Detection and analysis of nucleic acid in various biological samples of COVID-19 patients.
COVID-19 (corona virus disease 2019) is a kind of acute severe pneumonia caused by 2019-nCoV (2019-nCoV) infection. Since December 2019, it has been found in Wuhan, Hubei Province, and then spread to the whole country. Some parts of the world also showed an outbreak trend [1-3]. Real-time fluorescence quantitative reverse transcriptase polymerase chain reaction (reverse transcriptase-polymerase chain reaction,RT-PCR) and viral gene sequencing are the gold standard for the diagnosis of COVID-19. At present, upper respiratory tract nasopharyngeal swabs are mostly used as nucleic acid detection samples in China, but the positive rate is low. However, there are few reports on clinical application of 2019-nCoV nucleic acid detection in other biological samples.
| The East Section of Renmin Hospital of Wuhan University is a designated COVID-19 hospital in Wuhan City, Hubei Province, China. This observation study included 132 patients diagnosed with COVID-19 in the infectious disease areas of the East Section of Renmin Hospital of Wuhan University from 2020.1.31 to 2020.2.29. COVID-19 diagnostic criteria: according to China's ⟪pneumonia diagnosis and treatment Program of novel coronavirus infection (trial version 7) ⟫, in accordance with the relevant epidemiological and clinical manifestations, nasopharyngeal swabs real-time fluorescence RT-PCR detection of 2019-nCoV nucleic acid positive, COVID-19 cases were divided into mild, ordinary, severe and severe [1]. The nasopharyngeal swabs of 132 cases of COVID-19 were positive for 2019-nCoV nucleic acid on admission, including 72 males and 60 females, with an average age of 66.7 ± 9.1 years, including 80 cases of common type, 44 cases of severe type and 8 cases of critical type. During the period of admission, under the condition of tertiary protection, nasopharyngeal swabs, sputum, blood, feces and anal swabs of COVID-19 cases were collected many times in the isolation ward for 2019-nCoV nucleic acid detection. All biological samples are sealed and transferred to the laboratory in strict accordance with the standard process. The RT-PCR test kits (BioGerm) were recommended by the Chinese Center for Disease Control and Prevention. The same technician and brand of test kit was used for all RT-PCR testing reported; both internal controls and negative controls were routinely performed with each batch of tests.
| 132 the results of 2019-nCoV nucleic acid test of various biological samples during the treatment of confirmed COVID-19 cases are as follows: the positive rate of 2019-nCoV nucleic acid test of nasopharyngeal swab is 38.13% (180/472 times), the positive rate of 2019-nCoV nucleic acid test of sputum is 48.68% (148/304 times), the positive rate of blood 2019-nCoV nucleic acid test is 3.03% (4/132 times), and the positive rate of 2019-nCoV nucleic acid test of feces is 9.83% (24/244 times). The positive rate of 2019-nCoV nucleic acid detection in anal swabs is 10.00% (12/120 times).
In this study, it was found that the positive rate of 2019-nCoV nucleic acid in sputum of 132 patients with COVID-19 was higher than that of nasopharyngeal swabs, and viral nucleic acids were also detected in blood and digestive tract (fecal/anal swabs). Simple detection of nasopharyngeal swab 2019-nCoV nucleic acid detection positive rate is not high, multi-sample 2019-nCoV nucleic acid detection can improve the accuracy, reduce the false negative rate, better guide clinical treatment and evaluate the therapeutic effect.
Wu J
，Liu J
，Li S
，Peng Z
，Xiao Z
，Wang X
，Yan R
，Luo J
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Evaluation of simple nucleic acid extraction methods for the detection of SARS-CoV-2 in nasopharyngeal and saliva specimens during global shortage of extraction kits.
The severe shortage of nucleic acid extraction kits during the current COVID-19 pandemic represents a key limiting factor in testing capacity.
This study compared the results of SARS-CoV-2 RT-PCR using different simple nucleic acid extraction methods on nasopharyngeal and saliva specimens.
Fifty nasopharyngeal swab and saliva specimens previously tested positive for SARS-CoV-2 were retrieved. Three different methods of nucleic acid extraction were compared. The first method involves incubating the specimen with proteinase K, and then heat treatment at 98 °C for 5 min (PKH); the second method involves heat treatment at 98 °C for 5 min without proteinase K pre-incubation (heat only); the third method involves no pre-processing steps (direct). The products from all 3 methods were tested by SARS-CoV-2 RT-PCR.
PKH had significantly higher positive rate in SARS-CoV-2 RT-PCR (80 %) than those of heat only (58 %; P = 0.001) or direct (56 %; P = 0.002). The median Ct value was significantly earlier for PKH (median Ct: 37.0, IQR 31.7-40) than that of heat only (median Ct: 40, IQR 36.2-41; P < 0.0001) and direct (median Ct, 37.5; IQR 33.9-41.0; P = 0.0049). Subgroup analysis showed that PKH had higher detection rate, lower limit of detection and earlier Ct values than the other two groups for both NPS and saliva specimens.
PKH pre-processing resulted in the highest detection rate of SARS-CoV-2 by RT-PCR, and represents an alternative method for nucleic acid extraction when commercial extraction kits are not available.
Chu AW
，Chan WM
，Ip JD
，Yip CC
，Chan JF
，Yuen KY
，To KK
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ResearchGate
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