Effects of different homogenization and nucleic acid co-extraction methods on viral RNA and DNA extraction from sputum

Objective To evaluate the effects of different homogenization and nucleic acid coextraction methods on viral RNA and DNA co-extraction from sputum. Methods Sputum was liquefied by 0.1% Dithiothreitol (DTT) (group B) and 4% NaOH (group C) respectively, comparing with saline solutions (group A). TIANamp Virus DNA/RNA Kit (method 1) , KBW enzyme kit (method 2) , KBW univeral kit (method 3) and TAKARA extraction kit (method 4) were used to extract nucleic acid from each group. The purity of nucleic acid extracted by different methods was evaluated by nano ultramicro nucleic acid protein tester, while the concentration of nucleic acid was evaluated by multiple quantitative polymerase chain reactions (mqPCR). Results The nucleic acid purity: the ratio of DNA purity was slightly higher than 1.8 by method 2 and 3 in group A, method 1 and 2 in group B, and method 3 in group C; the ratio of RNA purity was higer than 2 by method 1 in each group, and was lower than 1.8 by method 4 in each group and method 3 in group C; and the rest results were within the normal range. The concentration of nucleic acid by mqPCR: the adenoviruse (DNA) Ct value: group A 0.05); method 1 0.05); the respiratory syncytial viruse (RNA) Ct value: group A 0.05). Conclusions The sputum homogenization methods have an adverse impact on nucleic acid extration. The co-extraction effects of by TIANamp Virus DNA/RNA Kit and KBW enzyme kit are better than the other methods. Key words: Sputum; Sputum homogenization; DNA and RNA co-extration; Multiple quantitative polymerase chain reaction (mqPCR); Adenoviruses; Respiratory syncytial viruses