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Microbiol Immunol 2009; 53: 375–383 doi:10.1111/j.1348-0421.2009.00145.x

ORIGINAL ARTICLE

Rapid, sensitive and simple detection method for koi herpesvirus using loop-mediated isothermal amplification Manabu Yoshino1 , Hajime Watari1 , Tadashi Kojima1 , Masanari Ikedo1 and Jun Kurita2 1

Biochemical Research Laboratory, Eiken Chemical Co., Ltd, Tochigi 329-0114, Japan, 2 Inland Station, National Research Institute of Aquaculture, Fisheries Research Agency, Mie 516-0193, Japan

ABSTRACT New methods were developed for the detection of koi herpesvirus (KHV, CyHV-3) by LAMP, which were compared with the PCR for specificity and sensitivity. We designed two primer sets targeting a specific sequence within the 9/5 PCR amplicon (9/5 LAMP) and the upper region of the SphI-5 PCR amplicon (SphI-5 LAMP), including a sequence highly conserved among the strains. The amplification was monitored in real-time based on the increase in turbidity, with magnesium pyrophosphate as the by-product. The reactions were carried out under isothermal conditions at 65◦ C for 60 min. The detection limit of both LAMP was six copies, equal to the modified SphI-5 PCR. No cross-reactivity with other fish pathogenic viruses and bacteria was observed. SphI-5 LAMP was found to have a quicker response in terms of the reaction velocity than 9/5 LAMP. Therefore, we consider SphI-5 LAMP to be superior for routine use. Additionally, LAMP was found applicable to crude extract from gills and other organs. LAMP methods are superior in terms of sensitivity, specificity, rapidity and simplicity, and are potentially a valuable diagnostic tool for KHV infections. Key words detection, koi herpesvirus, loop-mediated isothermal amplification (LAMP).

Since 2003, when the first outbreak of KHVD was reported in Japan (1), KHVD has become a prevalent infection nationwide, resulting in the massive death of carp in lakes and rivers. Two PCR methods (9/5 PCR (2) and modified SphI-5 PCR (3, 4)) have been adopted as routine diagnostic tools for KHV infection in Japan. However, the cost of the PCR equipment and extensive reaction time have slowed the implementation of PCR-based testing in diagnostic laboratories. To solve these problems, we developed a new diagnostic test for KHV infections using LAMP (5) and evaluated its efficacy. LAMP features the use of four different primers (FIP, BIP, F3 and B3) specifically designed to recognize six distinct regions on the target gene, and the reaction process is carried out at a constant temperature using a strand displacement reaction. Amplification and detection of a target gene can be completed in a single step by incu-

bating a mixture of the gene sample, primer set, DNA polymerase with strand displacement activity, and substrates at a constant temperature (approximately 65◦ C). In addition, loop primers can be designed to shorten the reaction time further and to increase the sensitivity (6). These primers provide high amplification efficiency, with DNA being amplified 109 –1010 times in 15–60 min. The LAMP reaction can be monitored in real-time by the increase in turbidity of a large amount of the by-product, magnesium pyrophosphate (7). In the present study, we designed two primer sets targeting different sequences of KHV to compare their reactivity and sensitivity. One of them, termed 9/5 LAMP (8), targets a specific sequence within the 9/5 PCR amplicon (AF411803). The other, SphI-5 LAMP, targets a sequence of the upper region of the gene which is amplified by SphI-5 PCR (3).

Correspondence Manabu Yoshino, 143 Nogi, Nogi-Machi, Shimotsuga-Gun, Tochigi 329-0114, Japan. Tel: +81-280-57-0717; fax: +81-280-57-0721; email: [email protected] Received 16 January 2009; revised 18 March 2009; accepted 26 March 2009. List of Abbreviations: KHV, koi herpesvirus; KHVD, koi herpesvirus disease; LAMP, loop-mediated isothermal amplification.

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Table 1 Viral and bacterial strains used in the present study and the specificity of each LAMP Fish-pathogenic viruses Species

Cell line

9/5 LAMP

SphI-5 LAMP

KHV (cyprinid herpesvirus 3) CHV (cyprinid herpesvirus 1) GFHNV (cyprinid herpesvirus 2) EHV (anguillid herpesvirus 1)

KF-1 FHM GFF EK-1

+ − − −

+ − − −

Bacteria Species

Reference

9/5 LAMP

SphI-5 LAMP

ATCC27061(type) NCTC9750(type) NCTC10396(type)

– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

Alcaligenes xylosoxidans subsp. Citrobacter freundii Edwardsiella tarda Enterobacter aerogenes Enterobacter cloacae Escherichia coli Haemophilus influenzae Hafnia alvei Klebsiella oxytoca Klebsiella pneumoniae Legionella bozemanii Legionella dumofrii Legionella dumoffii Legionella gormanii Legionella longbeachae Legionella micdadei Legionella pneumophila Listeria grayi Listeria innocua Listeria ivanovii Listeria monocyogenes Listeria monocyogenes Listeria seeligeri Morganella morganii Proteus mirabilis Proteus vulgaris Providencia alcalifaciens Providencia rettgeri Providencia stuartii Pseudomonas aeruginosa Pseudomonas fluorescens Salmonella Enteritidis Salmonella Paratyphi A Salmonella Typhi Salmonella Typhimurium Serratia marcescens Vibrio parahaemolyticus Vibrio parahaemolyticus(tdh+) Yersinia enterocolitica

ATCC13047(type) ATCC11775(type) ATCC9795 NCTC9540(type) ATCC13883(type) ATCC33217(type) ATCC33279(type) ATCC33343 ATCC33297(type) ATCC33462(type) ATCC33218(type) ATCC33152(type) ATCC25400 ATCC19119(type) JCM2873 ATCC35152 ATCC25830(type) ATCC13315 ATCC9886(type)

ATCC10145(type) ATCC13525(type) ATCC13076(type)

ATCC14028 ATCC8100 ATCC17802(type)

MATERIALS AND METHODS Viral and bacterial strains

In the present study, a supernatant culture of a koi fin (KF-1) cell line infected with KHV was used as the KHV 376

suspension (9). To determine the specificity with regard to similar fish pathogens, we used purified genomic DNA of carp herpesvirus (CyHV-1, isolated from the FHM cell line), goldfish hematopoietic necrosis virus (CyHV2, isolated from the GFF cell line) and eel herpesvirus (AngHV-1, isolated from the EK-1 cell line). These fish c 2009 The Societies and Blackwell Publishing Asia Pty Ltd 

Rapid detection of KHV using LAMP method

Table 2 Comparison of detection by each LAMP and PCR for 19 gills from NRIA 9/5 LAMP

SphI-5 LAMP

9/5 PCR

Modified SphI-5 PCR

Sample no.

PureGene

EXF

PureGene

EXF

PureGene

PureGene

A B C D E (decomposed) F (decomposed) G (decomposed) H (decomposed)

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + − + +

+ + + + + + +† +

2-A 2-B 2-C 2-D 2-E 2-F 2-G 2-H 2-I 2-J

+ + + + − + − + − −

+ + + + − + − + − −

+ + + + − + − + − −

+ + + + − + − + − −

− − − − − + − − − −

+ + + + − + − + − −

Normal













†An additional dilution of purified DNA was required.

pathogenic viruses were obtained from Dr Hideo Fukuda (Tokyo University of Marine Science and Technology, Faculty of Marine Science) and NRIA (National Research Institute of Aquaculture, Fisheries Research Agency). We also chose 24 bacterial strains considered to exist in the waters of rivers and lakes, such as Escherichia coli (Table 1).

Extraction from tissue specimens with the commercial kits

The DNA templates from tissue specimens were prepared using a PureGene Cell and Tissue Kit (Gentra System Co., Minneapolis, MN) or DNeasy Blood and Tissue Kit (QIAGEN Co., Hilden, Germany). Extraction from tissue specimens by an easy method

Tissue specimens from koi

Nineteen gill samples were obtained from NRIA. They comprised eight KHV-positive specimens as determined with both 9/5 PCR and modified SphI-5 PCR, 10 that had unclear results using the two PCR methods, and one healthy specimen (Table 2). Among the positive samples, some were showing signs of tissue decomposition. In order to compare the different types of tissue for KHV detection, gill samples and other internal organs samples, including liver, kidney and small intestine, individually from 30 carp, were obtained from a prefectural fisheries experimental station in Japan. These samples were separated into 15 KHV-positive samples (P1–P15) and 15 KHV-negative samples (N1–N15) as indicated from prior testing with the 9/5 PCR and modified SphI-5 PCR (Table 3). c 2009 The Societies and Blackwell Publishing Asia Pty Ltd 

An easier and faster extraction method than the use of the commercial kits for LAMP was examined. A slice of tissue (the size of a grain of rice) and 100 ul TE buffer (10 mM, pH 8.8) were placed in a 1.5-ml test tube and homogenized thoroughly with a pestle. Following the addition of 100 ul of extraction solution for food (EXF, pH12.5) contained R kits, the mixture was heated at 95◦ C for in the Loopamp 5 min. The boiled mixture was cooled on ice briefly, and 10 ul of 1 M Tris-HCl (pH 7.0) was added and the mixture was mixed well. After centrifugation at 2000 × g for 30 sec by a small benchtop centrifuge, 5 ul of the supernatant was used as a template for LAMP. Plasmid DNA

To determine the sensitivity of LAMP, we constructed a plasmid DNA containing the sequences which were amplified by 9/5 LAMP and SphI-5 LAMP, respectively. 377

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Table 3 Comparison of detection by each LAMP and modified SphI-5 PCR for gills and pooled internal organs obtained from 30 carp individually (A) gill

(B) pooled internal organs 9/5 LAMP

Modified SphI-5 PCR

SphI-5 LAMP

9/5 LAMP

Modified SphI-5 PCR

SphI-5 LAMP

Sample no.

QIAGEN

EXF

QIAGEN

EXF

QIAGEN

Sample no.

QIAGEN

EXF

QIAGEN

EXF

QIAGEN

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15

+ + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + +

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15

+ + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + +

N1 N2 N3 N4 N5 N6 N7 N8 N9 N10 N11 N12 N13 N14 N15

− − − − − − + − − − − − − − −

− − − − − − − − − − − − − − −

− − − − − − + − − − − − − − −

− − − − − − + − − − − − − − −

− − − − − − − − − − − − − − −

N1 N2 N3 N4 N5 N6 N7 N8 N9 N10 N11 N12 N13 N14 N15

− − − − − − − − − − − − − − −

− − − − − − − − − − − − − − −

− − − − − − + − − − − − − − −

− − − − − − − − − − − − − − −

− − − − − − − − − − − − − − −

LAMP assay

Details of 9/5LAMP and SphI-5 LAMP primer set with regard to their positions in the genomic sequence are shown in Figure 1a (9/5 LAMP) and Figure 1b (SphI-5 LAMP). For confirmation of the accuracy of the LAMP reaction, the sites of the restriction enzymes which recognize and digest the specific sequence from the genomic DNA were configured (BcnI for 9/5 LAMP, HhaI and PstI for SphI-5 LAMP). Both LAMP reactions were carried out in a 25-ul reR DNA amplification action volume using the Loopamp kit (Eiken Chemical Co. Ltd, Tokyo, Japan) containing 40 pmol each of the primers FIP and BIP, 10 pmol each of the primers F3 and B3, 20 pmol each of the primers loop primer F and B, 1.4 mM deoxynucleoside triphosphates, 0.8 M betaine, 0.1% Tween 20, 10 mM (NH 4 ) 2 SO 4 , 10 mM KCl, 8 mM MgSO 4 , 20 mM Tris-HCl (pH8.8), 378

8 units of Bst DNA polymerase (New England Biolabs, Ipswich, MA), and 5 ul DNA template heated at 95◦ C for 5 min prior. The real-time monitoring of the LAMP assay was accomplished by incubating the reaction mixture at R real-time turbidime65◦ C for 60 min in a Loopamp ter (LA-320C or RT-160C) (Eiken Chemical Co., Tokyo, Japan). PCR assay

For comparison with the LAMP, two types of PCR were used, the 9/5 PCR (484 bp product) and the modified SphI-5 PCR (292 bp product). The modified SphI-5 PCR reduced non-specific reactions and the total reaction time after optimization of the sequence of the reverse primer and the cycle conditions of the original assay (3). Both PCR reactions were carried out as previously described (2, 4). c 2009 The Societies and Blackwell Publishing Asia Pty Ltd 

Rapid detection of KHV using LAMP method

Fig. 1 Location and sequence of LAMP targets and priming sites of (a) 9/5 LAMP and (b) SphI-5 LAMP. DNA sequences used for LAMP primer designing are shown by solid arrows. PCR primers used as reference are shown by dotted arrows (a: 9/5 PCR (2), b: SphI-5 PCR (3, 4)), and the sequence-specific digestion sites of restriction enzymes are shown by the boxes, respectively.

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RESULTS

product had the sequence corresponding to the selected target.

Specificity of LAMP

To evaluate the specificity of both LAMP, we tested KHV, three non-KHV fish pathogenic viruses (CyHV-1, CyHV2 and AngHV-1) and 24 bacterial strains. Considerable amplification of the genomic DNA from KHV was observed with both the 9/5 LAMP and the SphI-5 LAMP. By contrast, none of the other viral or bacterial strains were amplified, even when excess amounts of template and a 60-min incubation period were used (Table 1). The products of both LAMP were evident upon agarose gel electrophoresis as a ladder-like pattern on the gel, which is characteristic of the LAMP reaction. The specificity of the amplification was confirmed by restriction enzyme digestion to ensure that the amplification

Sensitivity of LAMP

The sensitivity of each LAMP and PCR were compared using 10-fold serial dilutions of the KHV suspension (the supernatant of the KF-1 culture). The detection limit of both 9/5 LAMP and SphI-5 LAMP was 10−7 . On monitoring the LAMP reaction with the real-timer turbidimeter, SphI-5 LAMP was found to start more quickly than 9/5 LAMP. However, the detection limits of the 9/5 PCR and modified SphI-5 PCR were 10−2 and 10−7 , respectively (Fig. 2). To establish the detection limit of the LAMP assay, serial dilutions of each plasmid DNA which had been quantified by measuring the optical density at 260 nm were tested.

Fig. 2. Comparison of sensitivity of (a) 9/5 LAMP, (b) SphI-5 LAMP, (c) 9/5 PCR and (d) modified SphI-5 PCR using 10-fold serial dilutions of KHV suspension (the supernatant of KF-1 culture).

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Rapid detection of KHV using LAMP method

The detection limit of both 9/5 LAMP and SphI-5 LAMP was 6 copies in 60 min. In this experiment as well, SphI-5 LAMP exhibited a faster reactivity than 9/5 LAMP.

digestion, in the case of both 9/5 LAMP and SphI-5 LAMP (Fig. 3).

DISCUSSION Correlation between LAMP and PCR in the detection in tissue specimens

DNA templates from 19 gill samples from NRIA were extracted using a PureGene Cell and Tissue Kit and analyzed by each LAMP and PCR (Table 2). All 14 specimens positive by modified SphI-5 PCR were also found to be positive by both 9/5 LAMP and SphI-5 LAMP. The LAMP assay showed rapid amplification within 40 min in the 9/5 LAMP and 30 min in the SphI-5 LAMP. Although the determination by the modified SphI-5 PCR was in good agreement with the LAMP finding, an additional dilution was required in one decomposed gill sample for the correct detection by PCR. In contrast, only eight gills were detected as positive by 9/5 PCR. To test the use of crude extract to simplify the operation, we also tried an extraction method using EXF solution. The results of both LAMP were the same as the DNA samples extracted with the commercial kit. When the crude extracts from gill samples were also applied to PCR, however, numerous non-specific bands appeared. Furthermore, we investigated the optimal type of sample for detection. DNA templates from gill samples and other internal organs samples from 30 carp were extracted with two methods, DNeasy Blood and Tissue Kit (QIAGEN Co.) and the easy method using EXF, respectively, and analyzed by each LAMP and the modified SphI-5 PCR (except in combination with the crude extract obtained using the EXF solution). All of the samples of gills and internal organs from carp detected as KHV-positive by PCR beforehand were determined to be positive by both LAMP assays and the modified SphI-5 PCR regardless of the extraction methods (Table 3). Although none of the PCR-determined KHVnegative individuals displayed amplification by PCR in this study, one (N7) of them was detected as positive by LAMP. In the case of the gill sample of N7, the extract obtained with the QIAGEN kit was amplified in both LAMP assays, but the extract obtained with EXF was amplified in SphI-5 LAMP only. In the case of the internal organs of N7, only a combination of the extract obtained with the QIAGEN kit and SphI-5 LAMP gave a positive result. To ascertain whether the amplification from the extract of N7 was specific and correct, the LAMP product from N7 was subjected to an electrophoretic analysis with or without subsequent digestion by the restriction enzyme. The pattern on the gel was in good agreement with that from the positive control (plasmid DNA), with or without c 2009 The Societies and Blackwell Publishing Asia Pty Ltd 

The special features of the LAMP method are its high sensitivity and specificity despite a simple and rapid operation in which both the reaction and detection are carried out continuously in one tube under isothermal conditions. We examined the application of LAMP to the detection of KHV and whether more advantageous test methods than the conventional methods are feasible. In this study, 9/5 LAMP and SphI-5 LAMP had the same detection limit (6 copies/test) and an extremely high specificity. The sensitivity of both LAMP was 105 -fold higher than that of 9/5 PCR, and almost equivalent to that of the modified SphI-5 PCR. In the same way that the LAMP method did not undergo interference by inhibitive substances derived from the sample for testing, the application of crude extract using EXF was possible. The LAMP method did not require any additional dilution of the sample. Therefore, it is considered that the LAMP method combined with the extraction using EXF will allow quick and easy detection available on site. In the study with gills and internal organs, there was one discrepancy regarding the results of the detection between LAMP and PCR. It was guessed that KHV had been detected by the LAMP method only because the sample of N7 contained a very small amount of KHV. Although the infection spread of KHV from skin and gills toward numerous internal organs and nervous tissue was considered (10), it was thought that the gills that showed a high detection rate were preferable to other internal organs as the specimen material. Because the gills were organs that could be regenerated, this method suggested that it not only had the usefulness of the detection of KHV in dead fish, but also the possibility of its application as a method that can be substituted for conventional tests in the international fish trade. Although the extraction using EXF resulted in a lower sensitivity than with the highly purified DNA sample obtained with the commercial extraction kit, we consider it highly useful in detection in cases where the results are required immediately and/or reconfirmation of samples determined positive by PCR is needed. When the reaction velocity was measured, SphI-5 LAMP was found to have a quicker reaction response than 9/5 LAMP. We speculated that this was due to the base composition, GC content, and the formation of secondary structures of the primers and template, and because the SphI-5 LAMP was designed more appropriately than 9/5 LAMP. Although SphI-5 LAMP was superior in reaction velocity, 9/5 LAMP is currently the routine diagnostic tool used for KHV infections in Japan. However, we believe 381

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Fig. 3. Pattern on the agarose gel of product by (a) 9/5 LAMP and (b) SphI-5 LAMP from plasmid DNA as positive control and N7 sample.

that the method of diagnosing KHV infections by LAMP should be updated from 9/5 LAMP to SphI-5 LAMP as the more accurate and useful testing method.

ACKNOWLEDGMENTS This study was funded by a research project using advanced technologies in agriculture, forestry and fisheries (The Ministry of Agriculture, Forestry and Fisheries of Japan). We would like to thank Dr Kei Yuasa, Dr Motohiko Sano and Dr Takaji Iida of NRIA (National Research Institute of Aquaculture, Fisheries Research Agency, Japan), and Dr Keith Way of CEFAS (Centre for Environment, Fisheries and Aquaculture Science, UK) for advice and critical review of the manuscript. REFERENCES 1. Sano M., Ito T., Kurita J., Yanai T., Watanabe N., Miwa S., Iida T. (2004) First detection of koi herpesvirus in cultured carp Cyprinus carpio in Japan. Fish Pathol 39(3): 165–7.

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2. Gilad O., Yun S., Andree K.B., Adkison M.A., Zlotkin A., Bercovier H., Eldar A., Hedrick R.P. (2002) Initial characteristics of koi herpesvirus and development of a polymerase chain reaction assay to detect the virus in koi, Cyprinus carpio koi. Dis Aquat Organ 48(2): 101–8. 3. Gray W.L., Mullis L., LaPatra S.E., Groff J.M., Goodwin A. (2002) Detection of koi herpesvirus DNA in tissues of infected fish. J Fish Dis 25: 171–8. 4. Yuasa K., Sano M., Kurita J., Ito T., Iida T. (2005) Improvement of a PCR method with the SphI-5 primer set for the detection of Koi Herpesvirus (KHV). Fish Pathol 40: 37–9. 5. Notomi T., Okayama H., Masubuchi H., Yonekawa T., Watanabe K., Amino N., Hase T. (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28(12): E63. 6. Nagamine K., Hase T., Notomi T. (2002) Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes 16(3): 223–9. 7. Mori Y., Nagamine K., Tomita N., Notomi T. (2001) Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Biophys Res Commun 289(1): 150–4. 8. Yoshino M., Watari H., Kojima T., Ikedo M. (2006) Sensitive and rapid detection of Koi herpesvirus by LAMP method. Fish Pathol 41(1): 19–27.

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9. Gilad O., Yun S., Adkinson M.A., Way K., Willits N.H., Bercovier H., Hedrick R.P. (2003) Molecular comparison of isolates of an emerging fish pathogen, koi herpesvirus, and the effect of water temperature on mortality of experimentally infected koi. J Gen Virol 84: 2661–7.

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10. Gilad O., Yun S., Zagmutt-Vergara F.J., Leutenegger C.M., Bercovier H., Hedrick R.P. (2004) Concentration of a Koi herpesvirus (KHV) in tissues of experimentally infected Cyprinus carpio koi as assessed by real-time TaqMan PCR. Dis Aquat Org 60: 179–87.

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