Quantifying Birnavirus PiBV in Oysters

Since substantial amounts of the genes belong to a certain birnavirus genome were found only in the infected oysters with the comparative metatranscriptomics, we designated the virus as PiBV, as the putative causative agent of summer atrophy. To quantify the PiBV genome, we designed specific primers and a probe (Table 1) for RT-qPCR using primer3 (v. 0.4.0) (Untergasser et al., 2012 (link); Koressaar & Remm, 2007 (link)) based on the segment A sequence of PiBV. The total RNA was extracted from samples using TRIzol LS and dissolved in nuclease-free water. The PiBV genome was quantified using THUNDERBIRD Probe One-step qRT-PCR Kit (Toyobo, Osaka, Japan). The reaction mixture was prepared according to the manufacturer’s instructions with 2 μL of total RNA as a template, and LightCycler 96 (Roche, Basel, Switzerland) was used for the measurement. The RT-qPCR cycle was 50 °C for 10 min, 95 °C for 60 s, followed by 40 cycles of 95 °C for 15 s and 60 °C for 45 s. Standard curves were constructed using pCR2.1 TOPO vector (Thermo Fisher Scientific, Waltham, MA, USA) containing the segment A fragment (Table 1). The number of the PiBV gene in an individual specimen was estimated from the number of the virus gene detected, the amount of RNA extracted from each oyster, and the amount of RNA loaded to the reaction mixture.