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.

Fig 1. Schematic illustration of the detection of VP_AHPND using DNA-functionalized gold nanoparticles as colorimetric hybridization probes to detect complementary LAMP amplicons.

(1) Positive reaction for VP_AHPND. (2) Negative reaction for VP_AHPND.

Fig 2. Optimization of the LAMP assay for detection of VP_AHPND at different temperatures (60, 63 and 65°C) using 100 ng of DNA extracted from VP_AHPND isolate 5HP in duplicate tests (Lane P).

Lane M: 2 log DNA marker, Lane N: 100 ng of DNA extracted from healthy P. monodon (negative control).

Fig 3. Comparison of absorption spectra of colloidal AuNP and of the DNA-labelled AuNP probe.

Fig 4. Optimization of AuNP hybridization for detection of VP_AHPND amplicons.

(A) Effect of variation in the volume ratio of the AuNP probe solution (5 nM) and the VP_AHPND LAMP amplicon solution from 1:9 to 9:1 (gold probe:Lamp amplicon) followed by addition of 50 mM MgSO₄ and showing that 5:5 was the best ratio. (B) Effect of variation in MgSO₄ concentration (between 3 and 667 mM in a fixed volume) in tubes with a gold probe: Lamp amplicon ratio of 5:5 and containing either (1) VP_AHPND LAMP amplicon or (2) WSSV LAMP amplicon (non-complementary DNA target negative control) or (3) LAMP premix without DNA target (no-target negative control) and showing that 50 mM gave the best result.

Fig 5. Sensitivity of the LAMP-AuNP assay for the detection of VP_AHPND using 10-fold serial dilution of DNA extracted from a culture of VP_AHPND isolate 5HP (10⁷−1 CFU/ml).

(A) Colorimetric results of LAMP followed by AuNP probe assay. (B) UV-visible spectrum analysis corresponding to the individual tubes in Fig 5A (measured after salt addition). (C) AGE results of LAMP reactions. Lane M: 2 log DNA marker and N: 100 ng of DNA extracted from normal shrimp.

Fig 6. Comparison of a sensitivity test carried out using total DNA template as in Fig 5 with traditional PCR methods.

(A) Nested PCR followed by AGE (AP4 method). (B) 1-step PCR followed by AGE (AP3 method). Lane M: 2 log DNA marker and N: 100 ng of DNA extracted from normal shrimp.

Fig 7. Comparison of results obtained using the VP_AHPND AuNP hybridization probe with LAMP amplicons from VP_AHPND (Lane 1) and other common pathogens (Lanes 2–10).

(A) The result of agarose gel electrophoresis (AGE) of LAMP products from various pathogens. Lane M: 2 log DNA marker; Lane N: normal shrimp DNA as negative control; Lanes 2–10: TB, Plasmodium (Malaria), WSSV, YHV, IMNV, IHHNV, TSV, LSNV and PemoNPV, respectively. (B) Colorimetric result for the same LAMP products as in Fig 7A measured after salt addition. (C) UV-visible spectra analysis corresponding to the individual tubes in Fig 7B measured after salt addition.