Chen, F. and C.A. Suttle. 1994. Development of a PCR-based technique for detecting and quantifying algal viruses in aquatic environments. Abstracts American Society Limnology and Oceanography, and Phycological Society American, Miami, FL, June 1994.
Recently, the DNA polymerase gene of a lytic virus (MPV-SP1), which infects the photosynthetic picoflagellate Micromonas pusilla, has been sequenced and compared with two viruses that infect a Chlorella-like alga that is found as an endosymbiont in Paramecium bursaria. Based on the deduced amino acid sequences a pair of degenerate primers were designed for amplifying algal-virus DNA using the polymerase-chain-reaction (PCR). The downstream primer is specific for algal viruses; whereas, the upstream primer is a universal primer for B-family DNA polymerase genes. For MPV-SP1 the DNA fragment flanked by this set of primers contains 681 nucleotides. A second nested downstream primer that is also universal for B-family DNA polymerase genes (amino acid sequence YGDTDS-F) was used to confirm that the correct sequence was amplified. This primer set was used to amplify DNA from nine clonal isolates of viruses infecting M. pusilla. Each of the amplified DNA pieces has been cloned and preliminary sequence analysis indicates that the nucleotide sequences are identical except for 1 or 2 nucleotide mismatches per 200 base pairs. This is despite the fact that the genomic DNA from each of these viruses shows a unique and very different restriction fragment banding pattern. Given the high degree of conservation within the DNA polymerase gene of Micromonas viruses we are optimistic that non-degenerate and highly specific PCR primers can be designed that will permit detection and quantification of Micromonas viruses in infected cells or field samples.