Abstract
Vibrio alginolyticus is an important opportunistic pathogen in humans and marine animals. Culture-based methods and the traditional polymerase chain reaction (PCR) cannot quantify the pathogen with sufficient sensitivity. Thus, reliable, rapid, and accurate detection and quantification methods are essential to prevent and control V. alginolyticus. We developed a real-time PCR assay using SYBR Green I targeting the groEL gene to detect and quantify V. alginolyticus. A species-specific primer was designed based on the groEL gene. Primer specificity was confirmed using 3 V. alginolyticus strains and 32 other Vibrio and non-Vibrio strains. Only the V. alginolyticus strain showed a positive result in the specificity test. A melting curve analysis showed a specific peak with a melting temperature of 85.80 ± 0.15 °C. A standard curve was produced to permit quantification of the target organism. Detection sensitivity was 0.14 pg of genomic DNA (equivalent to 10 cells per ml) for a pure culture of V. alginolyticus. V. alginolyticus was also quantified in artificially inoculated shellfish and living shrimp. The results indicated that SYBR Green I-based quantitative real-time polymerase chain reaction targeting the groEL gene enabled accurate, sensitive, and rapid quantitative detection of V. alginolyticus in seawater, shellfish, and shrimp.
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References
Arya M, Sherqill IS, Williamson M, Gommersall L, Arya N, Patel HR (2005) Basic principles of real-time quantitative PCR. Expert Rev Mol Diagn 5:209–219
Austin B, Stobie M, Robertson AW, Glass HG, Stark JR (1993) Vibrio alginolyticus: the cause of gill disease leading to progressive low-level mortalities among juvenile turbot, Scophthalmus maximus L., in a Scottish aquarium. J Fish Dis 16:277–280
Austin B, Alsina M, Austin DA, Blanch AR, Grimont F, Grimont PAD, Jofre J, Koblavi S, Larsen JL, Pedersen K, Tiainen T, Verdonck L, Swings J (1995) Identification and typing of Vibrio anguillarum: a comparison of different methods. Syst Appl Microbiol 18:285–302
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) (1987) Current protocols in molecular biology. Wiley, New York
Aznar R, Ludwig W, Schleifer KH (1993) Ribotyping and randomly amplified polymorphic DNA analysis of Vibrio vulnificus biotypes. Syst Appl Microbiol 16:303–309
Bader JA, Shoemaker CA, Klesius PH (2003) Rapid detection of columnaris disease in channel catfish (Ictalurus punctatus) with a new species-specific 16S rRNA gene-based PCR primer for Flavobacterium columnare. J Microbiol Methods 52:209–220
Balebona MC, Andreu MJ, Bordas MA, Zorrilla I, Moriñigo MA, Borrego JJ (1998) Pathogenicity of Vibrio alginolyticus from cultured gilt-head sea bream (Sparus aurata L.). Appl Environ Microbiol 64:4269–4275
Ben K-NA, Chaieb K, Bakhrouf A (2009) Investigation of several virulence properties among V. alginolyticus strains isolated from diseased cultured fish in Tunisia. Dis Aquat Org 86:21–28
Campanelli A, Sanchez-Politta S, Saurat JH (2008) Cutaneous ulceration after an octopus bite: infection due to Vibrio alginolyticus, an emerging pathogen. Ann Dermatol Venereol 135:225–227
Chizhikov V, Rasooly A, Chumakov K, Levy DD (2001) Microarray analysis microbial virulence factors. Appl Environ Microbial 67:3258–3263
Colorni A, Paperna I, Gordin H (1981) Bacterial infections in gilt-head sea bream Sparus aurata cultured at Elat. Aquaculture 23:257–267
George MR, John KR, Iyappan T, Jeyaseelan MJ (2005) Genetic heterogeneity among Vibrio alginolyticus isolated from shrimp farms by PCR fingerprinting. Lett Appl Microbiol 40:369–372
Gomathi RS, Vinothkumar R, Arunagiri K (2013) Isolation and identification vibrios from marine seafood samples. Int J Curr Microbiol App Sci 2:36–43
Gómez-León J, Villamil L, Lemos ML, Novoa B, Figueras A (2005) Isolation of Vibrio alginolyticus and Vibrio splendidus from aquacultured carpet shell clam (Ruditapes decussatus) larvae associated with mass mortalities. Appl Environ Microbiol 71:98–104
Hameed ASS, Parameswaren V, Musthaq SS, Sudhakarn R, Balasubramanian G, Yoganandhan K (2005) A simple PCR procedure to detect white spot syndrome virus (WSSV) of shrimp, Penaeus monodon (Fabricious). Aquacult Int 13:441–450
Hervio-Heath D, Colwell RR, Derrien A, Robert-Pillot A, Fournier JM, Pommepuy M (2002) Occurrence of pathogenic vibrios in coastal areas of France. J Appl Microbiol 92:1123–1135
Hossain MT, Kim EY, Kim YR, Kim DG, Kong IS (2011) Application of groEL gene for the species-specific detection of Vibrio parahaemolyticus by PCR. Lett Appl Microbial 54:67–72
Hossain MT, Kim EY, Kim YR, Kim DG, Kong IS (2012) Development of a groEL gene-based species-specific multiplex polymerase chain reaction assay for simultaneous detection of Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus. J Appl Microbial 114:448–456
Hossain MT, Kim YR, Kim EY, Lee JM, Kong IS (2013a) Detection of Vibrio cholera, and Vibrio vulnificus by duplex PCR specific to the groEL gene. Fish Sci 79:335–340
Hossain MT, Kim YO, Kong IS (2013b) Multiplex PCR for the detection and differentiation of Vibrio parahaemolyticus strains using the groEL, tdh and trh genes. Mol Cell Probes 27:171–175
Izumiya H, Matsumoto K, Yahiro S, Lee J, Morita M, Yamamoto S, Arakawa E, Ohnishi M (2011) Multiplex PCR assay for identification of three major pathogenic Vibrio spp., Vibrio cholera, Vibrio parahaemolyticus and Vibrio vulnificus. Mol Cell Probes 25:174–176
Janakiram P, Jayasree L, Madhavi R (2000) Bacterial abundance in modified extensive and semi-intensive shrimp culture ponds of Penaeus monodon. Indian J Mar Sci 29:319–323
Jing-jing Z, Chang C, Peng L, Chun-hua R, Xiao J, Zhe Z, Chao-qun H (2011) SYBR Green I-based real-time PCR targeting the rpox gene for sensitive and rapid detection of Vibrio alginolyticus. Mol Cell Probes 25:137–141
Kim DG, Ahn SH, Kim LH, Park KJ, Hong YK, Kong IS (2008) Application of the rpoS gene for species-specific detection of isolation of Vibrio vulnificus by real-time PCR. J Microbiol Biotechnol 18:1841–1847
Kim DG, Kim YR, Kim EY, Cho HM, Ahn SH, Kong IS (2010) Isolation of the groESL cluster from Vibrio anguillarum and PCR detection targeting groEL gene. Fish Sci 76:803–810
Lalitha P, Siti SMN, Lim KL, Lee SY, Nur HAR, Chan YY, Ismail A, Zainuddin ZF, Ravichandran M (2008) Analysis of lolB gene sequence and its use in the development of a PCR assay for the detection of Vibrio cholera. J Microbiol Methods 75:142–144
Lee D-Y, Moon S-Y, Lee S-O, Yang H-Y, Lee H-J, Lee MS (2008) Septic shock due to Vibrio alginolyticus in a cirrhotic patient: the first case in Korea. Yonsei Med 49:329–332
Liu CH, Cheng W, Hsu JP, Chen JC (2004) Vibrio alginolyticus infection in the white shrimp Litopenaeus vannamei confirmed by polymerase chain reaction and 16S rDNA sequencing. Dis Aquat Organ 61:169–174
Luo P, Hu CQ (2008) Vibrio alginolyticus gyrB sequence analysis and gyrB-targeted PCR identification in environmental isolates. Dis Aquat Organ 82:209–216
Malinan E, Kassinen A, Rinttila T, Palva A (2003) Comparison of real-time PCR with SYBR Green I or 5′-nuclease assays and dot-blot hybridization with rDNA-targeted oligonucleotide probes in quantification of selected fecal bacteria. Microbiology 149:269–277
Mokhtari W, Nsaibia S, Gharbi A, Aouni M (2013) Real-time PCR using SYBR Green for the detection of Shigella spp. in food and stool samples. Mol Cell Probes 27:53–59
Mustapha S, Mustapha EM, Nozha C (2013) Vibrio alginolyticus: an emerging pathogen of food borne diseases. Int J Sci Tech 2:302–309
Neogi SB, Chowdhury N, Asakura M, Hinenoya A, Halder S, Saidi SM, Kogure K, Lara RJ, Yamasaki S (2010) A highly sensitive and specific multiplex PCR assay for simultaneous detection of Vibrio cholera, Vibrio parahaemolyticus and Vibrio vulnificus. Lett Appl Microbiol 51:293–300
Nhung PH, Shah MM, Ohkusu K, Noda M, Hata H, Sun XS, Iihara H, Goto K (2007) The dnaJ gene as a novel phylogenetic marker for identification of Vibrio species. Syst Appl Microbiol 30:309–315
Nishibuchi M (2006) The biology of vibrios. In: Thompson FL, Austin B, Swings J (eds) Molecular identification. American Society for Microbiology, Washington, DC
Oksuz L, Gurler N (2013) Sepsis due to Vibrio alginolyticus isolated from catheter of young patient with hypercholesterolemy: the first case from Turkey. Clin Med Res 2:37–39
Pascual J, Macián MC, Arahal DR, Garay E, Pujalte MJ (2010) Multilocus sequence analysis of the central clade of the genus Vibrio by using the 16S rRNA, recA, pyrH, rpoD, gyrB, rctB and toxR genes. Int J Syst Evol Microbiol 60:154–165
Reina J, Fernandez-Baca V, Lopez A (1995) Acute gastroenteritis caused by Vibrio alginolyticus in an immune competent patient. Clin Infect Dis 21:1044–1045
Ren C, Hu C, Jiang X, Sun H, Zhao Z, Chen C, Luo P (2013) Distribution and pathogenic relationship of virulence associated genes among Vibrio alginolyticus from the mariculture systems. Mol Cell Probes 27:164–168
Taylor R, McDonald M, Russ G, Carson M, Lukaczynski E (1981) Vibrio alginolyticus peritonitis associated with ambulatory peritoneal dialysis. Br Med J (Clin Res Ed) 283:275
Thompson FL, Gevers D, Thompson CC, Dawyndt P, Naser S, Hoste B, Munn CB, Swings J (2005) Phylogeny and molecular identification of vibrios on the basis of multilocus sequence analysis. Appl Environ Microbiol 71:5107–5115
Tran L, Nunan L, Redman RM, Mohney LL, Pantoja CR, Fitzsimmons K, Lightner DV (2013) Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp. Dis Aquat Org 105:45–55
Vantarakis A, Komininou G, Venieri D, Papapetropoulou M (2000) Development of a multiplex PCR for detection of Salmonella spp., and Shigella spp. in mussels. Lett Appl Microbiol 31:105–109
Wang D, Fang Z, Xie C, Liu Y (2013) Construction of method for rapid detection of Vibrio parahaemolyticus using the quantitative real-time PCR based on the toxR gene. Adv J Food Sci Technol 5:1022–1030
Xie ZY, Hu CQ, Chen C, Zhang LP, Ren CH (2005) Investigation of seven Vibrio virulence genes among Vibrio alginolyticus and Vibrio parahaemolyticus strains from the coastal mariculture systems in Guangdong, China. Lett Appl Microbiol 41:202–207
Yushan H, Lei L, Weijia L, Xiaoguang C (2010) Sequence analysis of the groEL gene and its potential application in identification of pathogenic bacteria. Afr J Microbiol Res 4:1733–1741
Zhao Z, Chen C, Hu CQ, Ren CH, Zhao JJ, Zhang LP (2010) The type III secretion system of Vibrio alginolyticus induces rapid apoptosis, cell rounding and osmotic lysis of fish cells. Microbiol 156:2864–2872
Zhao Z, Zhang LP, Ren CH, Zhao JJ, Chen C, Ziang X (2011) Autophagy is induced by the type III secretion system of Vibrio alginolyticus in several mammalian cell lines. Arch Microbiol 193:53–61
Zhou S, Hou Z, Li N, Qin Q (2007) Development of a SYBR Green I real-time PCR for quantitative detection of Vibrio alginolyticus in seawater and seafood. J Appl Microbiol 103:1897–1906
Acknowledgments
This work was supported by a Research Grant of Pukyong National University (2014 year). The authors are thankful to Korea International Co-operation Agency (KOICA), Korea for sponsoring Raju Ahmed.
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Ahmed, R., Rafiquzaman, S.M., Hossain, M.T. et al. Species-specific detection of Vibrio alginolyticus in shellfish and shrimp by real-time PCR using the groEL gene. Aquacult Int 24, 157–170 (2016). https://doi.org/10.1007/s10499-015-9916-5
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DOI: https://doi.org/10.1007/s10499-015-9916-5