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<p>Rapid and reliable on-site diagnostics, along with timely interventions, are vital for effective disease management. In this study, a multiplex LAMP-coupled lateral flow assay was developed for the simultaneous diagnosis of WSSV and EHP. The LAMP primers were designed to target the specific regions, envelope protein gene VP28 of WSSV and the spore wall protein gene (SWP) of EHP. To enable multiplex detection using lateral flow strips, EHP forward inner primer (FIP) and backward inner primer were labelled with biotin and FAM, at the 5’ end, respectively. While WSSV FIP and BIP were labelled with digoxigenin and FAM at the 5’ end. This multiplex LAMP was carried out using a simple dry bath and optimised at 65&#xa0;°C. Following LAMP assay, the reaction mix was applied to the lateral flow strip. This multiplex LAMP (mLAMP) coupled with lateral flow dipstick (LFD) demonstrated high sensitivity, detecting as few as 10 copies of WSSV and 10 copies of EHP. This assay was rapid and can detect the disease within 58&#xa0;min, including both amplification (55&#xa0;min) and lateral flow readout time (3&#xa0;min), excluding the DNA extraction step. This assay was observed to have 100% diagnostic sensitivity and specificity. This LAMP was highly specific and did not show any cross-amplification with other shrimp pathogens such as IMNV, IHHNV, <i>Vibrios</i> spp<i>.,</i> or with host genomic DNA of <i>Penaeus vannamei</i> and <i>Penaeus monodon</i>. This multiplex LAMP coupled with lateral flow offers a rapid, sensitive, specific point-of-care diagnostic assay which can be used for routine on-farm surveillance.</p>

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Point-of-care detection of shrimp pathogens WSSV and EHP using multiplex LAMP integrated lateral flow strips

  • 期刊论文

  • 发表日期：

    2026年1月13日

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  • T. Sathish Kumar,
  • S. Suvetha,
  • P. S. Meenakshi,
  • M. Makesh,
  • M. Shashi Shekhar,
  • Kuldeep K. lal

摘要

Rapid and reliable on-site diagnostics, along with timely interventions, are vital for effective disease management. In this study, a multiplex LAMP-coupled lateral flow assay was developed for the simultaneous diagnosis of WSSV and EHP. The LAMP primers were designed to target the specific regions, envelope protein gene VP28 of WSSV and the spore wall protein gene (SWP) of EHP. To enable multiplex detection using lateral flow strips, EHP forward inner primer (FIP) and backward inner primer were labelled with biotin and FAM, at the 5’ end, respectively. While WSSV FIP and BIP were labelled with digoxigenin and FAM at the 5’ end. This multiplex LAMP was carried out using a simple dry bath and optimised at 65 °C. Following LAMP assay, the reaction mix was applied to the lateral flow strip. This multiplex LAMP (mLAMP) coupled with lateral flow dipstick (LFD) demonstrated high sensitivity, detecting as few as 10 copies of WSSV and 10 copies of EHP. This assay was rapid and can detect the disease within 58 min, including both amplification (55 min) and lateral flow readout time (3 min), excluding the DNA extraction step. This assay was observed to have 100% diagnostic sensitivity and specificity. This LAMP was highly specific and did not show any cross-amplification with other shrimp pathogens such as IMNV, IHHNV, Vibrios spp., or with host genomic DNA of Penaeus vannamei and Penaeus monodon. This multiplex LAMP coupled with lateral flow offers a rapid, sensitive, specific point-of-care diagnostic assay which can be used for routine on-farm surveillance.