A rapid method for simultaneously diagnosing four shrimp diseases using PCR ‐ DNA chromatography method

Shrimp farming accounted for more than half of world production of shrimp in 2014 (Food and Agricultural Organization, 2017a). In shrimp farming, viral, bacterial, microspordium and fungal diseases cause severe losses, so rapid methods for detecting them are needed. Polymerase chain reaction (PCR)-based detection method is widely used as screening for many pathogenic bacteria due to its convenience and sensitivity. Common viral diseases include white spot disease (WSD) and infectious hypodermal and haematopoietic necrosis (IHHN). A common bacterial disease is acute hepatopancreas necrosis disease (AHPND). Conventional PCR detection methods targeting the agents that cause these diseases are recommended (World Organisation for Animal Health, 2016). Recently, Enterocytozoon hepatopenaei (EHP) was identified as the microsporidium that causes growth retardation in farmed shrimp (Tangprasittipap et al., 2013). Conventional PCR has also been used to detect EHP infection (Tang et al., 2015; Tangprasittipap et al., 2013). Conventional PCR requires agarose/acrylamide gel electrophoresis and a device for visualizing the gel. Another method for detecting the PCR product is single-strand tag hybridization (STH) chromatographic printed array strip (PAS) method (Monden et al., 2014; Ohshiro, Miyagi, Tamaki, Mizuno, & Ezaki, 2016; Tian et al., 2014). In this method, multiplex PCR products can be visualized with high sensitivity within 15 min. Moreover, these chromatography strips are portable and ideal for field testing, as it eliminates the need for an electrophoresis equipment and preparation of gels that needs also require gel documentation equipment for viewing the PCR results. The method consists of a multiplex PCR in which multiple primer sets are tagged with a specific linker and biotin. After obtaining the PCR products, the PCR products then rise by capillary action in a tiny DNA chromatography strip to a point determined and hybridized by tag linker, stained with streptavidin-coated blue latex, which is visible to the naked eye (Figure 1). In this study, we developed a detection system for four shrimp diseases (WSD, IHHN, AHPND and EHP infections) using multiplex PCR and STH chromatographic PAS, named PCR-DNA chromatography. A total of 89 shrimp DNA samples of Penaeus monodon and Litopenaeus vannamei were collected in Thailand for this study. Total genomic DNA of these samples was extracted using taco Nucleic Acid Automatic Extraction System (GeneReach Biotechnology Corp, Taichung City, Taiwan). For each of the DNA samples, each disease was detected by conventional PCR using KAPA2G Fast Multiplex Mix (Kapa Biosystems, Wilmington, MA, USA) or KAPA 2G Fast Hot Start Ready Mix with dye (Kapa Biosystems) referring to previous reports: WSD (Lo et al., 1996), IHHN (Tang, Navarro, & Lightner, 2007), AHPND (Tinwongger et al., 2014) and EHP infections (Tangprasittipap et al., 2013). We obtained 30 samples that were positive for white spot virus (WSV) which is the causative virus of WSD, seven samples that were positive for infectious hypodermal and haematopoietic necrosis virus (IHHNV) which is the causative virus of IHHN, 14 samples that were positive for toxin gene of a virulent strain of Vibrio parahaemolyticus of AHPND, 19 samples that were positive for EHP, 14 samples that were double-positive for IHHNV and EHP and samples that were negative for each of these diseases. To develop PCR-DNA chromatography, we targeted WSD, IHHN, AHPND and EHP infections and 16S rRNA/tRNA/12S rRNA mitochondrial region (an internal control) using primers previously reported for WSD (Kiatpathomchai et al., 2005), IHHN (Tang et al., 2007), AHPND (Tinwongger et al., 2014), EHP infections (Tangprasittipap et al., 2013) and 16S rRNA/tRNA/12S rRNA (Pascoal, Barros-Vel azquez, Cepeda, Gallardo, & Calo-Mata, 2008) *These authors contributed equally to this work. Received: 9 July 2017 | Revised: 22 August 2017 | Accepted: 23 August 2017 DOI: 10.1111/jfd.12732