Comparative Evaluation of Three Preprocessing Methods for Extraction and Detection of Influenza A Virus Nucleic Acids from Sputum

Yu, Fei; Qiu, Ting; Zeng, Ying; Wang, Yiyin; Zheng, Shufa; Chen, Xiao; Chen, Yu

During H7N9 virus epidemic, nearly 60% of the specimens received by laboratories to detect influenza virus were sputum. For these specimens, appropriate preprocess was essential. In this study, all of the sufficient sputum samples were preprocessed in three methods, IAV RNA extracted and tested by the same method, and comparison results were of important guiding significance. It was reported previously that treatment with N-acetyl-lcysteine and sodium citrate solution came out in whole homogenization of sputum samples, which was unable to destroy mRNA or rRNA of mycobacteria in sputum samples. However, for the purpose of detecting RNA targets in purulent sputum, this method was not appropriate (3, 9) . DTT and PK are often used for the homogenization processing of sputum specimens, which can fully digest mucous protein, release epithelial cells, and consequently increase the concentration of extracted RNA. Purulent sputum samples with treatment of DTT or PK can undergo automated extraction. Based on the experimental results of 217 specimens, our conclusion is that DTT and PK could be used to improve the homogenization effects of sputum and increase the positive rates by 5.53-6.91% higher than that of the NS group. Of the 17 missed specimens, 9 were genotyped as H7N9. Although these specimens were weakly positive, the missed detection of these highly pathogenic agents might result in serious consequences (1) . Vortex with NS is often adopted in respiratory viruses' culture from sputum, but the method is not appropriate for detection of IAV by rRT-PCR.