Multiplex-PCR protocol development for rapid screening of white spot syndrome virus (WSSV) in shrimp

Sharmin  Aktar; Md. Shahin  Parvez; H M Rakibul  Islam; Md. Nazmul  Ahsan

doi:10.17017/j.fish.230

Sharmin Aktar Department of Fisheries, Upazila Fisheries Office, Shalikha, Magura, Bangladesh https://orcid.org/0000-0002-5166-2800
Md. Shahin Parvez Fisheries and Marine Resource Technology Discipline, Khulna University, Khulna 9208, Bangladesh https://orcid.org/0000-0002-0153-6603
H M Rakibul Islam Bangladesh Fisheries Research Institute, Shrimp Research Station, Bagerhat, Bangladesh https://orcid.org/0000-0003-0067-4067
Md. Nazmul Ahsan Fisheries and Marine Resource Technology Discipline, Khulna University, Khulna 9208, Bangladesh https://orcid.org/0000-0003-0959-3247

DOI: https://doi.org/10.17017/j.fish.230

Keywords: faster, multiplex PCR, shrimp, simultaneous detection, single step, WSSV

Abstract

This study was aimed to develop a faster single step multiplex PCR protocol for the simultaneous detection of white spot syndrome virus (WSSV) with its host (i.e. shrimp) as internal positive control. To do so, four combinations of primer were tested (I. 16S rRNA+Lo F1R1; II. 16S rRNA+Lo F2R2; III. 16S rRNA+Lo F1R2; IV. 16S rRNA+Lo F2R1) which were selected based on two pairs of WSSV specific primer (Lo F1R1 and Lo F2R2) and one pair of shrimp specific primer (16S rRNA). DNA extracted from WSSV infected shrimp were amplified by PCR in a single tube using each of the primer combinations and the thermal cycling conditions as well as reagent compositions were optimized. All the primer combinations yielded their expected band sizes with stronger band resolution intensity that indicated the development of four multiplex PCR protocols. The developed multiplex protocols reduced the chance of cross contamination and these were found to be faster, single step and unique with less effort and resource use. Considering sensitivity and specificity, among the protocols, we suggested the protocols based on 16S rRNA+Lo F1R1 and/or 16S rRNA+Lo F2R2 primer combinations for rapid and routine screening of WSSV in shrimp PL, juvenile and adult.

References

Ayub F, Sarker MY, Alam MS (2008) Prevalence of white spot syndrome virus infection detected by one-step and nested PCR in selected tiger shrimp (Penaeus monodon) hatcheries. Aquaculture International 16: 405–415.

Chakraborty S, Ghosh U (2014) White spot syndrome virus (WSSV) in crustaceans: an overview of host-pathogen interaction. Journal of Marine Biology Oceanography 3(1): 1–17.

Chamberlain JS, Gibbs RA, Ranier JE, Nguyen PN, Caskey CT (1988) Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Research 16(23): 11141–11156.

Chou HY, Huang CY, Wang CH, Chiang HC, Lo CF (1995) Pathogenicity of a baculovirus infection causing white spot syndrome in cultured penaeid shrimp in Taiwan. Diseases of Aquatic Organisms 23: 165–173.

Dieu BT, Marks H, Siebenga JJ, Goldbach RW, Zuidema D, ... Vlak JM (2004) Molecular epidemiology of white spot syndrome virus within Vietnam. Journal of General Virology 85: 3607–3618.

Ganjoor M (2015) A short review on infectious viruses in cultural shrimps (Penaeidae family). Fisheries and Aquaculture Journal 6(3): 1–11.

Hossain MS, Chakraborty A, Joseph B, Otta SK, Karunasagar I, Karunasagar I (2001) Detection of new hosts for white spot syndrome virus of shrimp using nested polymerase chain reaction. Aquaculture 198: 1–11.

Hossain MS, Otta SK, Chakraborty A, Kumar HS, Karunasagar I, Karunasagar I (2004) Detection of WSSV in cultured shrimps, captured brooders, shrimp post-larvae and water samples in Bangladesh by PCR using different primers. Aquaculture 237: 59–71.

Islam MN, Hossain MA, Ahsan MN (2007) Development of a PCR based protocol for WSSV screening for major crustaceans inhabiting in cultured shrimp farm. International Journal of Sustainable Crop Production 2(4): 9–17.

Kanchanaphum P, Wongteerasupaya C, Sitidilokratana N, Boonsaeng V, Panyim S, ... Flegel TW (1998) Experimental transmission of white spot syndrome virus (WSSV) from crabs to shrimp Penaeus monodon. Disease of Aquatic Organism 34(1): 1–7.

Karim M, Sarwer RH, Brooks AC, Gregory R, Jahan KM, Belton B (2011) The incidence of suspected white spot syndrome virus in semi intensive and extensive shrimp farms in Bangladesh: implications for management. Aquaculture Research 43(9): 1357–1371.

Karunasagar I, Otta SK, Karunasagar I (1997) Histopathological and bacteriological study of white spot syndrome of Penaeus monodon along the west coast of India. Aquaculture 153: 9–13.

Kim CK, Kim PK, Sohn SG, Sim DS, Park MA, ... Jang KL (1998) Development of polymerase chain reaction (PCR) for the detection of baculovirus associated with white spot syndrome (WSBV) in penaeid shrimps. Journal of Fish Diseases 21: 11–17.

Lightner DV (1996) A handbook of shrimp pathology and diagnostic procedures for diseases of cultured penaeid shrimp. The World Aquaculture Society, Baton Rouge Louisiana, USA.

Lo CF, Ho CH, Peng SE, Chen CH, Hsu HC, ... Kou GH (1996) White spot syndrome baculovirus (WSBV) detected in cultured and captured shrimp, crabs and other arthropods. Diseases of Aquatic Organisms 27: 215–225.

Maeda M, Itami T, Furumoto A, Hennig O, Imamura T, ... Takahashi Y (1998) Detection of penaeid rod-shaped DNA virus (PRDV) in wild-caught shrimp and other crustaceans. Fish Pathology 33: 373–380.

Nunan LM, Lightner DV (2011) Optimized PCR assay for detection of white spot syndrome virus (WSSV). Journal of Virological Methods 171: 318–321.

OIE (2009) Diagnostic manual for aquatic animal diseases. 6th edition. Office International des Epizooties, Paris.

Otta SK, Shubha G, Joseph B, Chakraborty A, Karunasagar I, Karunasagar I (1999) Polymerase chain reaction (PCR) detection of white spot syndrome virus (WSSV) in cultured and wild crustaceans in India. Disease of Aquatic Organisms 38: 67–70.

Sahul Hameed AS, Murthi BLM, Rasheed M, Sathish S, Yoganandhan K, ... Jayaraman K (2002) An investigation of Artemia as a possible vector for white spot syndrome virus (WSSV) transmission to Penaeus indicus. Aquaculture 204(1–2): 1–10.

Sanchez-Martinez JG, Aguirre-Guzman G, Mejia-Ruiz H (2007) White spot syndrome virus in cultured shrimp: a review. Aquaculture Research 38: 1339–1354.

Sritunyalucksana K, Jiraporn S, McColl K, Nielsen L, Flegel TW (2006) Comparison of PCR testing methods for white spot syndrome virus (WSSV) infections in penaeid shrimp. Aquaculture 255: 95–104.

Strauss WM (1998) Preparation of genomic DNA from mammalian tissue. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, ... Struhl K (ed) Current protocols in molecular biology. John Wiley & Sons, New York.

Tang KFJ, Lightner DV (2000) Quantification of white spot syndrome virus DNA through a competitive polymerase chain reaction. Aquaculture 189: 11–21.

Vaseeharan B, Jayakumar R, Ramasamy P (2003) PCR-based detection of white spot syndrome virus in cultured and captured crustaceans in India. Letters in Applied Microbiology 37: 443–447.

Withyachumnarnkul B (1999) Results from black tiger shrimp Penaeus monodon culture ponds stocked with postlarvae PCR positive or -negative for white-spot syndrome virus (WSSV). Diseases of Aquatic Organisms 39: 21–27.

Yang B, Song XL, Huang J, Shi CY, Liu QH, Liu L (2006) A single step multiplex PCR for simultaneous detection of white spot syndrome virus and infectious hypodermal and haematopoietic necrosis virus in penaeid shrimp. Journal of Fish Diseases 29(5): 301–305.