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Journal of Virological Methods 162 (2009) 9195

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Journal of Virological Methods


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Immunocapture and direct binding loop mediated isothermal amplication simplify molecular diagnosis of Cyprinid herpesvirus-3
Hatem Soliman 1 , Mansour El-Matbouli
Fish Medicine and Livestock Management, University of Veterinary Medicine, Veterinaerplatz 1, A-1210 Vienna, Austria

a b s t r a c t
Article history: Received 26 April 2009 Received in revised form 20 July 2009 Accepted 23 July 2009 Available online 3 August 2009 Keywords: CyHV-3 Diagnosis LAMP PCR Immunocapture Direct binding

Loop mediated isothermal amplication (LAMP) assay is used for rapid diagnosis of Cyprinid herpesvirus3, formerly designated koi herpesvirus (KHV), with comparable sensitivity to PCR. To reduce the time required for the LAMP assay, an immunocapture (IC) and direct binding (DB) techniques were developed to exclude the DNA extraction step in molecular diagnostic procedures of the virus. Both techniques were evaluated by using PCR and CyHV-3-LAMP assays. The DB-LAMP/PCR assays were more sensitive (detecting 0.1 virus particles/ml) than the IC-LAMP/PCR assays (detecting 1 virus particle/ml). By using the SYBR Green I stain and the DB/LAMP assay the complete CyHV-3 diagnostic process can be achieved within 90 min compared to more than 5 h for the routine PCR assay. Both assays (IC/DB) could amplify successfully CyHV-3 from clinical samples which prove its application to diagnostic tests. The DB-LAMP assay is a simple, rapid, sensitive technique and applicable to the diagnosis of CyHV-3 in the eld. 2009 Elsevier B.V. All rights reserved.

1. Introduction Cyprinid herpes virus-3 (CyHV-3), formerly designated koi herpesvirus (KHV), threaten the worldwide production of common carp (Cyprinus carpio carpio) and koi (Cyprinus carpio koi) as an emerging highly contagious and extremely virulent virus with high mortality rate (Aoki et al., 2007; Costes et al., 2008; Ilouze et al., 2006). Since its emergence, CyHV-3 has caused severe economic and nancial losses in both the common carp and koi culture industries worldwide (Hedrick, 1996; Ronen et al., 2003). Koi shows, international trading and intensive culture of common carp have contributed to the rapid spread of the CyHV-3 (Haenen et al., 2004; Pikarsky et al., 2004; Sano et al., 2004). CyHV-3 infection is not only restricted to koi and the common carp sh but also to gold sh (Carassius auratus) and the crucian carp (Carassius carassius) (Haenen and Hedrick, 2006; El-Matbouli et al., 2007b; Sadler et al., 2008). Lethal viral disease is observed when water temperature ranged from 18 to 28 C which is called a permissive temperature (Gilad et al., 2003; Hedrick et al., 2000; Perelberg et al., 2003). The main clinical sign of the disease is severe necrosis of the gills. Mortality began 710 days after infection and reached 90% within 23

Corresponding author. Tel.: +43 1250775151; fax: +43 1250775192. E-mail addresses: hatemtoughan@hotmail.com (H. Soliman), Mansour.ElMatbouli@vu-wien.ac.at (M. El-Matbouli). 1 Permanent address: Veterinary Serum and Vaccine Research Institute, El-Sekka El-Beda St., P.O. Box 131 Abbasia, 11381 Cairo, Egypt. 0166-0934/$ see front matter 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2009.07.021

weeks (Hedrick et al., 2000). Due to the absence of available chemical treatment for viral infection the development of sensitive, rapid and specic tool for detection of viral pathogens is therefore critical for limiting their spread. Isolation of CyHV-3 from infected sh in cell cultures such as koi n (KF-1) cell line, with demonstrating the typical CPE (Hedrick et al., 2000), is considered the gold standard diagnostic method for CyHV-3 disease. This method is time consuming, only effective during the mortality phase of the disease and not suitable for detection of carrier sh (Gilad et al., 2002). Different improved diagnostic methods were developed to compensate the drawback of the virus isolation method. An enzyme linked immunosorbent assay (ELISA) for detection of antibodies to the koi herpesvirus was developed and evaluated (Adkison et al., 2005; Ronen et al., 2003; St-Hilaire et al., 2005). Different polymerase chain reactions (PCR), nested PCR (nPCR) and quantitative real-time PCR (qPCR) assays were described for rapid and sensitive detection of CyHV-3 DNA (Bercovier et al., 2005; El-Matbouli et al., 2007a; Gilad et al., 2002, 2004; Gray et al., 2002; Liu et al., 2002; Yuasa et al., 2005). An economic molecular diagnostic technique, loop mediate isothermal amplication (LAMP), which can be conducted under isothermal conditions ranging from 60 to 65 C was developed recently (Mori et al., 2001; Notomi et al., 2000). LAMP is based on the principle of autocycling strand displacement DNA synthesis performed by the Bst DNA polymerase for the detection of a specic DNA sequence (Notomi et al., 2000). Continuous amplication under isothermal conditions produces a very large amount of target DNA within 3060 min. Therefore, this method has high sensitivity and enables simple visual detection of DNA

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H. Soliman, M. El-Matbouli / Journal of Virological Methods 162 (2009) 9195 Table 1 Details of oligonucleotide primers used for CyHV-3-LAMP and PCR assays. Primer name F3 B3 FIP BIP LF LB KHV-TKf KHV-TKr Length 17-mer 18-mer 35-mer 35-mer 19-mer 22-mer 16-mer 17-mer Sequence (5 3 ) TGCAGCAGCCCTTCAAG GACACACCGCCTGGTAAG TGCACACCGCCGTCAGCT-CAGGTGACGGCGTTGGT GAAGTGCAAGATGCGCGACG-ACTCGGCGCCTCCAA GTCCAGCTTGTCCGCCATG CACCCTTCACCGTCAGAATCTC GGGTTACCTGTAC GAG CACCCAGTAGAT TATGC

amplication by a colour change of the reaction mixture (Iwamoto et al., 2003; Soliman and El-Matbouli, 2006). The LAMP assay was developed for rapid, specic and sensitive detection of CyHV-3 in tissues of infected sh (Gunimaladevi et al., 2004; Soliman and ElMatbouli, 2005). Immunocapture (IC) is a technique which uses specic anti-sera to capture the microorganism and this step concentrates and pre-puries the virus particles. PCR or LAMP assays are carried out using the nucleic acid released by heat denaturation from the trapped virions. Immunocapture-PCR (IC/PCR) and immunocapture-LAMP (IC/LAMP) were developed for detection of different viruses (Fukuta et al., 2004; Jansen et al., 1990; Le GallRecule et al., 2001; Wetzel et al., 1992). The aim of this study was to develop a new sample preparation technique, such as immunocapture or direct binding method which makes DNA extraction unnecessary, to simplify and accelerate the molecular diagnostic procedures of CyHV-3. The techniques were evaluated in conjunction with PCR and LAMP assays. The sensitivity, rapidity and feasibility of both techniques were assessed. 2. Materials and methods 2.1. CyHV-3 virus and anti-CyHV-3 polyclonal antibodies CyHV-3 virus was propagated in KF-1 cell line and the virus concentration was estimated by the 50% end point tissue culture infective dose (TCID50) analysis according to (Hedrick et al., 2000). The CyHV-3 virus and a rabbit polyclonal anti-CyHV-3 antiserum were provided by the Friedrich-Loefer Institute, Insel Riems, Germany. 2.2. Immunocapture (IC) of CyHV-3 viral particles An immunocapture of CyHV-3 viral particles prior to LAMP or PCR amplication was carried out using rabbit polyclonal antibodies against CyHV-3. Sterile DNase/RNase free polypropylene thin walled 0.2 ml microfuge tubes (Biozym Scientic GmbH, Hessisch Oldendorf, Germany) were coated with 25 l of CyHV-3 antibodies diluted with coating buffer (1.59 g sodium carbonate and 2.93 g sodium bicarbonate, pH 9.6) and incubated at 37 C for 2 h. Tubes were washed three times with 200 l phosphate buffer saline (PBS) containing 0.05% Tween-20 (PBS-T). Each tube received 25 l of CyHV-3 culture supernatant and incubated at 37 C for 2 h. The tubes were washed three times with 200 l PBS-T and dried briey and then subjected to LAMP or PCR assays. 2.3. Direct binding (DB) of CyHV-3 viral particles For experiments involving the direct binding of CyHV-3 particles to PCR tubes, virus culture supernatants were mixed with coating buffer and 25 l was placed in sterile polypropylene 0.2 ml thin walled tubes (without prior coating with anti-CyHV-3 antibodies) and incubated at 37 C for 2 h. Subsequent washes and further processing were carried out as described above. 2.4. Optimization of CyHV-3 immunocapture and direct binding techniques Different dilutions of CyHV-3 polyclonal antibodies in coating buffer were tested to select the lowest dilution which gives the best result. The effect of incubation temperature (4 C, room temperature, 37 C, 50 C) on the antibodies coating and virus particles binding efciency was appraised. Also the optimal time necessary for complete coating and binding of antibodies or virus particles to the tubes was assessed.

2.5. Immunocapture/direct binding CyHV-3-LAMP The LAMP reaction was carried out according to (unpublished data). Briey: for IC/DB-LAMP each tube received 24 l LAMP reaction mixture containing 20 mM TrisHCl (pH 8.8), 10 mM KCl, 7.5 mM MgSO4 , 10 mM (NH4 )2 SO4 , 0.1% Triton X-100, 1.6 M betaine (SigmaAldrich Chemie GmbH, Taufkirchen, Germany), 2.8 mM each deoxynucleotide triphosphates, 50 pmol each of the FIP and BIP primers, 5 pmol each of F3 and B3 primers, 25 pmol each of LF and LB primers, and appropriate amount of PCR grade water. The tubes were heat-denatured at 95 C for 5 min and then placed immediately on ice for 5 min. Each tube then received 8 units Bst DNA polymerase large fragment (New England BioLabs GmbH, Frankfurt, Germany) and incubated at 65 C for 1 h. The reaction was terminated by heating the reaction mixture to 85 C for 2 min. Details of LAMP primers are shown in Table 1. 2.6. Immunocapture/direct binding CyHV-3-PCR PCR was conducted according to Bercovier et al. (2005). Each IC/DB tube received 25 l reaction mixture containing: 2 Ready mix PCR Master mix (Thermo Scientic, Hamburg, Germany) containing:75 mM TrisHCl (pH 8.8), 20 mM (NH4 )2 SO4 , 1.5 mM MgCl2 , 0.01% Tween-20, 0.2 mM each nucleotide triphosphate, 1.25 U thermoprime plus DNA polymerase, and red dye for electrophoresis; 20 pmol each of forward (KHV-TKf) and reverse (KHV-TKr) primers, and appropriate amount of PCR grade water. The amplication was carried out in a Mastercycler Gradient thermocycler (Eppendorf). The cycling prole was: 95 C for 5 min, then 35 PCR cycles of 95 C for 30 s (DNA denaturation), 52 C for 30 s (primer annealing) and 72 C for 1 min (DNA extension), with a terminal extension step of 72 C for 10 min. Details of PCR primers are given in (Table 1). 2.7. Monitoring of immunocapture/direct binding CyHV-3 LAMP/PCR products LAMP and PCR products were separated by electrophoresis on 2% and 1.5% agarose gels, respectively, stained with GelRedTM Nucleic Acid Gel Stain, 10,000 in water (Biotrend Chemikalien GmbH, Kln, Germany) and visualised on a UV transilluminator. A TrackItTM 100 bp DNA ladder (Invitrogen GmbH, Karlsruhe, Germany) was used as molecular weight marker. For visual detection of the LAMP products, 1 l of 1:10 diluted SYBR Green I nucleic acid gel stain 10,000 concentration in DMSO (Cambrex BioScience, Rockland, Inc., ME, USA) was added to the reaction tube after the reaction termination. Any colour changes of the reaction mixture were noted. 2.8. Sensitivity of the immunocapture/direct binding techniques The CyHV-3 culture supernatant containing 104 viral particles/ml was diluted serially 10-fold diluted in culture medium or in coating buffer for testing the sensitivity of the IC and DB techniques, respectively, using the PCR and LAMP

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assays. For sensitivity comparison, 10 ng CyHV-3 DNA extracted from pure culture supernatants was diluted serially 10-fold for testing by the usual TK-PCR assay according to Bercovier et al. (2005). 2.9. Feasibility of immunocapture and direct binding techniques for CyHV-3 diagnosis The use of the IC and DB techniques for diagnosis of CyHV-3 disease was assessed. Tissue specimens from 60 clinical cases of koi and common carp which were tested previously by routine PCR were homogenised in ve times their weight of PBS in case of (IC) or coating buffer in case of (DB). After spinning of the homogenate at 1000 g for 5 min, 25 l supernatant was added to the tubes coated with CyHV-3 antibodies (IC) or placed in sterile CyHV-3 antibodies uncoated tubes (DB). Subsequent incubations, washes and further processing were carried out as described above for LAMP and PCR assays. 3. Results 3.1. Optimized immunocapture/direct binding techniques Different CyHV-3 antibodies dilutions, incubation times and temperatures were used for the IC assay. Using 1:10,000 CyHV-3 antibody dilution and incubation at 37 C for 2 h gave the expected results of the IC technique with both the LAMP and PCR assays (Fig. 1). In the case of DB technique, the LAMP and PCR products were obtained by incubating the virus diluted in coating buffer at 50 C for 15 min (Fig. 1). Positive CyHV-3 IC/DB-LAMP tubes changed to green after the addition of 1 l of 1:10 diluted SYBR Green I stain due to presence of LAMP products, while the negative tubes remained orange in colour due to the absence of LAMP products (Fig. 2). Results determined visually were the same as those obtained by gel electrophoresis. 3.2. Sensitivity of the immunocapture/direct binding techniques with LAMP and PCR assays DB/LAMP and DB/PCR have the ability to detect CyHV-3 virus to the dilution 109 which is equivalent to 0.1 virus particles/ml (Fig. 3). However, the IC/LAMP and the IC/PCR are able to detect CyHV-3 virus till the dilution 108 which is equivalent to one virus particle/ml (Fig. 3). The conventional TK-PCR assay detected CyHV3 DNA to the dilution of 107 which is equivalent to 30 virions (Fig. 4).

Fig. 2. Visual detection of immunocapture (IC) and direct binding (DB)-LAMP products by using SYBR Green I stain: tubes 1 and 3: positive sample exposing green colour which indicate presence of LAMP products in IC and DB, respectively; tubes 2 and 4: negative sample exposing orange colour which indicate absence of LAMP products in IC and DB, respectively. (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of the article.)

3.3. Feasibility of immunocapture and direct binding techniques for CyHV-3 diagnosis Both techniques (IC and DB) with both assays (LAMP and PCR) were able to detect CyHV-3 DNA in 52 out of 60 tested clinical homogenates of specimens while CyHV-3 DNA was not detected in the homogenate of the remaining rest 8 clinical specimens. The appropriate ratio of tissues to buffer to generate tissue homogenate was 1:5 (w/v) PBS (IC) or coating buffer (DB). 4. Discussion Sustainable solutions are required to decrease the magnitude of damage caused by CyHV-3 to the common carp and koi industry. One of these solutions is rapid, simple and sensitive molecular diagnosis of the CyHV-3 virus to full the preventive and control measures required which prevent the spread of the virus. Molecular diagnostic methods can detect viruses as long as genomic sequences exist, regardless of the integrity of the virus particle (Shirato et al., 2007). The main aim of all the previous diagnostic assays developed in our laboratory was to simplify the molecular diagnostic tests to be done in the small clinic or sh farms with increasing specicity, sensitivity and rapidity of the assay. Therefore, several LAMP assays for detection of different sh pathogens in 1 h with high specicity and sensitivity without the need for a sophisticated thermocycler were developed (El-Matbouli and Soliman, 2005a,b, 2006; Saleh et al., 2008a,b; Soliman and ElMatbouli, 2005, 2006; Soliman et al., 2009). But DNA extraction is still the step which is essential and requires well-trained personnel. Consequently, for simplifying the extraction procedure and to shorten the time needed from sampling to reading the result, the immunocapture and direct binding techniques were used. The immunocapture method is useful for detection of pathogens which are present either at low concentrations or in samples containing interfering substances since the technique cleans and concentrates the target pathogen which resulted in highly sensitive amplication assay (Fukuta et al., 2004; Wetzel et al., 1992). Both the IC and DB methods capture successfully the CyHV-3 particles and after simple heating at 95 C for 5 min the DNA was presented as a template for subsequent amplication by PCR or by the LAMP assay. The specicity of the rabbit polyclonal CyHV-3-antiserum was examined against Cyprinid herpesvirus-1 (CyHV-1) and Cyprinid herpesvirus-2 (CyHV-2), KF-1 cell lines and non-infected koi tissues in the case of the IC technique and proved to be specic for only CyHV-3 (data not shown). Also the specicity of both the IC and DB techniques was assured by the LAMP and PCR assays. Samples prepared by these methods were then subjected to PCR or LAMP assays which used specic primers which amplify only the CyHV-3 DNA. Therefore, no other DNA from other organisms, if captured, will be amplied by this assays which conrms the specicity of both techniques.

Fig. 1. Agarose gel electrophoresis demonstrating optimized immunocapture and direct binding LAMP and PCR results. Lane Mar: 100 bp DNA marker (Invitrogen); lane IC-LAMP: immunocapture LAMP products resulted from using anti-CyHV-3 antibodies in a dilution of 1:10,000 incubated at 37 C for 2 h; lane IC-PCR: immunocapture PCR products (409 bp) resulted from using anti-CyHV-3 antibodies in a dilution of 1:10,000 incubated at 37 C for 2 h; lane DB-LAMP: LAMP products of the CyHV-3/coating buffer bind directly to the tube wall after incubation at 50 C for 15 min; lane DB-PCR: PCR products (409 bp) of the CyHV-3/coating buffer bind directly to the tube wall after incubation at 50 C for 15 min; lane veco: no template control.

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Fig. 3. Sensitivity of direct binding (DB) and immunocapture (IC) LAMP and PCR assays in detection of CyHV-3 virus. DB-PCR/LAMP detected CyHV-3 virus till the dilution No. 6 which equal to virus dilution 109 , while IC-PCR/LAMP detected CyHV-3 virus till the dilution No. 5 which equal to virus dilution 108 . Lane Mar: 100 bp DNA marker (Invitrogen); lanes (1) to (8): serial dilution of 104 viral particles/ml CyHV-3 culture supernatant in coating buffer (DB) or in culture medium (IC); lane ve: no template control.

The two disposable plastic tubes available most commonly are manufactured from polypropylene or polystyrene, both of which give satisfactory results by the antibody-coating assay (Catt and Tregear, 1967). The result of this study demonstrated that, the least time required for CyHV-3 to bind to the plastic tube at 50 C is 15 min with a better sensitivity than the IC technique. This result is supported by the nding of Wang and Tschen (1994) who found that virus binding to a plastic tube was detectable after 1 min of incubation and reaches the maximum level after 10 min of incubation but after 1 h the amount of bound virus was not increases signicantly. The results of the sensitivity test demonstrated that IC and DB techniques had improved the sensitivity of the PCR and the LAMP assays, since they have the same lower detection limit (Soliman and El-Matbouli, unpublished data). The known lower detection limit of the TK-PCR assay was 30 virions (Bercovier et al., 2005) while by using IC and DB techniques the lower detection limit was one and 0.1 virions, respectively. This is explained by the fact that DB and IC methods concentrated and purify viral particles by

Fig. 4. Sensitivity of the conventional TK-PCR assay according to Bercovier et al. (2005) using agarose gel electrophoresis. The test detected CyHV-3 DNA till the dilution 107 which equivalent to 10 fg CyHV-3 DNA. Lane Mar: 100 bp DNA Marker (Invitrogen); lanes 110: 10-fold serial dilution of 10 ng CyHV-3 DNA; lane veco: no template control.

capture either directly or by specic antibodies and then the viral DNA released by heating from the trapped virus particles which then used as a template for PCR or LAMP assays. In this case the amount of released viral DNA is equivalent to the trapped viruses. While in the case of DNA extraction by conventional methods from infected tissues or from the virus culture supernatants, there is a loss of DNA during the consecutive DNA extraction steps. Also the extracted DNA contains not only the target viral DNA but also exogenous DNAs from tissues or cell culture debris. These exogenous DNAs can conceal partially the target viral DNA especially if it is present in small amounts. On the other hand, the DB methods were 10-fold more sensitive than the IC method. This can be elucidated by the fact that by the IC method the virus particles were captured by specic antibodies these antibodies were destroyed by heating, used to release the DNA of the captured viruses, and produce impurities which may conceal partially or hinder the target viral DNA from being available as a template for the PCR or LAMP assay. While with the DB method, the DNA released after heating is pure viral DNA without any impurities. The evaluation of the IC-LAMP/PCR and DB-LAMP/PCR assays for detection of viral DNA in clinical specimens has been validated using 60 samples of suspected infection of common carp and koi. Although the DB technique was more sensitive 10-fold than the IC technique; both methods, in combination with the LAMP/PCR assays, detected successfully CyHV-3 DNA in the tissue homogenates of 52 (out of 60) clinical samples (40 koi carp and 12 common carp samples) while CyHV-3 DNA was not detected, by both techniques, in the remaining 8 clinical samples (4 koi carp and 4 common carp). This result may be explained by the fact that those 52 clinical samples contain more than one CyHV-3 particles/ml in the tissues; therefore both assays could detect CyHV-3 DNA in these samples. On the other hand, these result were also comparable with the diagnostic results by the conventional PCR

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according to Bercovier et al. (2005) for the same clinical specimens since it detected 52 CyHV-3 positive samples (40 koi carp and 12 common carp samples) and 8 CyHV-3 negative samples (4 koi carp and 4 common carp). For sample preparation, DB is more advantageous than the IC technique since it is cost effective, did not require antibodies for primary coating, more rapid, needs only 15 min incubation, washing three times after virus attachment is needed, and 10-fold more sensitive than IC technique. Using of SYBR Green I stain for visual inspection of LAMP results reduce the time required for the LAMP assay and render it safer as it does not involve contact with ethidium bromide stain which has several health hazards (Iwamoto et al., 2003; El-Matbouli and Soliman, 2005a). Although the CyHV-3-LAMP assay has the same sensitivity of the PCR assay (Soliman and El-Matbouli, unpublished data), the LAMP assay is more rapid and cost effective, as it does not require the sophisticated PCR machine and the gel electrophoresis system, than the PCR assay. The combination of the DB technique, the LAMP assay and SYBR Green I stain requires only 90 min, from sample preparation to the end result, for detection of CyHV-3 with high sensitivity, specicity and efciency. Acknowledgments We thank Dr. D. Fichtner, National Reference Laboratory for Fish Diseases, Germany, for providing us CyHV-3 virus and rabbit antiCyHV-3 antibodies. References
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