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.
Highlights
Human infection with avian influenza A (H7N9) has been remaining persistent in China over the recent years
Of the 217 sputum specimens, 66 (30.41%) were positive in at least one group. 49 (22.58%), 64 (29.49%), and 61 (28.11%) were positive in the normal saline (NS), DTT, and proteinase K (PK) groups, respectively. 17 (25.76%), 2 (3.03%), and 5 (7.58%) positive specimens were missed in the NS, DTT, and PK groups, respectively
During H7N9 virus epidemic, nearly 60% of the specimens received by laboratories to detect influenza virus were sputum
Summary
Human infection with avian influenza A (H7N9) has been remaining persistent in China over the recent years. For laboratory diagnosis of H7N9 virus infection, related studies and guidelines have confirmed that the nucleic acid-positive rate of lower respiratory specimens, such as sputum, airway aspirates, and bronchoalveolar lavage fluid, is higher than that of upper respiratory specimens [1, 2]. Real-time reverse transcription PCR (rRT-PCR) of sputum specimens yields false-negative results owing to the difficulty of extracting RNA from sputum containing mucus [3]. Viscous sputum specimens usually cannot undergo automated extraction, and a pre-homogenization process is desirable before isolating nucleic acids for rRT-PCR. Some influenza A virus (IAV) detection kits lack homogenization reagents, and some clinical laboratories may have applied improper preprocessing methods, such as adding normal saline (NS) and mixing by vortex
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