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.

One-Pot Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) for Detecting MERS-CoV

“…LAMP has especially attracted considerable interest since it is a potentially comparable to the popular detection methods such as conventional PCR and real-time PCR, in terms of specificity and sensitivity. For example, Salmonella spp., Escherichia coli, Staphylococcus aureus and other pathogenic bacteria genome markers have been used for the LAMP-based amplification assay (Aoi et al, 2006;Arunrut et al, 2016). LAMP protocol can also be applied for the detection of RNA viruses, including the SARS corona virus (Hong et al, 2004), influenza viruses (Poon et al, 2005), and other infectious respiratory viruses, by merging the reverse transcription steps into the LAMP processes (Parida et al, 2005).…”

Polyclonal antibody‐based farmer‐friendly flow‐through test for the detection of acute hepatopancreatic necrosis disease in shrimp

“…Later, polymerase chain reaction (PCR)‐based detection methods were developed using primers like AP1 and AP2 (Flegel & Lo, 2013), AP3 (Thitamadee et al, 2015) and AP4 (Dangtipa et al, 2015). In addition, a new PCR‐based commercially diagnostic kit such as IQ2000 (Chung, 2014), IQ Plus TM AHPND/EMS Kit (POCKIT) (Zorriehzahra & Banaederakhshan, 2015) and other improved methods such as duplex PCR method (Han, Tang, Pantoj, Whit, & Lightner, 2015; Han, Tang, Tran, et al, 2015), qualitative PCR method (qPCR) (Han, Tang, Pantoj, et al, 2015), loop‐mediated isothermal amplification (LAMP) (Arunrut et al, 2016; Koiwai, Tinwongger, Nozaki, Kondo, & Hirono, 2016; Kongrueng et al, 2015), isothermal recombinase polymerase amplification (AHPND‐RPA)(Liu et al, 2017) and PCR‐DNA chromatography method (Koiwai et al, 2018) were available. However, PCR by AP3 primers targeting PirA gene for diagnosis of AHPND is considered to be promising tool (Soto‐Rodriguez, Gomez‐Gil, Lozano‐Olvera, Betancourt‐Lozano, & Morales‐Covarrubias, 2015).…”

Development of a rapid immunochromatographic strip test for the detection of Vibrio parahaemolyticus toxin B that cause acute hepatopancreatic necrosis disease

“…PCR methods for the detection of VP AHPND using various primer pairs have been reported, including AP1 and AP2 (Flegel & Lo, ; Lee et al, ), AP3 (Sirikharin et al, ), AP4 (Dangtip et al, ), TUMSAT‐Vp1, Vp2 and Vp3 (Tinwongger et al, ) and five other primer pairs (Han, Tang, Lightner, et al, ). Other highly sensitive molecular‐based techniques for VP AHPND detection have also developed, including qPCR (Han, Tang, Pantoja, White, & Lightner, ) and LAMP (Arunrut et al, ; Kongrueng et al, ) assays.…”