With this present study, we aimed to fabricate a lateral flow immunoassay (LFIA) strip to monitor the risks and rapid detection potential impacts of aquatic disease, particularly AHPND. at control area indicated a negative result. The limit of detection of LFIA was found to be 125?ng, which could be visually detected by naked attention within 15?min. There?was?no cross-reactivity observed with VPnon-AHPND. Furthermore, the level of sensitivity and specificity of LFIA were 94.0% and 98.0%, respectively. The formulated test strip could be a game changer for early and?in situ?analysis of AHPND. Keywords: AHPND, Anti-PirAvp, Anti-PirBvp, Colloidal platinum, Lateral circulation immunoassay strip Intro Acute hepatopancreatic necrosis disease (AHPND) is definitely a newly growing disease of cultured shrimp. AHPND was first appeared in China in 2009 2009, Viet Nam in 2010 2010, Malaysia in 2010 2010, Thailand in 2011, Mexico in 2013, the Philippines in 2015, South American countries in 2016, Bangladesh and the United States of America in 2017, Taiwan in 2018, South Korea in 2019, and Okinawa Prefecture of Japan in 2020 (FAO 2020). It causes high mortality in two of the most commercially cultured shrimp varieties including and in many countries. Economic losses caused by this disease were estimated over USD 7 billion each year (FAO 2020). The agent was known to lead to AHPND was strain of insect-related binary toxin (Lee et al. 2015; Prachumwat et al. 2018). PirAvpCPirBvp are secreted toxins that affect the hepatopancreas. The suggested part of PirAvp is as a receptor-binding part and the PirBvp is the main player in inducing hepatopancreatic necrosis, hence losing one of these Rabbit Polyclonal to RHPN1 toxins reduces or even completely abrogate the disease indications (Sirikharin et al. 2015; Lin et al. 2019). Standard indications of affected shrimp by AHPND include atrophied hepatopancreas, bare gut, and belly. Histopathological examination shows the hepatopancreatic tubule epithelial cells sloughing into the lumen and hemocytic infiltration (Lightner 2012). AHPND is definitely a contagious disease for the shrimp cultivation, therefore early detection of disease is an unmet need. PCR-based multiplex or simultaneous amplification of the PirAvpCPirBvp toxin genes are presently used as a reliable diagnostic or detection tool for AHPND (Kumar et al. 2020; Mai-Hoang et al. 2021). However, many things make it difficult for this method to apply in farmed ponds in situ such as time-consuming, expensive tools required, and experienced staff (Kumar et al. 2020). Unlike PCR, loop-mediated isothermal amplification (Light) is an alternative to PCR assay for the detection of at 65?C in very short time and requires only a water 3-Indoleacetic acid bath. LAMP may lead to a false positive test result because of nonspecific amplicons products and cross contamination (Hu et al. 2021; Xu et al. 2022). Recently, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has a potential for diagnostics. CRISPR combines nucleic acid pre-amplification with CRISPRCCas enzyme for specific recognition of target DNA or RNA (Kaminski et al. 2021; Li et al. 2021). This technique enhances the sensitive and specific, but time-consuming and expensive due to its dependence on fluorescence products. In instances of limited resources, it is very inconvenient to use this method (Xu et al. 2022). Consequently, antibody-dependent methods, e.g. ELISA, Western blot, and immunochromatographic strip test, to quickly detect AHPND and offer easy-to-use on-site for farmer was developed recently (Wangman et al. 2019; Mai et al. 2020). In addition, this method can 3-Indoleacetic acid combine with LAMP, CRISPR/Cas to offer a simple visualization (Hu et al. 2021; Xu et al. 2022). With this present study, we targeted to fabricate a lateral circulation immunoassay (LFIA) strip to monitor the risks and rapid detection potential effects of aquatic disease, particularly AHPND. LFIA method based on the basic principle of sandwich format. Binding event at reaction lines happens between colloidal goldCpolyclonal antibody conjugates, PirAvp and/or PirBvp protein(s) from analyzed sample, and immobilized antibodies onto a nitrocellulose membrane. Consequently, LFIA can detect both of PirAvp and PirBvp proteins simultaneously. Materials and methods Materials Bacterial strains VPAHPND XN89 and VPnon-AHPND XN8 were given by National Center for Genetic Executive and Biotechnology (BIOTEC), Thailand. Reagents Colloidal platinum solution (CG-020), backing cards (MIBA-020C60?mm) were purchased from DCNovations, USA. Membrane and pads (sample pad-81132250, conjugate pad-8133-2250, nitrocellulose membrane (NCM)10547004, and absorbent pad8116-2250), were offered from Cytiva Existence Sciences, Sweden. Goat anti-rabbit IgG antibody (R5506) was purchased from Sigma, USA. Tradition media were purchased from BD Difco. Additional reagents were of analytical grade. Polyclonal antibody preparation The process of antibody production was 3-Indoleacetic acid published elsewhere (Duong et al. 2021; Nguyen-Phuoc et al. 2021). All animal experiments adopted the regulations of the Directive 2010/63/EU guideline authorized by The Animal Care and Use Committee of University or college of Technology, VNU-HCM in Ho Chi Minh City (honest code 12/18-0599-01). Briefly, anti-PirAvp and anti-PirBvp polyclonal antibodies were produced from rabbits inoculated with the purified recombinant PirAvp/PirBvp protein. Then, these antibodies were purified by Hitrap protein G HP.