Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Mar 2:5:56.
doi: 10.3389/fmed.2018.00056. eCollection 2018.

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

Fei Yu  1 , Ting Qiu  2 , Ying Zeng  2 , Yiyin Wang  1 , Shufa Zheng  1 , Xiao Chen  1 , Yu Chen  2
Affiliations

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

Fei Yu et al. Front Med (Lausanne). .

Abstract

Viscous sputum specimens usually cannot undergo automated extraction, and thus, a pre-homogenization process is desirable before isolating nucleic acids for real-time reverse transcription PCR. In this study, we compared three preprocessing methods [preprocessing with normal saline (NS), dithiothreitol (DTT), and proteinase K (PK)] of sputum specimens on the extraction and detection of influenza A virus (IAV) nucleic acids. Based on the experimental results of 217 specimens, we found 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. Comparison of 49 positive specimens in all of the three groups demonstrated that the threshold cycle values of the DTT group and PK group were significantly lower and their nucleic acid concentration and A260/A280 ratio within 1.8-2.0 were higher than those of the NS group. Thus, sputum homogenization before nucleic acid extraction is essential for the accurate diagnosis of IAV infection.

Keywords: concentration; homogenization; influenza A virus; nucleic acid; purity; sputum.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Flowchart of experiment operation in this study.
Figure 2
Figure 2
Comparison of threshold cycle (Ct) value (x¯±s), nucleic acid concentration (median and P25–P75), and purity of 49 positive specimens in all three groups. Abbreviations: NS group, normal saline group; DTT, dithiothreitol group; PK group, proteinase K group.
See this image and copyright information in PMC

References

    1. Chen Y, Liang W, Yang S, Wu N, Gao H, Sheng J, et al. Human infections with the emerging avian influenza A H7N9 virus from wet market poultry: clinical analysis and characterisation of viral genome. Lancet (2013) 381:1916–25.10.1016/S0140-6736(13)60903-4 - DOI - PMC - PubMed
    1. National Health and Family Planning Commission of China (NHFPC). Diagnosis and Treatment Project of Human Infection with Avian Influenza (H7N9) Virus 2017. 1st ed Beijing: NHFPC; (2017).
    1. Sung H, Yong D, Ki CS, Kim JS, Seong MW, Lee H, et al. Comparative evaluation of three homogenization methods for isolating Middle East respiratory syndrome coronavirus nucleic acids from sputum samples for real-time reverse transcription PCR. Ann Lab Med (2016) 36:457–62.10.3343/alm.2016.36.5.457 - DOI - PMC - PubMed
    1. Sweeney PJ, Walker JM, Proteinase K. Enzymes of molecular biology. In: Burrell MM, editor. Methods in Molecular Biology. Totowa, NJ: Humana Press; (1993). p. 305–11. - PubMed
    1. Saraswathy VV, Sara GP, Jayasree K, Sujathan K. Comparative analysis of cell morphology in sputum samples homogenized with dithiothreitol, N-acetyl-L cysteine, Cytorich(®) red preservative and in cellblock preparations to enhance the sensitivity of sputum cytology for the diagnosis of lung cancer. Diagn Cytopathol (2015) 43:551–8.10.1002/dc.23266 - DOI - PubMed