title: Sensitive Visual Detection of AHPND Bacteria Using Loop-Mediated Isothermal Amplification Combined with DNA-Functionalized Gold Nanoparticles as Probes.
abstract: Acute hepatopancreatic necrosis disease (AHPND) is a component cause of early mortality syndrome (EMS) of shrimp. In 2013, the causative agent was found to be unique isolates of Vibrio parahaemolyticus (VPAHPND) that contained a 69 kbp plasmid (pAP1) carrying binary Pir-like toxin genes PirvpA and PirvpB. In Thailand, AHPND was first recognized in 2012, prior to knowledge of the causative agent, and it subsequently led to a precipitous drop in shrimp production. After VPAHPND was characterized, a major focus of the AHPND control strategy was to monitor broodstock shrimp and post larvae for freedom from VPAHPND by nucleic acid amplification methods, most of which required use of expensive and sophisticated equipment not readily available in a shrimp farm setting. Here, we describe a simpler but equally sensitive approach for detection of VPAHPND based on loop-mediated isothermal amplification (LAMP) combined with unaided visual reading of positive amplification products using a DNA-functionalized, ssDNA-labled nanogold probe (AuNP). The target for the special set of six LAMP primers used was the VPAHPND PirvpA gene. The LAMP reaction was carried out at 65°C for 45 min followed by addition of the red AuNP solution and further incubation at 65°C for 5 min, allowing any PirvpA gene amplicons present to hybridize with the probe. Hybridization protected the AuNP against aggregation, so that the solution color remained red upon subsequent salt addition (positive test result) while unprotected AuNP aggregated and underwent a color change from red to blue and eventually precipitated (negative result). The total assay time was approximately 50 min. The detection limit (100 CFU) was comparable to that of other commonly-used methods for nested PCR detection of VPAHPND and 100-times more sensitive than 1-step PCR detection methods (104 CFU) that used amplicon detection by electrophoresis or spectrophotometry. There was no cross reaction with DNA templates derived from non-AHPND bacteria commonly found in shrimp ponds (including other Vibrio species). The new method significantly reduced the time, difficulty and cost for molecular detection of VPAHPND in shrimp hatchery and farm settings.
authors.affiliation: Bioengineering and Sensing Technology Laboratory, BIOTEC, National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Phahonyothin Rd., Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand.
authors.forename: Narong
authors.initials: N
authors.lastname: Arunrut
authors.affiliation: Bioengineering and Sensing Technology Laboratory, BIOTEC, National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Phahonyothin Rd., Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand.
authors.forename: Jantana
authors.initials: J
authors.lastname: Kampeera
authors.affiliation: Bioengineering and Sensing Technology Laboratory, BIOTEC, National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Phahonyothin Rd., Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand.
authors.forename: Sarawut
authors.initials: S
authors.lastname: Sirithammajak
authors.affiliation: Center of Excellence for Shrimp Molecular Biology and Biotechnology (CENTEX Shrimp), Faculty of Science, Mahidol University, Rama VI road, Bangkok 10400, Thailand.
authors.forename: Piyachat
authors.initials: P
authors.lastname: Sanguanrut
authors.affiliation: Aquatic Animal Health Research Center, Charoen Pokphand Foods Pulic CO., LTD. Samutsakorn 74000, Thailand.
authors.forename: Porranee
authors.initials: P
authors.lastname: Proespraiwong
authors.affiliation: Bioengineering and Sensing Technology Laboratory, BIOTEC, National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Phahonyothin Rd., Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand.
authors.forename: Rungkarn
authors.initials: R
authors.lastname: Suebsing
authors.affiliation: Bioengineering and Sensing Technology Laboratory, BIOTEC, National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Phahonyothin Rd., Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand.
authors.forename: Wansika
authors.initials: W
authors.lastname: Kiatpathomchai
mesh_terms: D000818:Animals; D001412:Bacillus subtilis / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D006046:Gold / Q000737:chemistry; D057230:Limit of Detection; D053768:Metal Nanoparticles / Q000737:chemistry*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D000073718:Pancreatitis-Associated Proteins; D033561:Penaeidae / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D014736:Vibrio parahaemolyticus / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D017931:DNA Primers; D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human; D006046:Gold