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Ligation-mediated amplification for effective rapid determination of viral RNA sequences (RDV)
Shumpei Watanabe1, Tetsuya Mizutani, Kouji Sakai
1Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Background:
Emerging infectious diseases pose a significant risk to public health. Methods for rapid detection of pathogens are needed to effectively treat these diseases. Recently, we developed new methods for the rapid determination of viral RNA sequences, RDV ver1.0 and ver2.0. We demonstrated that these methods were able to simultaneously detect cDNA fragments of many different viruses without using sequence specific primers. However, some species of viruses, including the Yokose virus (YOKV), a flavivirus, could not be detected using the conventional procedures.
Objective:
The RDV method was further modified to reduce the candidate PCR primer sets.
Study Design:
Primer sets were reduced to 256 sets in the improved RDV ver3.0, and theoretically, all viral cDNA fragments ligated by two kinds of adaptors after digestion by two restriction enzymes could be amplified in the PCR step for direct sequencing.
Results:
We succeeded in obtaining 118 YOKV cDNA fragments of the 141 sequence fragments. The cDNA fragments covered diverse range of viral genome.
Conclusion:
We were able to reduce the combinations of PCR primer sets used in the RDV method. This RDV method ver3.0 has a potential to detect viral cDNA fragments of both known and unknown RNA viruses rapidly and conveniently.
