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Multiplex PCR using YeaD and 16S rRNA gene to identify major pathogens in vibriosis of Litopenaeus vannamei

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Abstract

The Vibrio species causing major diseases in Litopenaeus vannamei are Vibrio harveyi, Vibrio alginolyticus, and Vibrio parahaemolyticus. For multiplex PCR primers, YeaD was used to detect the three Vibrio species. Bioinformatic analysis such as MultiPLX and primer-BLAST was used to design stable and species-specific multiplex PCR primers. Multiplex PCR results showed clear band patterns with bands at 185 bp for V. alginolyticus, 396 bp for V. harveyi, 805 bp for V. arahaemolyticus, and 596 bp for common Vibrio species. The minimum concentration of DNA was measured by PCR; the value for V. alginolyticus was 0.1 ng, that of V. harveyi was 0.03 ng, and that of V. parahaemolyticus was 0.003 ng. Taken together, YeaD showed stability and specificity in identifying Vibrio species. Our multiplex PCR amplification method is an effective and inexpensive tool for identifying Vibrio species.

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References

  • Aftabuddin S, Roman WU, Hasan CK, Ahmed M, Rahman H, Siddique MAM (2017) First incidence of loose-shell syndrome disease in the giant tiger shrimp Penaeus monodon from the brackish water ponds in Bangladesh. J Appl Anim Res 46:1–8

    Google Scholar 

  • Aguirre-Guzmán G, Vázquez-Juárez R, Ascencio F (2001) Differences in the susceptibility of American white shrimp larval substages (Litopenaeus vannamei) to four Vibrio species. J Invertebr Pathol 78:215–219

    Article  PubMed  Google Scholar 

  • Ananda RR, Sridhar R, Balachandran C, Palanisammi A, Ramesh S, Nagarajan K (2017) Pathogenicity profile of Vibrio parahaemolyticus in farmed Pacific white shrimp, Penaeus vannamei. Fish Shellfish Immunol 67:368–381

    Article  CAS  Google Scholar 

  • Annam RA (2015) Analysis of engine test and emission test of seaweed biodiesel for sustainable energy. J Chem Pharm Res 7(2):755–760

    CAS  Google Scholar 

  • Biju VN, Gunalan B (2016) Prevalence of Vibrio infection in Penaeus (Litopenaeus) vannamei farms. Int J Sci Invent Today 5:485–493

    Google Scholar 

  • Castroverde CDM, Luis BBS, Monsalud RG, Hedreyda CT (2006) Differential detection of vibrios pathogenic to shrimp by multiplex PCR. J Gen Appl Microbiol 52:273–280

    Article  CAS  PubMed  Google Scholar 

  • Chittori S, Simanshu DK, Savithri HS, Murthy MRN (2007) Structure of the putative mutarotase YeaD from Salmonella typhimurium: structural comparison with galactose mutarotases. Biol Crystallogr 63:197–205

    Article  CAS  Google Scholar 

  • Danna KJ, Sack GH, Nathans D (1973) Studies of Simian virus 40 DNA. VII. A cleavage map of the SV40 genome. J Mol Biol 78:363–376

    Article  CAS  PubMed  Google Scholar 

  • FAO (2016) The state of world fisheries and aquaculture. Food and Agriculture Organization of the United Nations, Rome

    Google Scholar 

  • Goarant C, Reynaud Y, Ansquer D, de Decker S, Merien F (2007) Sequence polymorphism-based identification and quantification of Vibrio nigripulchritudo at the species and subspecies level targeting an emerging pathogen for cultured shrimp in New Caledonia. J Microbiol Methods 70:30–38

    Article  CAS  PubMed  Google Scholar 

  • Gomez-Gil B, Tron-Mayen L, Roque AF, Turnbull J, Inglis V, Guerra-Flores AL (1998) Species of Vibrio isolated from hepatopancreas, haemolymph and digestive tract of a population of healthy juvenile Penaeus vannamei. Aquaculture 163:1–9

    Article  Google Scholar 

  • Gomez-Gil B, Soto-Rodriguez S, Garcia-Gasca A, Roque A, Vazquez-Juaarez R, Thompson FL, Swings J (2004) Molecular identification of Vibrio harveyi-related isolates associated with diseased aquatic organisms. Microbiology 150:1769–1777

    Article  CAS  PubMed  Google Scholar 

  • Haldar S, Chatterjee S, Asakura M, Vijayakumaran M, Yamasaki S (2007) Isolation of Vibrio parahaemolyticus and Vibrio cholerae (Non-O1 and O139) from moribund shrimp (Penaeus monodon) and experimental challenge study against post larvae and juveniles. Ann Microbiol 57:55–60

    Article  CAS  Google Scholar 

  • Halder S, Neogi SB, Kogure K, Chatterjee S, Chowdhury N, Hinenoya A, Asakura M, Yamasaki S (2009) Development of a haemolysin gene-based multiplex PCR for simultaneous detection of Vibrio campbellii, Vibrio harveyi. and Vibrio parahaemolyticus. Lett Appl Microbiol 50:146–152

    Article  CAS  Google Scholar 

  • Heenatigala PPM, Fernando MUL (2016) Occurrence of bacteria species responsible for vibriosis in shrimp pond culture systems in Sri Lanka and assessment of the suitable control measures. Sri Lanka J Aquat Sci 21:1–17

    Article  Google Scholar 

  • 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 Microbiol 114:448–456

    Article  CAS  PubMed  Google Scholar 

  • Jayasree L, Janakiram P, Madhavi R (2006) Characterization of Vibrio spp. Associated with diseased shrimp from culture ponds of Andhra Pradesh. J World Aquac Soc 37:523–532

    Article  Google Scholar 

  • Jung SH, Choi HS, Do JW, Kim SM, Kwon MG, Seo JS, Jee YH, Kim SR, Cho YR, Kim JD, Park MA, Jee BY, Cho MY, Kim JW (2012) Monitoring of bacteria and parasites in cultured olive flounder, black rockfish, red sea bream and shrimp during summer period in Korea from 2007 to 2011. J Fish Pathol 25:231–241

    Article  Google Scholar 

  • Johannessen J-Ar, Olsen B, Olaisen J (1999) Aspects of innovation theory based on knowledge-management. Int J Inf Manage 19(2):121–139

    Article  Google Scholar 

  • Kaplinski L, Andreson R, Puurand T, Remm M (2004) MultiPLX: automatic grouping and evaluation of PCR primers. Bioinform Appl Note 21:1701–1702

    Article  CAS  Google Scholar 

  • Kinne RK (1993) The role of organic osmolytes in osmoregulation: from bacteria to mammals. J Exp Zool Suppl 265(4):346–355

    Article  CAS  Google Scholar 

  • Kim HJ, Ryu JO, Lee SY, Kim ES, Kim HY (2015) Multiplex PCR for detection of the Vibrio genus and five pathogenic Vibrio species with primer sets designed using comparative genomics. BioMed Cent Microbiol. https://doi.org/10.1186/s12866-015-0577-3

    Article  Google Scholar 

  • Kim JK, Lee JB, Huh YR, Jang HA, Kim CH, Yoo JW, Lee BL (2015) Burkholderia gut symbionts enhance the innate immunity of host Riptortus pedestris. Dev Comp Immunol 53(1):265–269

    Article  CAS  PubMed  Google Scholar 

  • Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Evolut Genet Anal 33:1870–1874

    CAS  Google Scholar 

  • Lan D, Lin B, Xiong X, Xiaonong Y, Li J (2016) Identification and characteristics analysis of toll-like receptors family genes in yak. Genes Genom 38:429–438

    Article  CAS  Google Scholar 

  • Lee TW, Delongchamp RR, Kim WK, Reis RJS (2016) Use of p-value plots to diagnose and remedy problems with statistical analysis of microarray data. Genes Genom 38:45–52

    Article  CAS  Google Scholar 

  • Lu X, Luan S, Cao B, Meng X, Sui J, Dai P, Luo K, Shi X, Hao D, Han G, Kong J (2017) Estimation of genetic parameters and genotype-by-environment interactions related to acute ammonia stress in Pacific white shrimp (Litopenaeus vannamei) juveniles at two different salinity levels. PLoS ONE. https://doi.org/10.1371/journal.pone.0173835

    Article  PubMed  PubMed Central  Google Scholar 

  • Mašková J (2011) Supercoiling (supercoiling of E. Coli). http://www.wikilectures.eu/w/File:Supercoiling.jpg#filelinks

  • Min JR, Na K, Chong HJ, Jeong HS (2015) Bactericidal efficacy of a monopersulfate compound against Vibrio harveyi and toxicity to Litopenaeus vannamei. Korean J Fish Aquat Sci 48:5 661–667

    Google Scholar 

  • Pinto AD, Ciccarese G, Tantillo G, Catalano D, Forte VT (2005) A collagenase-targeted multiplex PCR assay for identification of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus. J Food Prot 68:150–153

    Article  PubMed  Google Scholar 

  • Pourmozaffar S, Hajimoradloo A, Miandare HK (2017) Dietary effect of apple cider vinegar and propionic acid on immune related transcriptional responses and growth performance in white shrimp, Litopenaeus vannamei. Fish Shellfish Immunol 60:65–71

    Article  CAS  PubMed  Google Scholar 

  • Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    CAS  Google Scholar 

  • Sawabe T, Ogura Y, Matsumura Y, Feng G, Amin AKMR, Mino S, Nakagawa S, Sawabe T, Kumar R, Fukui Y, Satomi M, Matsushima R, Thompson FL, Gomez-Gil B, Christen R, Maruyama F, Kurokawa K, Hayashi T (2013) Updating the Vibrio clades defined by multilocus sequence phylogeny: proposal of eight new clades, and the description of Vibrio tritonius sp. nov. Front Microbiol 4:414, 1–14

    Article  Google Scholar 

  • Shanmugasundaram S, Mayavu P, Manikandarajan M, Suriya A, Anbarasu ER (2015) Isolation and identification of Vibrio sp. in the Hepatopancreas of cultured white pacific shrimp (Litopenaeus vannamei). Int Lett Nat Sci 46:52–59

    Google Scholar 

  • Sirirustananun N, Chen JC, Lin YC, Yeh ST, Liou CH, Chen LL, Sim SS, Chiew SL (2011) Dietary administration of a Gracilaria tenuistipitata extract enhances the immune response and resistance against Vibrio alginolyticus and white spot syndrome virus in the white shrimp Litopenaeus vannamei. Fish Shellfish Immunol 31:848–855

    Article  PubMed  Google Scholar 

  • Soto-Rodriguez SA, Gomez-Gil B, Lozano R (2010) ‘Bright-red’ syndrome in Pacific white shrimp Litopenaeus vannamei is caused by Vibrio harveyi. Dis Aquat Org 92:11–19

    Article  CAS  PubMed  Google Scholar 

  • Staroscik A (2004) Calculator for determining the number of copies of a template. http://www.uri.edu/research/gsc/resources/cndna.html

  • Stokke C, Waldminghaus T, Skarstad K (2011) Replication patterns and organization of replication forks in Vibrio cholerae. Microbiology 157:695–708

    Article  CAS  PubMed  Google Scholar 

  • Sun K, Hu YH, Zhang XH, Bai FF, Sun L (2009) Identification of vhhP2, a novel genetic marker of Vibrio harveyi, and its application in the quick detection of V. harveyi from animal specimens and environmental samples. J Appl Microbiol 107:1251–1257

    Article  CAS  PubMed  Google Scholar 

  • Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple; sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acid Res 22:4673–4680

    Article  CAS  PubMed  Google Scholar 

  • Vallone PM, Butler JM (2004) AutoDimer: a screening tool for primer-dimer and hairpin structures. Biotechniques 37:226–231

    Article  CAS  PubMed  Google Scholar 

  • Vandenberghe J, Verdonck L, Robles-Arozarena R, Rivera G, Bolland A, Balladares M, Gomez-Gil B, Calderon J, Sorgeloos P, Swings J (1999) Vibrios associated with Litopenaeus vannamei larvae, postlarvae, broodstock, and hatchery probionts. Appl Environ Microbiol 65:2592–2597

    CAS  PubMed  PubMed Central  Google Scholar 

  • Wang Z, Shi X, Sun L, Bai Y, Zhang D, Tang B (2017a) Evolution of mitochondrial energy metabolism genes associated with hydrothermal vent adaption of Alvinocaridid shrimps. Genes & Genomics 39(12):1367–1376

    Article  CAS  Google Scholar 

  • Wang Z, Wang B, Chen G, Lu Y, Jian J, Wu Z (2017b) Identification and comparative analysis of the pearl oyster Pinctada fucata hemocytes microRNAs in response to Vibrio alginolyticus infection. Genes & Genomics 39(10):1069–1081

    Article  CAS  Google Scholar 

  • Wei S, Zhao H, Xian Y, Hussain MA, Wu X (2014) Multiplex PCR assays for the detection of Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae with an internal amplification control. Diagn Microbiol Infect Dis 79:115–118

    Article  CAS  PubMed  Google Scholar 

  • Xue M, Wu L, He Y, Liang H, Wen C (2018) Biases during DNA extraction affect characterization of the microbiota associated with larvae of the Pacific white shrimp. Peer J 6:e5257

    Article  PubMed  Google Scholar 

  • Yatip P, Nitin CTD, Flegel TW, Soowannayan C (2018) Extract from the fermented soybean product Natto inhibits Vibrio biofilm formation and reduces shrimp mortality from Vibrio harveyi infection. Fish Shellfish Immunol 72:348–355

    Article  CAS  PubMed  Google Scholar 

  • Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL (2012) Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinform 13:134

    Article  CAS  Google Scholar 

  • You W, Qiu X, Zhang Y, Ma J, Gao Y, Zhang X, Niu L, Teng M (2010) Crystallization and preliminary Xray diffraction analysis of the putative aldose 1-epimerase YeaD from Escherichia coli. Struct Biol Cryst Commun 66:951–953

    Article  CAS  Google Scholar 

  • Zhao C, Fan S, Qiu L (2018) Identification of MicroRNAs and Their Target Genes Associated with Ovarian Development in Black Tiger Shrimp (Penaeus monodon) Using High-Throughput Sequencing. Scientific Reports 8(1):11602

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang Y, Han Z, Gao T, Shi H (2018) Genetic structure analysis of mantis shrimp Oratosquilla oratoria based on mitochondrial DNA control region sequence. Genes & Genomics 40(9):1001–1009

    Article  CAS  Google Scholar 

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Acknowledgements

This research was a part of the project titled “Omics based on fishery disease control technology development and industrialization (20150242),” funded by the Ministry of Oceans and Fisheries, Korea.

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Authors and Affiliations

  1. Department of Biological Sciences, College of Natural Sciences, Pusan National University, Busan, 46241, Republic of Korea

    Yeong-Jong Han, Ara Jo, So-Won Kim, Hee-Eun Lee & Heui-Soo Kim

  2. Institute of Systems Biology, Pusan National University, Busan, 46241, Republic of Korea

    Ara Jo, So-Won Kim, Hee-Eun Lee & Heui-Soo Kim

  3. Department of Aquatic Life Medicine, Pukyong National University, 599-1 Dae Yeon Dong Nam Ku, Pusan, 608-737, Republic of Korea

    Young Chul Kim & Hyun Do Jeong

  4. Department of Biochemistry, College of Korean Medicine, Dongeui University, Busan, 47227, Republic of Korea

    Yung Hyun Choi

  5. Department of Chemistry, College of Natural Sciences, Pusan National University, Busan, 46241, Republic of Korea

    Suhkmann Kim

  6. Departments of Parasitology and Genetics, College of Medicine, Kosin University, Busan, 49267, Republic of Korea

    Hee-Jae Cha

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  1. Yeong-Jong Han
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Han, YJ., Jo, A., Kim, SW. et al. Multiplex PCR using YeaD and 16S rRNA gene to identify major pathogens in vibriosis of Litopenaeus vannamei. Genes Genom 41, 35–42 (2019). https://doi.org/10.1007/s13258-018-0736-7

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