Improved Detection Sensitivity of Spring Viremia of Carp Virus by Substituting a Two-Step with a One-Step Nested Reverse Transcription Polymerase Chain Reaction Method

Spring viremia of carp (SVC) is a highly contagious disease that affects cyprinids, resulting in systemic hemorrhage, abdominal distension, exophthalmia, and high mortality in juveniles. This can lead to significant losses in the aquaculture industry. The World Organization for Animal Health (WOAH) recommends a two-step semi-nested reverse transcription polymerase chain reaction (RT-PCR) method for diagnosis. However, this method is labor-intensive, requires large reagent volumes, and is prone to carry-over contamination. Here, we evaluated the detection sensitivity of one-step semi-nested RT-PCR (combining RT and primary amplification in a single tube, followed by a second nested PCR step) against conventional two-step semi-nested RT-PCR. SVC virus (SVCV) subgroup Ia was tested using cell culture, RT-quantitative PCR, and one-step RT-PCR. The two-step semi-nested PCR method detected viral RNA up to a 10^-2 dilution, whereas one-step semi-nested RT-PCR detected it up to a 10^-5 dilution, showing a 1000-fold improvement in sensitivity. Moreover, detection rates increased from 84.2% with two-step semi-nested RT-PCR to 91.7% with one-step semi-nested RT-PCR in fish tissue samples. One-step semi-nested RT-PCR reduces processing time, minimizes handling steps, and contamination risk, and enhances analytical sensitivity. This supports its adoption as a practical, high-throughput diagnostic tool for SVCV and consideration for future WOAH guidelines.