Comparative study on nucleic acid amplification based detection methods for acute hepatopancreatic necrosis in Penaeus vannamei

Acute hepatopancreatic necrosis disease (AHPND), primarily caused by certain strain of Vibrio parahaemolyticus, refers to severe mortality rate up to 100 %, and posed a significant threat to the global shrimp aquaculture. Currently, various molecular diagnostic techniques for AHPND, such as, conventional polymerase chain reaction (PCR), nested PCR, quantitative real-time PCR (qPCR), loop-mediated Isothermal amplification (LAMP), and recombinase polymerase amplification (RPA), have been developed and continuously optimized. However, comparative studies on the performance of these methods are lacking. This study compared various published molecular diagnostic approaches, in terms of sensitivity, specificity, stability, and practical efficacy. A total of five kinds of methods were compared in this study. The sensitivity of PCR-1 and PCR-2 achieved 1.77 × 10² copies/μL and 1.77 × 10³ copies/μL, respectively; qPCR-1, qPCR-2, and qPCR-3 had limits of 1.77 × 10² copies/μL, 1.77 × 10¹ copies/μL, and 1.77 × 10³ copies/μL, respectively; nested PCR reached 1.77 × 10⁰ copies/μL. In isothermal amplification methods, LAMP-1 and LAMP-2 reached 1.77 × 10³ copies/μL, while RPA-1, RPA-2, and RPA-3 detected 1.77 × 10¹ copies/μL, 1.77 × 10¹ copies/μL, and 1.77 × 10³ copies/μL, respectively. All methods reliably detected the target gene, exhibiting robust specificity. Stability assessments showed minimal interference at low DNA concentrations, while at high concentrations, both conventional PCR and three RPA methods remained unaffected. In terms of detection accuracy, conventional PCR showed detection rates of 75 % for PCR-1 and 87.5 % for PCR-2; qPCR reached 100 % detection for qPCR-1, qPCR-2, and qPCR-3; nested PCR had an 87.5 % detection rate, LAMP achieved 75 % and 100 % for LAMP-1 and LAMP-2, respectively; and RPA reported 100 % detection for RPA-1 and RPA-2, with RPA-3 at 75 %. Notably, qPCR and nested PCR performed well. Generally, RPA-1 has good sensitivity (1.77 × 10¹ copies/μL), specificity, stability, and the detection rate of actual samples reaches 100 %. At the same time, RPA had the shortest reaction time and did not require expensive reaction equipment.