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Sensitive and rapid identification of Vibrio vulnificus by loop-mediated isothermal amplification
ARTICLE IN PRESS Microbiological Research 164 (2009) 514—521
www.elsevier.de/micres
Sensitive and rapid identification of Vibrio vulnificus by loop-mediated isothermal amplification Chun-Hua Ren, Chao-Qun Hu, Peng Luo, Qing-Bai Wang The Key Laboratory of Applied Marine Biology of Guangdong Province and Chinese Academy of Sciences, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, PR China Received 6 March 2008; received in revised form 28 April 2008; accepted 18 May 2008
KEYWORDS Identification; Vibrio vulnificus; LAMP
Summary Vibrio vulnificus is a serious bacterial pathogen for humans and aquatic animals. We developed a rapid, sensitive and specific identification method for V. vulnificus using loop-mediated isothermal amplification (LAMP) technique. A set of primers, composed of two outer primers and two inner primers, was designed based on the cytolysin gene sequence of V. vulnificus. The LAMP reaction was processed in a heat block at 65 1C for 60 min. The amplification products were detected by visual inspection using SYBR Green I, as well as by electrophoresis on agarose gels. Our results showed that the LAMP reaction was highly specific to V. vulnificus. This method was 10-fold more sensitive than conventional PCR. In conclusion, the LAMP assay was extremely rapid, simple, cost-effective, sensitive and specific for the rapid identification of V. vulnificus. & 2008 Elsevier GmbH. All rights reserved.
Introduction Vibrio vulnificus is a Gram-negative pathogen that was first isolated by the US Centers for Disease Control (CDC) in 1964 (Strom and Paranjpye, 2000) and designated as V. vulnificus in 1980 (Farmer, 1980). This halophilic bacterium is widely distribCorresponding author. Tel.: +86 020 89023216;
fax: +86 020 89023218. E-mail address: [email protected] (C.-Q. Hu).
uted in estuarine and coastal waters around the world (Oliver et al., 1983). Associated with the consumption of raw or undercooked seafood (especially oysters), V. vulnificus opportunistically causes primary septicemia in susceptible individuals with a history of immunosuppression, hemochromatosis, cirrhosis or alcoholism. The mortality rate of primary septicemia caused by V. vulnificus is over 50% within days (Strom and Paranjpye, 2000). In addition to the primary septicemia, wound infections are incurred via contamination of
0944-5013/$ - see front matter & 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2008.05.002
ARTICLE IN PRESS Sensitive and rapid identification of Vibrio vulnificus preexisting wounds with seawater or seafood products where the organism is present, with a mortality rate of about 25% (Harwood et al., 2004). V. vulnificus is pathogenic not only for humans but also for aquatic animals. Under certain environmental conditions V. vulnificus can infect marine fish (especially eels) and shellfish, causing high mortality and serious economic losses (Dalsgaard et al., 1998). Thus, in order to decrease the occurrence of the bacterial infection to human beings and prevent the outbreak of aquaculture diseases caused by this pathogen, it is necessary to establish rapid and effective methods for the identification and detection of V. vulnificus. PCR and real-time PCR have been successfully used to detect V. vulnificus (Brauns et al., 1991; Hill et al., 1991; Coleman et al., 1996; Lee et al., 1999; Chen et al., 2005; Kumar et al., 2006; Wang and Levin, 2006; Gordon et al., 2008). PCR is a powerful tool for the detection of V. vulnificus because of its relatively rapid speed and sensitivity compared with conventional methods. However, an expensive thermal cycler is necessary for PCR amplification. A novel isothermal DNA amplification method termed loop-mediated isothermal amplification (LAMP) was invented by Notomi et al. (2000). This method relies on auto-cycling strand displacement DNA synthesis performed by the Bst DNA polymerase large fragment (with high-strand displacement activity) and a set of two specially designed inner and two outer primers that recognizes a total of six distinct sequences on the target DNA. An extremely large amount of DNA is produced by the cycling reaction in less than an hour under isothermal conditions ranging from 60 to 65 1C in a heat block or water bath. The final products are stem-loop DNA with several inverted repeats of the target and exhibit cauliflower-like structures with multiple loops. LAMP is highly specific, efficient and rapid (Notomi et al., 2000). In addition, the LAMP reaction products can be simply judged with eye inspection by the color change of a mixture with SYBR Green I (Iwamoto et al., 2003) or a white turbidity of magnesium pyrophosphate (Mori et al., 2001). LAMP has been successfully used to detect DNA viruses such as human herpesvirus (Yoshikawa et al., 2004; Kuhara et al., 2007) and infectious hypodermal and hematopoietic necrosis virus (Sun et al., 2006); pathogenic bacteria such as Mycobacterium tuberculosis (Iwamoto et al., 2003), Edwardsiella ictaluri (Yeh et al., 2005) and Salmonella (Wang et al., 2008); protozoa (El-Matbouli and Soliman, 2005; Njiru et al., 2008) and fungus (Endo et al., 2004; Gadkar and Rillig, 2008). RT-LAMP has been used for the detection of RNA
515 viruses such as SARS virus (Hong et al., 2004), West Nile virus (Prida et al., 2004), Norovirus (Fukuda et al., 2006) and septicemia virus (Soliman and El-Matbouli, 2006). In the present study, a rapid, sensitive and specific method for identification of V. vulnificus was developed based on LAMP technique.
Materials and methods Bacterial strains and DNA extraction The bacterial strains used in this study are listed in Table 1. All strains were identified by standard biochemical tests (FDA, 2001). Two methods were used for the preparation of DNA for LAMP. In method I, DNA was extracted by using the TaKaRa MiniBEST Bacterial Genomic DNA Extraction Kit (Takara Bio. Dalian Co. Ltd.) according to the manufacturer’s protocol. In method II, DNA was extracted using the boiling method as described by Mohran et al. (1998).
Design of primers for LAMP Based on the cytolysin gene sequence of V. vulnificus (M34670; Yamamoto et al., 1990), a
Table 1. Bacterial strains used to test the specificity of LAMP reaction in this study Species
Sourcesa
Number of strains tested
V. vulnificus (strain 1.1758) V. vulnificus V. cholerae V. parahaemolyticus V. alginolyticus V. fluvialis V. mimicus V. harveyi V. anguillarum V. splendidus V. nereis V. pelagia Aeromonas sobria Aeromonas hydrophila Pseudomonas aeruginosa Pseudomonas fluorescens Shewanella algae
IMCAS EI GDIM, EI SYSU, EI EI IMCAS, EI IMCAS, EI SCSFI, EI SHFU, SCSFI IMCAS IMCAS IMCAS SHFU SHFU SHFU SYSU EI
1 3 2 4 4 3 2 2 2 1 1 1 1 1 1 1 1
a
EI ¼ environmental isolate, GDIM ¼ Guangdong Institute of Microbiology, IMCAS ¼ Institute of Microbiology of Chinese Academy of Sciences, SCSFI ¼ South China Sea Fisheries Institute of Chinese Academy of Fishery Sciences, SHFU ¼ Shanghai Fisheries University, SYSU ¼ Sun Yat-Sen University.
ARTICLE IN PRESS 516 set of four primers composed of two outer primers which initiate strand displacement and two inner primers which structure ‘‘the loop’’ through the reaction was designed using Primer Explorer version 3 (https://primerexplorer.jp/lamp3.0.0/ index.html). The forward inner primer (FIP) consisted of the complementary sequence of F1 (22 nt), a T TTT spacer and F2 (18 nt). The backward inner primer (BIP) consisted of B1c (22 nt), a TTTT spacer and the complementary sequence of B2c (20 nt). The outer primers consisted of the forward outer primer F3 (18 nt) and the backward outer primer B3 (the complementary sequence of B3c, 20 nt). The positions of the LAMP primers used in this study are shown in Figure 1.
LAMP reaction The LAMP reaction was carried out according to the method described by Notomi et al. (2000). LAMP was performed in a 25 ml reaction volume containing the following reagents: 0.8 mM each of FIP and BIP, 0.2 mM each of F3 and B3, 400 mM dNTP,
C.-H. Ren et al. 1 M betaine (Sigma), 20 mM Tris–HCl (pH 8.8), 10 mM KCl, 10 m M (NH4)2SO4, 4 mM MgSO4, 0.1% Triton X-100, and 1 ml of target DNA or 1 ml distilled water (negative control). In a heat block, the reaction mixture was heated at 95 1C for 5 min, then cooled on ice. Finally, 8 U Bst DNA polymerase large fragment (New England Biolabs, Beverly, MA) was added. Subsequently, the mixture was incubated at 65 1C for 60 min and then heated at 80 1C for 3 min to terminate the reaction.
Detection of LAMP products After the reaction, 1 ml of 10-fold diluted original SYBR Green I (Molecular Probes Inc.) was added to 25 ml LAMP products. The solution turned green in the presence of LAMP amplification products, while it remained orange in the absence of amplicon. For further confirming the presence of amplicons, 3 ml of LAMP products were electrophoresed on a 2.0% agarose gel stained with GoldViewTM Nucleic Acid Stain and photographed using gel documentation system (Vilber Lourmat, France).
Figure 1. (A) Locations and nucleotide sequence of the cytolysin gene of V. vulnificus (M34670) used for designing the inner and outer primers. The DNA sequences of primer sites were underlined. (B) Names and sequences of LAMP primers used for specific amplification of V. vulnificus gene. B3, F1c and B2 represent complementary sequence to B3c, F1 and B2c, respectively.
ARTICLE IN PRESS Sensitive and rapid identification of Vibrio vulnificus
517 templates were extracted by method I and II. Two parallel experiments were made.
Specificity of LAMP identification To determine the specificity of LAMP method, LAMP was carried out with the different DNA templates (extracted by method I) from the 31 strains in the Vibrionacea family (Table 1) under the conditions described above. Each strain was examined at least twice.
Sensitivity of LAMP identification In order to test the sensitivity of the cytolysin gene primers in LAMP identification of V. vulnificus, DNA from V. vulnificus 1.1758 (extracted by method I) was serially 10-fold diluted. The diluted DNA templates were tested by LAMP and conventional PCR. In conventional PCR, three sets of primers (see Table 2) anchoring various target sequences were adopted. Using primers F3 and B3, PCR amplification was carried out with the TaKaRa TaqTM (Takara Bio. Dalian Co. Ltd.) according to the manufacturer’s protocol by adding 1 ml of DNA template and 20 pmol of each primer in a 25 ml total reaction volume. Amplification conditions consisted of an initial denaturation of 94 1C for 3 min and 40 cycles of 60 s at 95 1C, 60 s at 55 1C, 90 s at 72 1C and a final extension of 10 min at 72 1C in PTC-100 (Bio-Rad, USA). Using the other two sets of primers, reactions were performed according to the amplification conditions described by Chen et al. (2005) and Kumar et al. (2006), respectively. Distilled water was used as negative control for both PCR and LAMP. Two parallel experiments were made.
Results LAMP reaction The mechanism and expected reaction steps of LAMP were shown in the illustration finished by Notomi et al. (2000). The LAMP reaction includes three steps: (1) starting material producing step; (2) cycling amplification step; (3) elongation and recycling step. The reaction time of the LAMP method is about 1 h, whereas 2–4 h is required for conventional PCR. The final LAMP products are mixtures of DNA with various lengths, showing many ladder-like pattern bands on agarose gel due to its characteristic structure.
Specificity of LAMP identification As shown in Figure 2, only when the DNA from V. vulnificus was present the LAMP reaction was positive and the amplification products were detected, showing a typical ladder-like pattern on gel electrophoresis which indicated that stem-loop DNA with inverted repeats was formed (Notomi et al., 2000), whereas LAMP reaction was negative for distilled water (negative control) and the other strains tested (Table 1), indicating that the LAMP reaction was highly specific to V. vulnificus strains.
Applicability of the LAMP assay
Sensitivity of LAMP identification
In order to evaluate the practicability of the LAMP assay in identification of V. vulnificus, a total of 50 strains of environmental bacteria grown on TCBS agar plates (Vibrio Selective Agar) were identified for the presence of V. vulnificus by the LAMP assay, and the results were compared with those of conventional PCR using two sets of primers (F3/B3 and gyr-vv1/gyr-vv2, respectively). DNA
As shown in Figure 3A, the detection limit of LAMP for V. vulnificus could be reached at 107 dilution level. With two sets of primers (F3/B3 and gyr-vv1/gyr-vv2, respectively), the detection limit of PCR could be reached at 106 dilution level (Figure 3B and D), whereas with primer set vvbfpP1/vvbfp-P2 the detection limit could be reached only at 104 dilution (Figure 3C). These results
Table 2.
Primers used in conventional PCR
Primer
Primer target gene
Sequence
Designers
F3 B3 vvbfp-P1 vvbfp-P2 gyr-vv1 gyr-vv2
Cytolysin Cytolysin Blue fluorescent protein Blue fluorescent protein Gyrase B subunit Gyrase B subunit
50 -TGGTTCGGTTAACGGCTG-30 50 -GCCATCAACATAGCGGCTAA-30 50 -GGATCACAAAATGAAAAAATTAGTCG-30 50 -CGTTGTTGACTAATACGTCC-30 50 -GTCCGCAGTGGAATCCTTCA-30 50 -TGGTTCTTACGGTTACGGCC-30
Present study Chen et al. (2005) Kumar et al. (2006)
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Figure 2. Identification specificity of the LAMP assay for V. vulnificus. Samples were electrophoresed on 2.0% agarose gels. LAMP was carried out with the different sources of DNA template from the 31 strains in the Vibrionacea family. (A) Lane M, 100 bp DNA ladder; Lane 1, distilled water (negative control); Lane 2, V. vulnificus 1.1758; Lanes 3–5, V. vulnificus; Lanes 6 and 7, V. cholerae; Lanes 8–11, V. parahaemolyticus; Lanes 12–15, V. alginolyticus; Lane 16, V. fluvialis; (B) Lane M, 100 bp DNA ladder; Lanes 17 and 18, V. fluvialis; Lanes 19 and 20, V. mimicus; Lanes 21 and 22, V. harveyi; Lanes 23 and 24, V. anguillarum; Lane 25, V. splendidus; Lane 26, V. nereis; Lane 27, V. pelagia; Lane 28, Aeromonas sobria; Lane 29, Aeromonas hydrophila; Lane 30, Pseudomonas aeruginosa; Lane 31, Pseudomonas fluorescens; Lane 32, Shewanella algae.
revealed that the LAMP assay was 10-fold sensitive than the PCR assay.
Applicability of the LAMP assay Our results showed that 16 out of 50 strains tested were positive by LAMP. The results of LAMP by visual inspection with diluted SYBR Green I agreed with those of gel electrophoresis. When SYBR Green I was added, all the amplification products that were positive examined by electrophoresis turned green, while all the reaction products that were negative analyzed by electrophoresis remained orange (Figure 4). Identical results were obtained through conventional PCR using both sets of primers (F3/B3 and gyr-vv1/gyr-vv2, respectively). No sample that was negative by the LAMP assay was positive by the PCR test, and vice versa.
Furthermore, it was found that the difference of DNA extraction methods (by kit or by boiling) did not affect the identification result of V. vulnificus by LAMP.
Discussion In this study, we used LAMP to identify V. vulnificus, a serious pathogen for humans and aquatic animals. Specificity of the LAMP primers was tested using V. vulnificus, V. cholerae, V. parahaemolyticus, V. alginolyticus, V. fluvialis, V. mimicus, V. harveyi, V. anguillarum, V. splendidus, V. nereis, V. pelagia, Aeromonas sobria, Aeromonas hydrophila, Pseudomonas aeruginosa, Pseudomonas fluorescens and Shewanella algae. The use of the four specific primers that recognize six distinct regions on the target cytolysin gene of
ARTICLE IN PRESS Sensitive and rapid identification of Vibrio vulnificus
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Figure 4. Detection of LAMP-amplified products by SYBR Green I. The solution turned green in the presence of LAMP amplification products or remained orange in the absence of amplicon. Tubes 1–4, negative samples; Tubes 5–8, positive samples.
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Figure 3. Sensitivity of V. vulnificus identification by LAMP and conventional PCR. Each sample was electrophoresed on a 2.0% agarose gel. (A) LAMP products; (B) conventional PCR products using primers F3 and B3. A band of 208 bp was seen with positive samples. (C) Conventional PCR products using primers vvbfp-P1and vvbfp-P2. A band of 251 bp was seen with positive samples. (D) Conventional PCR products using primers gyr-vv1and gyr-vv2. A band of 285 bp was seen with positive samples. Lane M, 100 bp DNA ladder; Lanes 1–8, amplification products using 10-fold serial dilutions of template DNA (101, 102, 103, 104, 105, 106, 107 and 108, respectively); Lane 9, distilled water.
V. vulnificus ensured high specificity of the template DNA amplification. Though one of the V. vulnificus strains listed in Table 1 had been proved to be a pathogenic strain (data not shown), the LAMP reaction was positive for all the V. vulnificus strains and negative for the other species tested (Table 1), demonstrating that these
primers were specific for identification of V. vulnificus regardless of pathogenic or non-pathogenic strains. The specificity of LAMP in identification of V. vulnificus was in conformity with other studies about specificity of LAMP reaction in bacterial detection (Iwamoto et al., 2003; Endo et al., 2004; Yeh et al., 2005). In addition to its higher specificity, the LAMP assay in identification of V. vulnificus showed sufficient sensitivity. The sensitivity of LAMP was found to be 10 times more than that of conventional PCR assay, which was in complete accordance with the results reported by Yano et al. (2007) and Wang et al. (2008) for detection of Escherichia coli and Salmonella. Horisaka et al. (2004) reported that LAMP was 100 times more sensitive than PCR in detecting Yersinia pseudotuberculosis. Identical sensitivity was obtained by Kamachi et al. (2006) for diagnosis of Bordetella pertussis infection. The greater sensitivity was due to the high amplification efficiency of the LAMP method. There is no time loss for thermal change under isothermal conditions in LAMP. Besides, it was known that PCR inhibitors in samples inhibited PCR amplification and reduced the sensitivity of PCR (Wilson, 1997), while inhibition reactions in LAMP were occurred less compared with PCR (Mori et al., 2001). Due to the high amplification efficiency of LAMP (Notomi et al., 2000; Mori et al., 2001) and the high binding affinity of SYBR Green I to DNA (Karlsen et al., 1995), the sensitivity of LAMP detection using SYBR Green I was very high. The result of visual inspection with SYBR Green I was found to match with gel electrophoresis. During visual inspection, only 1 min was required. Thereby, the visual judgment of LAMP amplification products by using SYBR Green I instead of gel electrophoresis made the LAMP test more rapid and simple. In addition, our results indicated that both DNA extraction methods were suitable for LAMP identification of V. vulnificus. Since the boiling method was simple and rapid, the time of identification of V. vulnificus was reduced.
ARTICLE IN PRESS 520 As discussed above, the LAMP assay in this study was extremely specific, sensitive and rapid for identification of V. vulnificus. Besides the advantages of reaction rapidity and sensitivity, LAMP does not require a high-precision thermal cycler in comparison with PCR and other nucleic acid-based techniques. The only device required for the LAMP reaction is a simple water bath or heating block, resulting in a low cost for the assay. Therefore, the LAMP assay is potential for field detection of V. vulnificus. This method could also be applied for the rapid identification of other Vibrio species.
Acknowledgments This study was funded by Projects under the Major State Basic Research Development Program of China, Grant 2006CB101803.
References Brauns LA, Hudson MC, Oliver JD. Use of the polymerase chain reaction in detection of culturable and nonculturable Vibrio vulnificus cells. Appl Environ Microbiol 1991;57:2651–5. Chen YC, Su JH, Chang WT, Chang JK. Specific primers for rapid and sensitive identification of Vibrio vulnificus. Chia-Nan Annu Bull 2005;31:19–27. Coleman SS, Melanson DM, Biosca EG, Oliver JD. Detection of Vibrio vulnificus biotypes 1 and 2 in eels and oysters by PCR amplification. Appl Environ Microbiol 1996;62:1378–82. Dalsgaard I, Høi L, Siebeling RJ, Dalsgaard A. Indolepositive Vibrio vulnificus isolated from disease outbreaks on a Danish eel farm. Dis Aquat Org 1998;35: 187–94. El-Matbouli M, Soliman H. Rapid diagnosis of Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease (PKD) in salmonid fish by a novel DNA amplification method, loop-mediated isothermal amplification (LAMP). Parasitol Res 2005;96: 277–84. Endo S, Komori T, Ricci G, Sano A, Yokoyama K, Ohori A, et al. Detection of gp43 of Paracoccidioides brasiliensis by the loopmediated isothermal amplification (LAMP) method. FEMS Microbiol Lett 2004;234:93–7. Farmer JJ. Revival of the name Vibrio vulnificus. Int J Syst Bacteriol 1980;30:656. FDA. Bacteriological analytical manual of food and drug administration, 7th ed. Arlington, VA: AOAC International; 2001. p. 111–140. Fukuda S, Takao S, Kuwayama M, Shimazu Y, Miyazaki K. Rapid detection of norovirus from fecal specimens by real-time reverse transcription-loop-mediated isothermal amplification assay. J Clin Microbiol 2006;44: 1376–81.
C.-H. Ren et al. Gadkar V, Rillig MC. Evaluation of loop-mediated isothermal amplification (LAMP) to rapidly detect arbuscular mycorrhizal fungi. Soil Biol Biochem 2008;40: 540–3. Gordon KV, Vickery MC, DePaola A, Staley C, Harwood VJ. Real-time PCR assays for quantification and differentiation of Vibrio vulnificus strains in oysters and water. Appl Environ Microbiol 2008;74:1704–9. Harwood VJ, Gandhi JP, Wright AC. Methods for isolation and confirmation of Vibrio vulnificus from oysters and environmental sources: a review. J Microbiol Methods 2004;59:301–16. Hill WE, Keasler SP, Trucksess MW, Feng P, Kaysner CA. Lampel KAPolymerase chain reaction identification of Vibrio vulnificus in artificially contaminated oysters. Appl Environ Microbiol 1991;57:707–11. Hong TC, Mai QL, Cuong DV, Parida M, Minekawa H, Notomi T, et al. Development and evaluation of a novel loop-mediated isothermal amplification of method for rapid detection of severe acute respiratory syndrome coronavirus. J Clin Microbiol 2004;42: 1956–61. Horisaka T, Taniguchi T, Honda E, Yokomizo Y, Hayashidani H. Sensitive and specific detection of Yersinia pseudotuberculosis by loop-mediated isothermal amplification. J Clin Microbiol 2004;42:5349–52. Iwamoto T, Sonobe T, Hayashi K. Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M. avium, and M. intracellulare in sputum samples. J Clin Microbiol 2003;41:2616–22. Kamachi K, Toyoizumi-Ajisaka T, Toda K, Soeung SC, Sarath S, Nareth Y, et al. Development and evaluation of a loop-mediated isothermal amplification method for rapid diagnosis of Bordetella pertussis infection. J Clin Microbiol 2006;44:1899–902. Karlsen F, Steen H, Nesland J. SYBR Green I DNA staining increases the detection sensitivity of viruses by polymerase chain reaction. J Virol Methods 1995;55: 153–6. Kuhara T, Yoshikawa T, Ihira M, Watanabe D, Tamada Y, Katano H, et al. Rapid detection of human herpesvirus 8 DNA using loop-mediated isothermal amplification. J Virol Methods 2007;144:79–85. Kumar HS, Parvathi A, Karunasagar I, Karunasagar I. A gyrB-based PCR for the detection of Vibrio vulnificus and its application for direct detection of this pathogen in oyster enrichment broths. Int J Food Microbiol 2006;111:216–20. Lee JY, Bang YB, Rhee JH, Choi SH. Two stage nested PCR effectiveness for direct detection of Vibrio vulnificus in natural samples. J Food Sci 1999;64:157–62. Mohran ZS, Arthur RR, Oyofo BA, Peruski LF, Wasfy MO, Ismail TF, et al. Differentiation of Campylobacter isolates on the basis of sensitivity to boiling in water as measured by PCR-detectable DNA. Appl Environ Microbiol 1998;64:363–5. Mori Y, Nagamine K, Tomita N, Notomi T. Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate
ARTICLE IN PRESS Sensitive and rapid identification of Vibrio vulnificus formation. Biochem Biophys Res Commun 2001;289: 150–4. Njiru ZK, Mikosza ASJ, Matovu E, Enyaru JCK, Ouma JO, Kibona SN, et al. African trypanosomiasis: sensitive and rapid detection of the sub-genus Trypanozoon by loop-mediated isothermal amplification (LAMP) of parasite DNA. Int J Parasitol 2008;38:589–99. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 2000;28: e63. Oliver JD, Warner RA, Cleland DR. Distribution of Vibrio vulnificus and other lactose fermenting Vibrios in the marine environment. Appl Environ Microbiol 1983;45: 985–98. Prida M, Posadas G, Inoue S, Hasebe F, Morita K. Realtime reverse transcription loop-mediated isothermal amplification for rapid detection of West Nile virus. J Clin Microbiol 2004;42:257–63. Soliman H, El-Matbouli M. Reverse transcription loopmediated isothermal amplification (RT-LAMP) for rapid detection of viral hemorrhagic septicaemia virus (VHS). Vet Microbiol 2006;114:205–13. Strom MS, Paranjpye RN. Epidemiology and pathogenesis of Vibrio vulnijkus. Microbes Infect 2000;2: 177–88. Sun ZF, Hu CQ, Ren CH, Shen Q. Sensitive and rapid detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimps by loop-mediated
521 isothermal amplification. J Virol Methods 2006;131: 41–6. Wang S, Levin RE. Rapid quantification of Vibrio vulnificus in clams (Protochaca staminea) using real-time PCR. Food Microbiol 2006;23:757–61. Wang L, Shi L, Alam MJ, Geng YH, Li L. Specific and rapid detection of foodborne Salmonella by loop-mediated isothermal amplification method. Food Res Int 2008; 41:69–74. Wilson IG. Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol 1997;63:3741–51. Yamamoto K, Wright AC, Kaper JB, Morris JG. The cytolysin gene of Vibrio vulnificus: sequence and relationship to the Vibrio cholerae E1 Tor hemolysin gene. Infect Immun 1990;58:2706–9. Yano A, Ishimaru R, Hujikata R. Rapid and sensitive detection of heat-labile I and heat-stable I enterotoxin genes of enterotoxigenic Escherichia coli by loopmediated isothermal amplification. J Microbiol Methods 2007;68:414–20. Yeh HY, Shoemaker CA, Klesius PH. Evaluation of a loopmediated isothermal amplification method for rapid detection of channel catfish Ictalurus punctatus important bacterial pathogen Edwardsiella ictaluri. J Microbiol Methods 2005;63:36–44. Yoshikawa T, Ihira M, Akimoto S, Usui C, Miyake F, Suga S, et al. Detection of human herpesvirus 7 DNA by loopmediated isothermal amplification. J Clin Microbiol 2004;42:1348–52.
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Sensitive and rapid identification of Vibrio vulnificus by loop-mediated isothermal amplification Chun-Hua Ren, Chao-Qun Hu, Peng Luo, Qing-Bai Wang The Key Laboratory of Applied Marine Biology of Guangdong Province and Chinese Academy of Sciences, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, PR China Received 6 March 2008; received in revised form 28 April 2008; accepted 18 May 2008
KEYWORDS Identification; Vibrio vulnificus; LAMP
Summary Vibrio vulnificus is a serious bacterial pathogen for humans and aquatic animals. We developed a rapid, sensitive and specific identification method for V. vulnificus using loop-mediated isothermal amplification (LAMP) technique. A set of primers, composed of two outer primers and two inner primers, was designed based on the cytolysin gene sequence of V. vulnificus. The LAMP reaction was processed in a heat block at 65 1C for 60 min. The amplification products were detected by visual inspection using SYBR Green I, as well as by electrophoresis on agarose gels. Our results showed that the LAMP reaction was highly specific to V. vulnificus. This method was 10-fold more sensitive than conventional PCR. In conclusion, the LAMP assay was extremely rapid, simple, cost-effective, sensitive and specific for the rapid identification of V. vulnificus. & 2008 Elsevier GmbH. All rights reserved.
Introduction Vibrio vulnificus is a Gram-negative pathogen that was first isolated by the US Centers for Disease Control (CDC) in 1964 (Strom and Paranjpye, 2000) and designated as V. vulnificus in 1980 (Farmer, 1980). This halophilic bacterium is widely distribCorresponding author. Tel.: +86 020 89023216;
fax: +86 020 89023218. E-mail address: [email protected] (C.-Q. Hu).
uted in estuarine and coastal waters around the world (Oliver et al., 1983). Associated with the consumption of raw or undercooked seafood (especially oysters), V. vulnificus opportunistically causes primary septicemia in susceptible individuals with a history of immunosuppression, hemochromatosis, cirrhosis or alcoholism. The mortality rate of primary septicemia caused by V. vulnificus is over 50% within days (Strom and Paranjpye, 2000). In addition to the primary septicemia, wound infections are incurred via contamination of
0944-5013/$ - see front matter & 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2008.05.002
ARTICLE IN PRESS Sensitive and rapid identification of Vibrio vulnificus preexisting wounds with seawater or seafood products where the organism is present, with a mortality rate of about 25% (Harwood et al., 2004). V. vulnificus is pathogenic not only for humans but also for aquatic animals. Under certain environmental conditions V. vulnificus can infect marine fish (especially eels) and shellfish, causing high mortality and serious economic losses (Dalsgaard et al., 1998). Thus, in order to decrease the occurrence of the bacterial infection to human beings and prevent the outbreak of aquaculture diseases caused by this pathogen, it is necessary to establish rapid and effective methods for the identification and detection of V. vulnificus. PCR and real-time PCR have been successfully used to detect V. vulnificus (Brauns et al., 1991; Hill et al., 1991; Coleman et al., 1996; Lee et al., 1999; Chen et al., 2005; Kumar et al., 2006; Wang and Levin, 2006; Gordon et al., 2008). PCR is a powerful tool for the detection of V. vulnificus because of its relatively rapid speed and sensitivity compared with conventional methods. However, an expensive thermal cycler is necessary for PCR amplification. A novel isothermal DNA amplification method termed loop-mediated isothermal amplification (LAMP) was invented by Notomi et al. (2000). This method relies on auto-cycling strand displacement DNA synthesis performed by the Bst DNA polymerase large fragment (with high-strand displacement activity) and a set of two specially designed inner and two outer primers that recognizes a total of six distinct sequences on the target DNA. An extremely large amount of DNA is produced by the cycling reaction in less than an hour under isothermal conditions ranging from 60 to 65 1C in a heat block or water bath. The final products are stem-loop DNA with several inverted repeats of the target and exhibit cauliflower-like structures with multiple loops. LAMP is highly specific, efficient and rapid (Notomi et al., 2000). In addition, the LAMP reaction products can be simply judged with eye inspection by the color change of a mixture with SYBR Green I (Iwamoto et al., 2003) or a white turbidity of magnesium pyrophosphate (Mori et al., 2001). LAMP has been successfully used to detect DNA viruses such as human herpesvirus (Yoshikawa et al., 2004; Kuhara et al., 2007) and infectious hypodermal and hematopoietic necrosis virus (Sun et al., 2006); pathogenic bacteria such as Mycobacterium tuberculosis (Iwamoto et al., 2003), Edwardsiella ictaluri (Yeh et al., 2005) and Salmonella (Wang et al., 2008); protozoa (El-Matbouli and Soliman, 2005; Njiru et al., 2008) and fungus (Endo et al., 2004; Gadkar and Rillig, 2008). RT-LAMP has been used for the detection of RNA
515 viruses such as SARS virus (Hong et al., 2004), West Nile virus (Prida et al., 2004), Norovirus (Fukuda et al., 2006) and septicemia virus (Soliman and El-Matbouli, 2006). In the present study, a rapid, sensitive and specific method for identification of V. vulnificus was developed based on LAMP technique.
Materials and methods Bacterial strains and DNA extraction The bacterial strains used in this study are listed in Table 1. All strains were identified by standard biochemical tests (FDA, 2001). Two methods were used for the preparation of DNA for LAMP. In method I, DNA was extracted by using the TaKaRa MiniBEST Bacterial Genomic DNA Extraction Kit (Takara Bio. Dalian Co. Ltd.) according to the manufacturer’s protocol. In method II, DNA was extracted using the boiling method as described by Mohran et al. (1998).
Design of primers for LAMP Based on the cytolysin gene sequence of V. vulnificus (M34670; Yamamoto et al., 1990), a
Table 1. Bacterial strains used to test the specificity of LAMP reaction in this study Species
Sourcesa
Number of strains tested
V. vulnificus (strain 1.1758) V. vulnificus V. cholerae V. parahaemolyticus V. alginolyticus V. fluvialis V. mimicus V. harveyi V. anguillarum V. splendidus V. nereis V. pelagia Aeromonas sobria Aeromonas hydrophila Pseudomonas aeruginosa Pseudomonas fluorescens Shewanella algae
IMCAS EI GDIM, EI SYSU, EI EI IMCAS, EI IMCAS, EI SCSFI, EI SHFU, SCSFI IMCAS IMCAS IMCAS SHFU SHFU SHFU SYSU EI
1 3 2 4 4 3 2 2 2 1 1 1 1 1 1 1 1
a
EI ¼ environmental isolate, GDIM ¼ Guangdong Institute of Microbiology, IMCAS ¼ Institute of Microbiology of Chinese Academy of Sciences, SCSFI ¼ South China Sea Fisheries Institute of Chinese Academy of Fishery Sciences, SHFU ¼ Shanghai Fisheries University, SYSU ¼ Sun Yat-Sen University.
ARTICLE IN PRESS 516 set of four primers composed of two outer primers which initiate strand displacement and two inner primers which structure ‘‘the loop’’ through the reaction was designed using Primer Explorer version 3 (https://primerexplorer.jp/lamp3.0.0/ index.html). The forward inner primer (FIP) consisted of the complementary sequence of F1 (22 nt), a T TTT spacer and F2 (18 nt). The backward inner primer (BIP) consisted of B1c (22 nt), a TTTT spacer and the complementary sequence of B2c (20 nt). The outer primers consisted of the forward outer primer F3 (18 nt) and the backward outer primer B3 (the complementary sequence of B3c, 20 nt). The positions of the LAMP primers used in this study are shown in Figure 1.
LAMP reaction The LAMP reaction was carried out according to the method described by Notomi et al. (2000). LAMP was performed in a 25 ml reaction volume containing the following reagents: 0.8 mM each of FIP and BIP, 0.2 mM each of F3 and B3, 400 mM dNTP,
C.-H. Ren et al. 1 M betaine (Sigma), 20 mM Tris–HCl (pH 8.8), 10 mM KCl, 10 m M (NH4)2SO4, 4 mM MgSO4, 0.1% Triton X-100, and 1 ml of target DNA or 1 ml distilled water (negative control). In a heat block, the reaction mixture was heated at 95 1C for 5 min, then cooled on ice. Finally, 8 U Bst DNA polymerase large fragment (New England Biolabs, Beverly, MA) was added. Subsequently, the mixture was incubated at 65 1C for 60 min and then heated at 80 1C for 3 min to terminate the reaction.
Detection of LAMP products After the reaction, 1 ml of 10-fold diluted original SYBR Green I (Molecular Probes Inc.) was added to 25 ml LAMP products. The solution turned green in the presence of LAMP amplification products, while it remained orange in the absence of amplicon. For further confirming the presence of amplicons, 3 ml of LAMP products were electrophoresed on a 2.0% agarose gel stained with GoldViewTM Nucleic Acid Stain and photographed using gel documentation system (Vilber Lourmat, France).
Figure 1. (A) Locations and nucleotide sequence of the cytolysin gene of V. vulnificus (M34670) used for designing the inner and outer primers. The DNA sequences of primer sites were underlined. (B) Names and sequences of LAMP primers used for specific amplification of V. vulnificus gene. B3, F1c and B2 represent complementary sequence to B3c, F1 and B2c, respectively.
ARTICLE IN PRESS Sensitive and rapid identification of Vibrio vulnificus
517 templates were extracted by method I and II. Two parallel experiments were made.
Specificity of LAMP identification To determine the specificity of LAMP method, LAMP was carried out with the different DNA templates (extracted by method I) from the 31 strains in the Vibrionacea family (Table 1) under the conditions described above. Each strain was examined at least twice.
Sensitivity of LAMP identification In order to test the sensitivity of the cytolysin gene primers in LAMP identification of V. vulnificus, DNA from V. vulnificus 1.1758 (extracted by method I) was serially 10-fold diluted. The diluted DNA templates were tested by LAMP and conventional PCR. In conventional PCR, three sets of primers (see Table 2) anchoring various target sequences were adopted. Using primers F3 and B3, PCR amplification was carried out with the TaKaRa TaqTM (Takara Bio. Dalian Co. Ltd.) according to the manufacturer’s protocol by adding 1 ml of DNA template and 20 pmol of each primer in a 25 ml total reaction volume. Amplification conditions consisted of an initial denaturation of 94 1C for 3 min and 40 cycles of 60 s at 95 1C, 60 s at 55 1C, 90 s at 72 1C and a final extension of 10 min at 72 1C in PTC-100 (Bio-Rad, USA). Using the other two sets of primers, reactions were performed according to the amplification conditions described by Chen et al. (2005) and Kumar et al. (2006), respectively. Distilled water was used as negative control for both PCR and LAMP. Two parallel experiments were made.
Results LAMP reaction The mechanism and expected reaction steps of LAMP were shown in the illustration finished by Notomi et al. (2000). The LAMP reaction includes three steps: (1) starting material producing step; (2) cycling amplification step; (3) elongation and recycling step. The reaction time of the LAMP method is about 1 h, whereas 2–4 h is required for conventional PCR. The final LAMP products are mixtures of DNA with various lengths, showing many ladder-like pattern bands on agarose gel due to its characteristic structure.
Specificity of LAMP identification As shown in Figure 2, only when the DNA from V. vulnificus was present the LAMP reaction was positive and the amplification products were detected, showing a typical ladder-like pattern on gel electrophoresis which indicated that stem-loop DNA with inverted repeats was formed (Notomi et al., 2000), whereas LAMP reaction was negative for distilled water (negative control) and the other strains tested (Table 1), indicating that the LAMP reaction was highly specific to V. vulnificus strains.
Applicability of the LAMP assay
Sensitivity of LAMP identification
In order to evaluate the practicability of the LAMP assay in identification of V. vulnificus, a total of 50 strains of environmental bacteria grown on TCBS agar plates (Vibrio Selective Agar) were identified for the presence of V. vulnificus by the LAMP assay, and the results were compared with those of conventional PCR using two sets of primers (F3/B3 and gyr-vv1/gyr-vv2, respectively). DNA
As shown in Figure 3A, the detection limit of LAMP for V. vulnificus could be reached at 107 dilution level. With two sets of primers (F3/B3 and gyr-vv1/gyr-vv2, respectively), the detection limit of PCR could be reached at 106 dilution level (Figure 3B and D), whereas with primer set vvbfpP1/vvbfp-P2 the detection limit could be reached only at 104 dilution (Figure 3C). These results
Table 2.
Primers used in conventional PCR
Primer
Primer target gene
Sequence
Designers
F3 B3 vvbfp-P1 vvbfp-P2 gyr-vv1 gyr-vv2
Cytolysin Cytolysin Blue fluorescent protein Blue fluorescent protein Gyrase B subunit Gyrase B subunit
50 -TGGTTCGGTTAACGGCTG-30 50 -GCCATCAACATAGCGGCTAA-30 50 -GGATCACAAAATGAAAAAATTAGTCG-30 50 -CGTTGTTGACTAATACGTCC-30 50 -GTCCGCAGTGGAATCCTTCA-30 50 -TGGTTCTTACGGTTACGGCC-30
Present study Chen et al. (2005) Kumar et al. (2006)
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Figure 2. Identification specificity of the LAMP assay for V. vulnificus. Samples were electrophoresed on 2.0% agarose gels. LAMP was carried out with the different sources of DNA template from the 31 strains in the Vibrionacea family. (A) Lane M, 100 bp DNA ladder; Lane 1, distilled water (negative control); Lane 2, V. vulnificus 1.1758; Lanes 3–5, V. vulnificus; Lanes 6 and 7, V. cholerae; Lanes 8–11, V. parahaemolyticus; Lanes 12–15, V. alginolyticus; Lane 16, V. fluvialis; (B) Lane M, 100 bp DNA ladder; Lanes 17 and 18, V. fluvialis; Lanes 19 and 20, V. mimicus; Lanes 21 and 22, V. harveyi; Lanes 23 and 24, V. anguillarum; Lane 25, V. splendidus; Lane 26, V. nereis; Lane 27, V. pelagia; Lane 28, Aeromonas sobria; Lane 29, Aeromonas hydrophila; Lane 30, Pseudomonas aeruginosa; Lane 31, Pseudomonas fluorescens; Lane 32, Shewanella algae.
revealed that the LAMP assay was 10-fold sensitive than the PCR assay.
Applicability of the LAMP assay Our results showed that 16 out of 50 strains tested were positive by LAMP. The results of LAMP by visual inspection with diluted SYBR Green I agreed with those of gel electrophoresis. When SYBR Green I was added, all the amplification products that were positive examined by electrophoresis turned green, while all the reaction products that were negative analyzed by electrophoresis remained orange (Figure 4). Identical results were obtained through conventional PCR using both sets of primers (F3/B3 and gyr-vv1/gyr-vv2, respectively). No sample that was negative by the LAMP assay was positive by the PCR test, and vice versa.
Furthermore, it was found that the difference of DNA extraction methods (by kit or by boiling) did not affect the identification result of V. vulnificus by LAMP.
Discussion In this study, we used LAMP to identify V. vulnificus, a serious pathogen for humans and aquatic animals. Specificity of the LAMP primers was tested using V. vulnificus, V. cholerae, V. parahaemolyticus, V. alginolyticus, V. fluvialis, V. mimicus, V. harveyi, V. anguillarum, V. splendidus, V. nereis, V. pelagia, Aeromonas sobria, Aeromonas hydrophila, Pseudomonas aeruginosa, Pseudomonas fluorescens and Shewanella algae. The use of the four specific primers that recognize six distinct regions on the target cytolysin gene of
ARTICLE IN PRESS Sensitive and rapid identification of Vibrio vulnificus
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Figure 4. Detection of LAMP-amplified products by SYBR Green I. The solution turned green in the presence of LAMP amplification products or remained orange in the absence of amplicon. Tubes 1–4, negative samples; Tubes 5–8, positive samples.
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Figure 3. Sensitivity of V. vulnificus identification by LAMP and conventional PCR. Each sample was electrophoresed on a 2.0% agarose gel. (A) LAMP products; (B) conventional PCR products using primers F3 and B3. A band of 208 bp was seen with positive samples. (C) Conventional PCR products using primers vvbfp-P1and vvbfp-P2. A band of 251 bp was seen with positive samples. (D) Conventional PCR products using primers gyr-vv1and gyr-vv2. A band of 285 bp was seen with positive samples. Lane M, 100 bp DNA ladder; Lanes 1–8, amplification products using 10-fold serial dilutions of template DNA (101, 102, 103, 104, 105, 106, 107 and 108, respectively); Lane 9, distilled water.
V. vulnificus ensured high specificity of the template DNA amplification. Though one of the V. vulnificus strains listed in Table 1 had been proved to be a pathogenic strain (data not shown), the LAMP reaction was positive for all the V. vulnificus strains and negative for the other species tested (Table 1), demonstrating that these
primers were specific for identification of V. vulnificus regardless of pathogenic or non-pathogenic strains. The specificity of LAMP in identification of V. vulnificus was in conformity with other studies about specificity of LAMP reaction in bacterial detection (Iwamoto et al., 2003; Endo et al., 2004; Yeh et al., 2005). In addition to its higher specificity, the LAMP assay in identification of V. vulnificus showed sufficient sensitivity. The sensitivity of LAMP was found to be 10 times more than that of conventional PCR assay, which was in complete accordance with the results reported by Yano et al. (2007) and Wang et al. (2008) for detection of Escherichia coli and Salmonella. Horisaka et al. (2004) reported that LAMP was 100 times more sensitive than PCR in detecting Yersinia pseudotuberculosis. Identical sensitivity was obtained by Kamachi et al. (2006) for diagnosis of Bordetella pertussis infection. The greater sensitivity was due to the high amplification efficiency of the LAMP method. There is no time loss for thermal change under isothermal conditions in LAMP. Besides, it was known that PCR inhibitors in samples inhibited PCR amplification and reduced the sensitivity of PCR (Wilson, 1997), while inhibition reactions in LAMP were occurred less compared with PCR (Mori et al., 2001). Due to the high amplification efficiency of LAMP (Notomi et al., 2000; Mori et al., 2001) and the high binding affinity of SYBR Green I to DNA (Karlsen et al., 1995), the sensitivity of LAMP detection using SYBR Green I was very high. The result of visual inspection with SYBR Green I was found to match with gel electrophoresis. During visual inspection, only 1 min was required. Thereby, the visual judgment of LAMP amplification products by using SYBR Green I instead of gel electrophoresis made the LAMP test more rapid and simple. In addition, our results indicated that both DNA extraction methods were suitable for LAMP identification of V. vulnificus. Since the boiling method was simple and rapid, the time of identification of V. vulnificus was reduced.
ARTICLE IN PRESS 520 As discussed above, the LAMP assay in this study was extremely specific, sensitive and rapid for identification of V. vulnificus. Besides the advantages of reaction rapidity and sensitivity, LAMP does not require a high-precision thermal cycler in comparison with PCR and other nucleic acid-based techniques. The only device required for the LAMP reaction is a simple water bath or heating block, resulting in a low cost for the assay. Therefore, the LAMP assay is potential for field detection of V. vulnificus. This method could also be applied for the rapid identification of other Vibrio species.
Acknowledgments This study was funded by Projects under the Major State Basic Research Development Program of China, Grant 2006CB101803.
References Brauns LA, Hudson MC, Oliver JD. Use of the polymerase chain reaction in detection of culturable and nonculturable Vibrio vulnificus cells. Appl Environ Microbiol 1991;57:2651–5. Chen YC, Su JH, Chang WT, Chang JK. Specific primers for rapid and sensitive identification of Vibrio vulnificus. Chia-Nan Annu Bull 2005;31:19–27. Coleman SS, Melanson DM, Biosca EG, Oliver JD. Detection of Vibrio vulnificus biotypes 1 and 2 in eels and oysters by PCR amplification. Appl Environ Microbiol 1996;62:1378–82. Dalsgaard I, Høi L, Siebeling RJ, Dalsgaard A. Indolepositive Vibrio vulnificus isolated from disease outbreaks on a Danish eel farm. Dis Aquat Org 1998;35: 187–94. El-Matbouli M, Soliman H. Rapid diagnosis of Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease (PKD) in salmonid fish by a novel DNA amplification method, loop-mediated isothermal amplification (LAMP). Parasitol Res 2005;96: 277–84. Endo S, Komori T, Ricci G, Sano A, Yokoyama K, Ohori A, et al. Detection of gp43 of Paracoccidioides brasiliensis by the loopmediated isothermal amplification (LAMP) method. FEMS Microbiol Lett 2004;234:93–7. Farmer JJ. Revival of the name Vibrio vulnificus. Int J Syst Bacteriol 1980;30:656. FDA. Bacteriological analytical manual of food and drug administration, 7th ed. Arlington, VA: AOAC International; 2001. p. 111–140. Fukuda S, Takao S, Kuwayama M, Shimazu Y, Miyazaki K. Rapid detection of norovirus from fecal specimens by real-time reverse transcription-loop-mediated isothermal amplification assay. J Clin Microbiol 2006;44: 1376–81.
C.-H. Ren et al. Gadkar V, Rillig MC. Evaluation of loop-mediated isothermal amplification (LAMP) to rapidly detect arbuscular mycorrhizal fungi. Soil Biol Biochem 2008;40: 540–3. Gordon KV, Vickery MC, DePaola A, Staley C, Harwood VJ. Real-time PCR assays for quantification and differentiation of Vibrio vulnificus strains in oysters and water. Appl Environ Microbiol 2008;74:1704–9. Harwood VJ, Gandhi JP, Wright AC. Methods for isolation and confirmation of Vibrio vulnificus from oysters and environmental sources: a review. J Microbiol Methods 2004;59:301–16. Hill WE, Keasler SP, Trucksess MW, Feng P, Kaysner CA. Lampel KAPolymerase chain reaction identification of Vibrio vulnificus in artificially contaminated oysters. Appl Environ Microbiol 1991;57:707–11. Hong TC, Mai QL, Cuong DV, Parida M, Minekawa H, Notomi T, et al. Development and evaluation of a novel loop-mediated isothermal amplification of method for rapid detection of severe acute respiratory syndrome coronavirus. J Clin Microbiol 2004;42: 1956–61. Horisaka T, Taniguchi T, Honda E, Yokomizo Y, Hayashidani H. Sensitive and specific detection of Yersinia pseudotuberculosis by loop-mediated isothermal amplification. J Clin Microbiol 2004;42:5349–52. Iwamoto T, Sonobe T, Hayashi K. Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M. avium, and M. intracellulare in sputum samples. J Clin Microbiol 2003;41:2616–22. Kamachi K, Toyoizumi-Ajisaka T, Toda K, Soeung SC, Sarath S, Nareth Y, et al. Development and evaluation of a loop-mediated isothermal amplification method for rapid diagnosis of Bordetella pertussis infection. J Clin Microbiol 2006;44:1899–902. Karlsen F, Steen H, Nesland J. SYBR Green I DNA staining increases the detection sensitivity of viruses by polymerase chain reaction. J Virol Methods 1995;55: 153–6. Kuhara T, Yoshikawa T, Ihira M, Watanabe D, Tamada Y, Katano H, et al. Rapid detection of human herpesvirus 8 DNA using loop-mediated isothermal amplification. J Virol Methods 2007;144:79–85. Kumar HS, Parvathi A, Karunasagar I, Karunasagar I. A gyrB-based PCR for the detection of Vibrio vulnificus and its application for direct detection of this pathogen in oyster enrichment broths. Int J Food Microbiol 2006;111:216–20. Lee JY, Bang YB, Rhee JH, Choi SH. Two stage nested PCR effectiveness for direct detection of Vibrio vulnificus in natural samples. J Food Sci 1999;64:157–62. Mohran ZS, Arthur RR, Oyofo BA, Peruski LF, Wasfy MO, Ismail TF, et al. Differentiation of Campylobacter isolates on the basis of sensitivity to boiling in water as measured by PCR-detectable DNA. Appl Environ Microbiol 1998;64:363–5. Mori Y, Nagamine K, Tomita N, Notomi T. Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate
ARTICLE IN PRESS Sensitive and rapid identification of Vibrio vulnificus formation. Biochem Biophys Res Commun 2001;289: 150–4. Njiru ZK, Mikosza ASJ, Matovu E, Enyaru JCK, Ouma JO, Kibona SN, et al. African trypanosomiasis: sensitive and rapid detection of the sub-genus Trypanozoon by loop-mediated isothermal amplification (LAMP) of parasite DNA. Int J Parasitol 2008;38:589–99. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 2000;28: e63. Oliver JD, Warner RA, Cleland DR. Distribution of Vibrio vulnificus and other lactose fermenting Vibrios in the marine environment. Appl Environ Microbiol 1983;45: 985–98. Prida M, Posadas G, Inoue S, Hasebe F, Morita K. Realtime reverse transcription loop-mediated isothermal amplification for rapid detection of West Nile virus. J Clin Microbiol 2004;42:257–63. Soliman H, El-Matbouli M. Reverse transcription loopmediated isothermal amplification (RT-LAMP) for rapid detection of viral hemorrhagic septicaemia virus (VHS). Vet Microbiol 2006;114:205–13. Strom MS, Paranjpye RN. Epidemiology and pathogenesis of Vibrio vulnijkus. Microbes Infect 2000;2: 177–88. Sun ZF, Hu CQ, Ren CH, Shen Q. Sensitive and rapid detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimps by loop-mediated
521 isothermal amplification. J Virol Methods 2006;131: 41–6. Wang S, Levin RE. Rapid quantification of Vibrio vulnificus in clams (Protochaca staminea) using real-time PCR. Food Microbiol 2006;23:757–61. Wang L, Shi L, Alam MJ, Geng YH, Li L. Specific and rapid detection of foodborne Salmonella by loop-mediated isothermal amplification method. Food Res Int 2008; 41:69–74. Wilson IG. Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol 1997;63:3741–51. Yamamoto K, Wright AC, Kaper JB, Morris JG. The cytolysin gene of Vibrio vulnificus: sequence and relationship to the Vibrio cholerae E1 Tor hemolysin gene. Infect Immun 1990;58:2706–9. Yano A, Ishimaru R, Hujikata R. Rapid and sensitive detection of heat-labile I and heat-stable I enterotoxin genes of enterotoxigenic Escherichia coli by loopmediated isothermal amplification. J Microbiol Methods 2007;68:414–20. Yeh HY, Shoemaker CA, Klesius PH. Evaluation of a loopmediated isothermal amplification method for rapid detection of channel catfish Ictalurus punctatus important bacterial pathogen Edwardsiella ictaluri. J Microbiol Methods 2005;63:36–44. Yoshikawa T, Ihira M, Akimoto S, Usui C, Miyake F, Suga S, et al. Detection of human herpesvirus 7 DNA by loopmediated isothermal amplification. J Clin Microbiol 2004;42:1348–52.