Abstract
In 5–40% of respiratory infections in children, the diagnostics remain negative, suggesting that the patients might be infected with a yet unknown pathogen. Virus discovery cDNA-AFLP (VIDISCA) is a virus discovery method based on recognition of restriction enzyme cleavage sites, ligation of adaptors and subsequent amplification by PCR. However, direct discovery of unknown pathogens in nasopharyngeal swabs is difficult due to the high concentration of ribosomal RNA (rRNA) that acts as competitor. In the current study we optimized VIDISCA by adjusting the reverse transcription enzymes and decreasing rRNA amplification in the reverse transcription, using hexamer oligonucleotides that do not anneal to rRNA. Residual cDNA synthesis on rRNA templates was further reduced with oligonucleotides that anneal to rRNA but can not be extended due to 3′-dideoxy-C6-modification. With these modifications >90% reduction of rRNA amplification was established. Further improvement of the VIDISCA sensitivity was obtained by high throughput sequencing (VIDISCA-454). Eighteen nasopharyngeal swabs were analysed, all containing known respiratory viruses. We could identify the proper virus in the majority of samples tested (11/18). The median load in the VIDISCA-454 positive samples was 7.2 E5 viral genome copies/ml (ranging from 1.4 E3–7.7 E6). Our results show that optimization of VIDISCA and subsequent high-throughput-sequencing enhances sensitivity drastically and provides the opportunity to perform virus discovery directly in patient material.
Highlights
Respiratory tract infection is the most common cause of hospitalization of children below the age of 5 years [1,2]
It is relatively easy to decrease the influence of background bacterial or human DNA and mRNA by centrifugation and DNase/RNase treatment, but ribosomal RNA is difficult to eliminate because the ribosomal proteins protect the rRNA inside the ribosomes
The method can be adjusted at several levels: 1) non-rRNA-annealing-primers can be used during reverse transcription 2) a choice for certain restriction enzymes can be made to diminish the chance of rRNA digestion and subsequent amplification, and 3) rRNA-blocking oligos can be used during the reverse transcription to halt cDNA synthesis on an rRNA template
Summary
Respiratory tract infection is the most common cause of hospitalization of children below the age of 5 years [1,2]. In 5– 40% of these hospitalizations no infectious agent can be identified but it is suspected that a viral infection is involved [3,4,5]. In these cases a yet unknown virus might be the cause of respiratory illness. At least 1 E6 genome copies/ml of a virus in a background that is low in competitor RNA/DNA are needed. These conditions are generally only met when virus culture supernatant is used. In particular RNA viruses are difficult to discover since in these cases a reverse transcription is needed, which will enable rRNA to act as competiting nucleic acid sequences
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