Development of colorimetric triplex reverse transcription loop-mediated isothermal amplification for rapid and sensitive detection of shrimp yellow head virus (YHV)

Outbreaks of yellow head virus (YHV) have a significant impact on shrimp farming, leading to rapid shrimp mortality within 3 to 5 days of initial symptoms. Current disease identification methods are hindered by sophisticated workflows that require high-cost instruments and specialized expertise, posing a critical challenge on the accessibility of tests. We introduce a cost-effective detection method using triplex reverse transcription loop-mediated isothermal amplification (tRT-LAMP) by exploiting a pH-sensitive dye, xylenol orange (XO), for field-deployable colorimetric detection. To address the need for on-farm testing, a combination of extremely sensitive LAMP primer sets and a rapid RNA extraction strategy were utilized to achieve a detection limit of 100 fg for total RNA and at least 10 copies for in vitro RNA transcript, which is comparable to nested RT-PCR. By overcoming the complexity and cost barrier of the standard PCR-based method, our triplex LAMP-XO offers rapid, field-friendly YHV detection to mediate timely interventions and proper shrimp farm management during outbreaks.