Multiplex reverse transcription quantitative PCR detection of a single-stranded RNA virus HcRNAV infecting the bloom-forming dinoflagellate Heterocapsa circularisquama

Abstract

Real-time PCR potentially allows for the accurate, rapid, and sensitive quantification of infectious agents in natural samples. Heterocapsa circularisquama single-stranded RNA virus (HcRNAV) causes a lytic infection of the bloom-forming dinoflagellate H. circularisquama. We established a multiplex reverse transcription quantitative PCR (mRT-qPCR) system for quantifying total HcRNAV and determined the applicability of this method to environmental samples. Two primer-probe sets targeting separate conserved regions in the HcRNAV genome enabled highly specific and sensitive detection of HcRNAV. The accuracy of the new system was evaluated using three typical HcRNAV clones by comparing the enumeration results of the mRT-qPCR with two conventional methods, transmission electron microscopy (TEM) and a most-probable-number (MPN) assay, which are used for direct and indirect virus quantification, respectively. Estimates obtained via the mRT-qPCR method were consistent with those obtained by TEM, indicating that it is useful for accurately quantifying Heterocapsa circularisquama single-stranded RNA virus (HcRNAV). Estimates obtained by MPN assay were lower than those obtained by mRT-qPCR. Whereas the TEM and MPN assays require 2-7 d, the entire mRT-qPCR method can be completed in only 2 h. Recovery efficiencies of HcRNAV clones from natural seawater were quite high with mRT-qPCR, which recovered almost all of original inoculation. We advocate that this new quantification method could be a powerful tool for monitoring of HcRNAV in vitro and natural seawater. Furthermore, total numbers and titer of HcRNAV by the mRT-qPCR together with MPN would lead to the collection of essential data to further understand the biology of H. circularisquama blooms and HcRNAV infection.

Quantitative real‐time polymerase chain reaction (PCR) and droplet digital PCR duplex assays for detecting Zostera marina DNA in coastal sediments

“…The detection and quantification of eDNA and RNA in water and sediment samples are performed using quantitative real‐time PCR (qPCR) (Takahara et al ; Thomsen et al ; Pilliod et al ; Turner et al ; Nakayama and Hamaguchi ). In recent years, a third‐generation PCR called droplet digital PCR (ddPCR) was developed (Hindson et al ) to provide the absolute quantification of a target DNA without using a standard reference curve, and this technique is more accurate and reliable than conventional qPCR.…”

Chemical Ecology of the Benthic Dinoflagellate Genus Ostreopsis: Review of Progress and Future Directions

“…To our knowledge, there has been no research on the interactions between Ostreopsis species and viruses while viruses have been proposed as natural agents to control HABs. This is the case of the virus HcRNAV, specific of the dinoflagellate Heterocapsa circularisquama (Nagasaki and Yamaguchi, 1997;Nagasaki et al, 2004;Nakayama and Hamaguchi, 2016).…”

Visualization of RNA virus infection in a marine protist with a universal biomarker

“…RNA dependent RNA polymerase (RdRp) is a gene shared by all orthornavirans and thus has potential as a molecular marker for RNA-virus infected virocells. Yet, although viral RdRp proteins have a deeply conserved polymerase function, their genes exhibit vast sequence divergence 30 , limiting gene-based detections to select lineages at a time 31 – 33 . Nevertheless, RdRp generates a biomarker for RNA virus infection that is universally conserved: long, double-stranded RNA (dsRNA).…”