrio harveyi infected fish muscle samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5385493&req=5

f7: CV responses of different antigens modified GCE/GO@MB-Ab1-BSA-Ag-Ab2-HRP without and with 500 μM H2O2 containing pH 7 PBS at v = 10 mV s−1; (A) Enterocytozoon Hepatopenaei, (B) Infectious Hypodermal and Haematapoietic Necrosis Virus, (C) Fish intestine and (D) Fish Muscle. (E) Agarose gel showing PCR amplification of WSSV vp28 gene. M-100 bp DNA Marker, NC-Negative Control, PC-positive control-WSSV (white Spot Syndrome Virus), 2-IHHNV (Infectious Hypodermal and Haematopoietic necrosis Virus) DNA, 3-EHP (Enterocytozoon Hepatopenaei) DNA, 4-vibrio parahaemolyticus infected Fish intestine, and 5-vibrio harveyi infected fish muscle samples.
Mentions: Electrochemical immunosensor specificity is major concern in the real sample analysis. Figure 7(A–D) is CV response of the GCE/GO@MB for various cross-reactivity samples based on different aquatic pathogens such as EHP, IHHNV, Vibrio parahaemolyticus and Vibrio harveyi. For the preparation of different aquatic pathogen modified GCE/GO@MB electrodes, procedure mentioned in Fig. 1 and Fig. 2G was adopted. Interestingly, these immunosensor systems failed to show any signal for H2O2 reduction current, unlike to the WSSV-vp28, postulating the selectiveness of the present sensor for the WSSV. The specificity study was cross-confirmed with discreet PCR as well. Fig. 7(E) is the typical PCR analysis result of WSSV-infected positive DNA (PC, vp28) and healthy animals-negative DNA (NC) samples. The symbol “M” in the PCR photograph is the standard commercial 100 base pair DNA ladder. As can be seen, there were no target specific bands observed with the cross-reactivity samples when compared with the controls.

View Article: PubMed Central - PubMed

ABSTRACT

White spot syndrome virus (WSSV) is a major devastating virus in aquaculture industry. A sensitive and selective diagnostic method for WSSV is a pressing need for the early detection and protection of the aquaculture farms. Herein, we first report, a simple electrochemical immunosensor based on methylene blue dye (MB) immobilized graphene oxide modified glassy carbon electrode (GCE/GO@MB) for selective, quick (35 ± 5 mins) and raw sample analysis of WSSV. The immunosensor was prepared by sequential modification of primary antibody, blocking agent (bovine serum album), antigen (as vp28 protein), secondary antibody coupled with horseradish peroxidase (Ab2-HRP) on the GCE/GO@MB. The modified electrode showed a well-defined redox peak at an equilibrium potential (E1/2), −0.4 V vs Ag/AgCl and mediated H2O2 reduction reaction without any false positive result and dissolved oxygen interferences in pH 7 phosphate buffer solution. Under an optimal condition, constructed calibration plot was linear in a range of 1.36 × 10−3 to 1.36 × 107 copies μL−1 of vp28. It is about four orders higher sensitive than that of the values observed with polymerase chain reaction (PCR) and western blot based WSSV detection techniques. Direct electrochemical immunosensing of WSSV in raw tissue samples were successfully demonstrated as a real sample system.

No MeSH data available.


Related in: MedlinePlus