Detection of Acute Hepatopancreatic Necrosis Disease in Pacific White Shrimp (Penaeus vannamei) by Recombinase Polymerase Amplification
JIN Dong-Sheng1,2, LOU Yi-Rong2, WANG Yao-Hua3, DUAN Li-Jun2, LI Qi-Ang2, CHEN Chen3, YAN Mao-Cang3, CHEN Jiong1,2, ZHOU Qian-Jin1,2,*
1 State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Ningbo University, Ningbo 315211, China; 2 School of Marine Science, Ningbo University, Ningbo 315832, China; 3 Zhejiang Key Laboratory of Exploitation and Preservation of Coastal Bio-resource, Zhejiang Mariculture Research Institute, Wenzhou 325005, China
Abstract：Acute hepatopancreatic necrosis disease (AHPND) is a newly emerged bacterial disease in shrimps (Penaeidae), causing significant economic losses in the global shrimp farming industry. AHPND is primarily caused by Vibrio spp. carrying virulent pirA and pirB. In this study, specific primers and probes were designed and screened for pirA and pirB, and a recombinase polymerase amplification (RPA) method was developed. This RPA reaction ran in 39 ℃ for 20 min. The results revealed successful screening of primer/probe combinations for both pirA and pirB genes. Through a series of tests using the RPA method, these primer/probe combinations demonstrated specific detection of the target virulence genes, without amplification of genomic DNA from 13 microorganisms, such as Vibrio alginolyticus, V. parahaemolyticus, and Enterocytozoon hepatomegaly. The detection limit of the RPA method was 0.01 and 0.1 fg/μL of recombinant plasmid pirAB-pMD19, when the primers and probes targeting pirA or pirB were used. The primer concentrations were optimized at 0.4 µmol/L each, while the optimal concentration for the probe was 0.16 µmol/L. The RPA method was used to detect suspicious tissues of Penaeus vannamei using optimized sets of primers and probe. The results showed that the RPA method could accurately detect pirA and pirB from shrimp tissues, which was consistent with the PCR method recommended by the World Organization for Animal Health (WOAH). In summary, this PRA methods targeting pirA and pirB respectively could rapid and accurately detect ANPND-causing bacteria with easy operation and short time-consumption, making it a promising supplementary tool for routine monitoring and rapid detection of AHPND.
[1] Arunrut N, Kampeera J, Sirithammajak S, et al.2016. Sensitive visual detection of AHPND bacteria using loop-mediated isothermal amplification combined with DNA-functionalized gold nanoparticles as probes[J]. PLOS ONE, 11(3): e0151769. [2] Dangtip S, Sirikharin R, Sanguanrut P, et al.2015. AP4 method for two-tube nested PCR detection of AHPND isolates of Vibrio parahaemolyticus[J]. Aquaculture Reports, 2: 158-162. [3] De Schryver P, Defoirdt T, Sorgeloos P.2014. Early mortality syndrome outbreaks: A microbial management issue in shrimp farming?[J]. PLOS Pathogens, 10(4): e1003919. [4] Dong X, Chen J, Song J, et al.2019b. Evidence of the horizontal transfer of pVA1-type plasmid from AHPND-causing V. campbellii to non-AHPND V. owensii[J]. Aquaculture, 503: 396-402. [5] Dong X, Song J, Chen J, et al.2019a. Conjugative transfer of the pva1-type plasmid carrying the pirABvp genes results in the formation of new AHPND-causing Vibrio[J]. Frontiers in Cellular and Infection Microbiology, 9: 195. [6] Dong X, Wang H, Xie G, et al.2017. An isolate of Vibrio campbellii carrying the pir(VP) gene causes acute hepatopancreatic necrosis disease[J]. Emerging Microbes & Infections, 6(1): e2. [7] Du Y, Xu W, Wu T, et al.2022. Enhancement of growth, survival, immunity and disease resistance in Litopenaeus vannamei, by the probiotic, Lactobacillus plantarum Ep-M17[J]. Fish & Shellfish Immunology, 129: 36-51. [8] FAO.2013. Report of the FAO/MARD technical workshop on early mortality syndrome (EMS) or acute hepatopancreatic necrosis syndrome (AHPNS) of cultured shrimp (Under TCP/VIE/3304)[R]. FAO Fisheries and Aquaculture report no, 1053: 54. [9] FAO.2023. Fishery and aquaculture statistics - Yearbook 2020[M]. FAO Fisheries and Aquaculture Division. Rome, pp. 119-122. [10] Han J E, Tang K F J, Pantoja C R, et al.2015. qPCR assay for detecting and quantifying a virulence plasmid in acute hepatopancreatic necrosis disease (AHPND) due to pathogenic Vibrio parahaemolyticus[J]. Aquaculture, 442: 12-15. [11] Hong X, Lu L, Xu D.2016. Progress in research on acute hepatopancreatic necrosis disease (AHPND)[J]. Aquaculture International, 24(2): 577-593. [12] Jiang H J, Rong T A N, Min J I N, et al.2022. Visual detection of Vibrio parahaemolyticus using combined CRISPR/Cas12a and recombinase polymerase amplification[J]. Biomedical and Environmental Sciences, 35(6): 518-527. [13] Joshi J, Srisala J, Truong V H, et al.2014. Variation in Vibrio parahaemolyticus isolates from a single Thai shrimp farm experiencing an outbreak of acute hepatopancreatic necrosis disease (AHPND)[J]. Aquaculture, 428-429: 297-302. [14] Kondo H, Van Phan T, Dang Lua T, et al.2015. Draft genome sequence of non-Vibrio parahaemolyticus acute hepatopancreatic necrosis disease strain KC13.17.5, isolated from diseased shrimp in Vietnam[J]. Genome Announcements, 3(5): e00978-15. [15] Kumar R, Ng T H, Wang H-C.2020. Acute hepatopancreatic necrosis disease in penaeid shrimp[J]. Reviews in Aquaculture, 12(3): 1867-1880. [16] Lai H-C, Ng T H, Ando M, et al.2015. Pathogenesis of acute hepatopancreatic necrosis disease (AHPND) in shrimp[J]. Fish & Shellfish Immunology, 47(2): 1006-1014. [17] Lee C-T, Chen I T, Yang Y-T, et al.2015. The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin[J]. Proceedings of the National Academy of Sciences of the USA, 112(34): 10798-10803. [18] Li C, Lin N, Feng Z, et al.2022. CRISPR/Cas12a based rapid molecular detection of acute hepatopancreatic necrosis disease in shrimp[J]. Frontiers in Veterinary Science, 8: 819681. [19] Lightner D V, Redman R M, Pantoja C R, et al.2012. Early mortality syndrome affects shrimp in Asia[J]. Global Aquaculture Advocate, 15: 40. [20] Liu L, Jiang L, Yu Y, et al.2017. Rapid diagnosis of Vibrio owensii responsible for shrimp acute hepatopancreatic necrosis disease with isothermal recombinase polymerase amplification assay[J]. Molecular and Cellular Probes, 33: 4-7. [21] Liu L, Xiao J, Zhang M, et al.2018. A Vibrio owensii strain as the causative agent of AHPND in cultured shrimp, Litopenaeus vannamei[J]. Journal of Invertebrate Pathology, 153: 156-164. [22] Phiwsaiya K, Charoensapsri W, Taengphu S, et al.2017. A natural Vibrio parahaemolyticus ΔpirAVppirBVp+ mutant kills shrimp but produces neither PirVp toxins nor acute hepatopancreatic necrosis disease lesions[J]. Applied and Environmental Microbiology, 83(16): e00680-00617. [23] Stedtfeld R D, Tourlousse D M, Seyrig G, et al.2012. Gene-Z: A device for point of care genetic testing using a smartphone[J]. Lab on a Chip, 12(8): 1454-1462. [24] Theethakaew C, Nakamura S, Motooka D, et al.2017. Plasmid dynamics in Vibrio parahaemolyticus strains related to shrimp acute hepatopancreatic necrosis syndrome (AHPNS)[J]. Infection, Genetics and Evolution, 51: 211-218. [25] Tinwongger S, Proespraiwong P, Thawonsuwan J, et al.2014. Development of PCR diagnosis for shrimp acute hepatopancreatic necrosis disease (AHPND) strain of Vibrio parahaemolyticus[J]. Fish Pathology, 49(4): 159-164. [26] Wang P, Yang L, Guo B, et al.2023a. Highly sensitive detection of white spot syndrome virus with an RPA-CRISPR combined one-pot method[J]. Aquaculture, 567, 739296. [27] Wang P, Zhang X, Shen H, et al.2023b. A one-pot RPA-CRISPR detection method for point-of-care testing of Enterocytozoon hepatopenaei infection in shrimp[J]. Sensors and Actuators B: Chemical, 374: 132853. [28] Wood S A, Pochon X, Laroche O, et al.2019. A comparison of droplet digital polymerase chain reaction (PCR), quantitative PCR and metabarcoding for species-specific detection in environmental DNA[J]. Molecular Ecology Resources, 19(6): 1407-1419. [29] Xia X, Yu Y, Hu L, et al.2015. Rapid detection of Infectious hypodermal and hematopoietic necrosis virus (IHHNV) by real-time, isothermal recombinase polymerase amplification assay[J]. Archives of Virology, 160(4): 987-994. [30] Xia X, Yu Y, Weidmann M, et al.2014. Rapid detection of shrimp White spot syndrome virus by real time, isothermal recombinase polymerase amplification assay[J]. PLOS ONE, 9(8): e104667. [31] Zhou S, Wang M, Liu M, et al.2020. Rapid detection of Enterocytozoon hepatopenaei in shrimp through an isothermal recombinase polymerase amplification assay[J]. Aquaculture, 521: 734987. [32] Zorriehzahra M, Banaederakhshan R.2015. Early mortality syndrome (EMS) as new emerging threat in shrimp industry[J]. Advances in Animal and Veterinary Sciences, 3(2s): 64-72. [33] Zwetlana A, Tanwer P, Evans D, et al.2023. An Indian perspective on the infection and diagnostic landscape of shrimp aquaculture[J]. ACS Agricultural Science & Technology, 3(4): 305-317.
