Comparison of nucleic acid amplification based white spot syndrome virus (WSSV) detection methods in Penaeus vannamei

The white spot syndrome virus (WSSV) posed a serious threat to shrimp farming, which caused enormous economic losses due to its 100 % mortality rate and rapid spread. At present, a lot of molecular diagnostic methods are utilized to diagnose WSSV, including classic polymerase chain reaction (PCR), nested PCR, loop-mediated isothermal amplification (LAMP), quantitative real-time PCR (qPCR), and recombinant polymerase amplification (RPA). In this study, we compared 17 published molecular diagnostic methods and their primers/probes, including sensitivity, specificity, stability, and practicality. The sensitivities of PCR-1, PCR-2, PCR-3, and PCR-4 were 102 copies/mu L, 101 copies/mu L, 103 copies/mu L, and 104 copies/mu L, respectively. The sensitivity of nested PCR was 100 copies/mu L. The sensitivities of qPCR-1, qPCR-2, qPCR-3, qPCR-4, qPCR-5, and qPCR-6 had sensitivities of 102 copies/mu L, 100 copies/mu L, 100 copies/mu L, 100 copies/mu L, 102 copies/mu L, and 102 copies/mu L, respectively. Among the isothermal amplification methods, LAMP-1, LAMP-2 and LAMP-3 were 102 copies/mu L, 101 copies/mu L and 100 copies/mu L, respectively. While RPA-1, RPA-2, and RPA-3 were 103 copies/mu L, 101 copies/mu L, and 100 copies/mu L, respectively. All methods can specifically detect the target gene. Based on the stability assessment analysis, each detection method was essentially unaffected by low concentration background DNA, whereas the four PCR methods, nested PCR, qPCR-1, qPCR-5, qPCR-6, RPA-1, RPA-2, LAMP-2, and LAMP-3, were not affected by high background DNA concentrations. The detection accuracy was 62.5 % for PCR-3, PCR-4, qPCR-1, qPCR-6 and LAMP-1. The PCR-1 and PCR-2 had an accurate rate of 75 %. The detection rates of qPCR-3, qPCR-4, RPA-1, RPA-2 and RPA-3 were 87.5 %. The detection rates of nested-PCR, qPCR-2, qPCR-5, LAMP-2 and LAMP-3 were 100 %. In all the compared methods, the RPA-3 has a sensitivity of 100 copies/mu L, high specificity, and the actual detection rate of 87.5 %. RPA also requires no additional technicians or costly response equipment, and it offers the fastest reaction time. Therefore, RPA, as an emerging assay, seems to be more suitable for low-cost field pathogen detection.