5 ABSTRACT
The present study describes the optimization of Rolling Circle Amplification (RCA) assays to detect members of the family Anelloviridae from pig and human sera.
Standard RCA involves the prolonged elongation of random primers bound to a circular DNA template by Phi29 DNA polymerase. The RCA products, that are tandem repeat copies of the viral complete genome, were used for the Sequence Independent Single Primer Amplification (SISPA) method, based on the use of endonuclease restriction of target sequences. These products were separated on agarose gel and different length products were ligated to specific linkers followed by PCR amplification and sequencing. With this combined approach it was possible to detect Torque teno sus virus (TTSuV) 2 in swine.
Afterwards, a specific RCA technique was optimized. RCA was first carried out using short Anellovirus specific primers based on a highly conserved region among available anellovirus full-length genomes. Then, products from RCA were used as templates to amplify full-length genomes with Anellovirus specific PCR.
Amplification products were run on agarose gel and full-length genomes were isolated based on the expected size. With this novel Anello-RCA/PCR approach it was possible to detect TTSuV 1 and 2 in swine and Torque teno virus (TTV) in human serum samples, which tested previously negative by conventional PCR.
Based on these results, we demonstrated that combined RCA-SISPA previously set up for other species is applicable also for TTV DNA detection in swine, and Anello- RCA/PCR is a useful technique to discover members of the genus Iotatorquevirus and Alphatorquevirus. In comparison with the standard RCA combined with the SISPA
6 approach, the Anello-RCA/PCR assay is faster and cheaper, resulting in full-length genomic sequences of Anelloviruses. Furthermore, the technique could be applicable to other species whose TTVs have not been fully characterized as yet, increasing the probability to obtain new viral species belonging to the family Anelloviridae.
