Novel One-Step Single-Tube Nested Quantitative Real-Time PCR Assay
for Highly Sensitive Detection of SARS-CoV-2

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Abstract

Coronavirus disease 2019 (COVID-19) has become a public health emergency. The reverse transcriptase real-time quantitative PCR (qRT-PCR) test is currently considered as the gold standard in the laboratory for the etiological detection of COVID-19. However, qRT-PCR results could be false-negative due to the inadequate sensitivity of qRT-PCR. In this study, we have developed and evaluated a novel one-step single-tube nested quantitative real-time PCR (OSN-qRT-PCR) assay for the highly sensitive detection of SARS-CoV-2 targeting the ORF1ab and N genes. The sensitivity of the OSN-qRT-PCR assay was 1 copy/reaction and 10-fold higher than that of the commercial qRT-PCR kit (10 copies/reaction). The clinical performance of the OSN-qRT-PCR assay was evaluated using 181 clinical samples. Among them, 14 qRT-PCR-negative samples (7 had no repetitive results and 7 had no cycle threshold (CT) values) were detected by OSN-qRT-PCR. Moreover, the 7 qRT-PCR-positives in the qRT-PCR gray zone (CT values of ORF1ab ranged from 37.48 to 39.07, and CT values of N ranged from 37.34 to 38.75) were out of the gray zone and thus were deemed to be positive by OSN-qRT-PCR, indicating that the positivity of these samples is confirmative. Compared to the qRT-PCR kit, the OSN-qRT-PCR assay revealed higher sensitivity and specificity, showing better suitability to clinical applications for the detection of SARS-CoV-2 in patients with low viral load.

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Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China

Chaolin Huang, Yeming Wang, Xingwang Li … (2020)

Summary Background A recent cluster of pneumonia cases in Wuhan, China, was caused by a novel betacoronavirus, the 2019 novel coronavirus (2019-nCoV). We report the epidemiological, clinical, laboratory, and radiological characteristics and treatment and clinical outcomes of these patients. Methods All patients with suspected 2019-nCoV were admitted to a designated hospital in Wuhan. We prospectively collected and analysed data on patients with laboratory-confirmed 2019-nCoV infection by real-time RT-PCR and next-generation sequencing. Data were obtained with standardised data collection forms shared by WHO and the International Severe Acute Respiratory and Emerging Infection Consortium from electronic medical records. Researchers also directly communicated with patients or their families to ascertain epidemiological and symptom data. Outcomes were also compared between patients who had been admitted to the intensive care unit (ICU) and those who had not. Findings By Jan 2, 2020, 41 admitted hospital patients had been identified as having laboratory-confirmed 2019-nCoV infection. Most of the infected patients were men (30 [73%] of 41); less than half had underlying diseases (13 [32%]), including diabetes (eight [20%]), hypertension (six [15%]), and cardiovascular disease (six [15%]). Median age was 49·0 years (IQR 41·0–58·0). 27 (66%) of 41 patients had been exposed to Huanan seafood market. One family cluster was found. Common symptoms at onset of illness were fever (40 [98%] of 41 patients), cough (31 [76%]), and myalgia or fatigue (18 [44%]); less common symptoms were sputum production (11 [28%] of 39), headache (three [8%] of 38), haemoptysis (two [5%] of 39), and diarrhoea (one [3%] of 38). Dyspnoea developed in 22 (55%) of 40 patients (median time from illness onset to dyspnoea 8·0 days [IQR 5·0–13·0]). 26 (63%) of 41 patients had lymphopenia. All 41 patients had pneumonia with abnormal findings on chest CT. Complications included acute respiratory distress syndrome (12 [29%]), RNAaemia (six [15%]), acute cardiac injury (five [12%]) and secondary infection (four [10%]). 13 (32%) patients were admitted to an ICU and six (15%) died. Compared with non-ICU patients, ICU patients had higher plasma levels of IL2, IL7, IL10, GSCF, IP10, MCP1, MIP1A, and TNFα. Interpretation The 2019-nCoV infection caused clusters of severe respiratory illness similar to severe acute respiratory syndrome coronavirus and was associated with ICU admission and high mortality. Major gaps in our knowledge of the origin, epidemiology, duration of human transmission, and clinical spectrum of disease need fulfilment by future studies. Funding Ministry of Science and Technology, Chinese Academy of Medical Sciences, National Natural Science Foundation of China, and Beijing Municipal Science and Technology Commission.

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Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study

Nanshan Chen, Min Zhou, Xuan Dong … (2022)

Summary Background In December, 2019, a pneumonia associated with the 2019 novel coronavirus (2019-nCoV) emerged in Wuhan, China. We aimed to further clarify the epidemiological and clinical characteristics of 2019-nCoV pneumonia. Methods In this retrospective, single-centre study, we included all confirmed cases of 2019-nCoV in Wuhan Jinyintan Hospital from Jan 1 to Jan 20, 2020. Cases were confirmed by real-time RT-PCR and were analysed for epidemiological, demographic, clinical, and radiological features and laboratory data. Outcomes were followed up until Jan 25, 2020. Findings Of the 99 patients with 2019-nCoV pneumonia, 49 (49%) had a history of exposure to the Huanan seafood market. The average age of the patients was 55·5 years (SD 13·1), including 67 men and 32 women. 2019-nCoV was detected in all patients by real-time RT-PCR. 50 (51%) patients had chronic diseases. Patients had clinical manifestations of fever (82 [83%] patients), cough (81 [82%] patients), shortness of breath (31 [31%] patients), muscle ache (11 [11%] patients), confusion (nine [9%] patients), headache (eight [8%] patients), sore throat (five [5%] patients), rhinorrhoea (four [4%] patients), chest pain (two [2%] patients), diarrhoea (two [2%] patients), and nausea and vomiting (one [1%] patient). According to imaging examination, 74 (75%) patients showed bilateral pneumonia, 14 (14%) patients showed multiple mottling and ground-glass opacity, and one (1%) patient had pneumothorax. 17 (17%) patients developed acute respiratory distress syndrome and, among them, 11 (11%) patients worsened in a short period of time and died of multiple organ failure. Interpretation The 2019-nCoV infection was of clustering onset, is more likely to affect older males with comorbidities, and can result in severe and even fatal respiratory diseases such as acute respiratory distress syndrome. In general, characteristics of patients who died were in line with the MuLBSTA score, an early warning model for predicting mortality in viral pneumonia. Further investigation is needed to explore the applicability of the MuLBSTA score in predicting the risk of mortality in 2019-nCoV infection. Funding National Key R&D Program of China.

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A new coronavirus associated with human respiratory disease in China

Fan Wu, Su Zhao, Bin Yu … (2020)

Emerging infectious diseases, such as severe acute respiratory syndrome (SARS) and Zika virus disease, present a major threat to public health 1–3 . Despite intense research efforts, how, when and where new diseases appear are still a source of considerable uncertainty. A severe respiratory disease was recently reported in Wuhan, Hubei province, China. As of 25 January 2020, at least 1,975 cases had been reported since the first patient was hospitalized on 12 December 2019. Epidemiological investigations have suggested that the outbreak was associated with a seafood market in Wuhan. Here we study a single patient who was a worker at the market and who was admitted to the Central Hospital of Wuhan on 26 December 2019 while experiencing a severe respiratory syndrome that included fever, dizziness and a cough. Metagenomic RNA sequencing 4 of a sample of bronchoalveolar lavage fluid from the patient identified a new RNA virus strain from the family Coronaviridae, which is designated here ‘WH-Human 1’ coronavirus (and has also been referred to as ‘2019-nCoV’). Phylogenetic analysis of the complete viral genome (29,903 nucleotides) revealed that the virus was most closely related (89.1% nucleotide similarity) to a group of SARS-like coronaviruses (genus Betacoronavirus, subgenus Sarbecovirus) that had previously been found in bats in China 5 . This outbreak highlights the ongoing ability of viral spill-over from animals to cause severe disease in humans.

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Author and article information

Journal

Journal ID (nlm-ta): Anal Chem

Journal ID (iso-abbrev): Anal. Chem

Journal ID (publisher-id): ac

Journal ID (coden): ancham

Title: Analytical Chemistry

Publisher: American Chemical Society

ISSN (Print): 0003-2700

ISSN (Electronic): 1520-6882

Publication date (Electronic): 22 May 2020

Electronic Location Identifier: acs.analchem.0c01884

Affiliations

[† ]National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention , Beijing 102206, China

[‡ ]Hubei Center for Disease Control and Prevention , Wuhan 430000, China

[§ ]Hebei General Hospital , Shijiazhuang 050051, China

[∥ ]Hebei Medical University , Shijiazhuang 050031, China

[⊥ ]Center for Biosafety Mega-Science, Chinese Academy of Sciences , Wuhan 430071, China

Author notes

[* ]Email: 575652423@ 123456qq.com .

[* ]Email: wenbo_xu1@ 123456aliyun.com .

[* ]Tel: +86-010-58900810. Email: maxj@ 123456ivdc.chinacdc.cn .

Article

DOI: 10.1021/acs.analchem.0c01884

PMC ID: 7299395

PubMed ID: 32438806

SO-VID: 71240c69-6239-4eb7-91b7-0e61b791545a

License:

This article is made available via the PMC Open Access Subset for unrestricted RESEARCH re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.

History

Date received : 02 May 2020

Date accepted : 22 May 2020

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Novel One-Step Single-Tube Nested Quantitative Real-Time PCR Assay for Highly Sensitive Detection of SARS-CoV-2

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Abstract

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Novel Coronavirus Disease COVID-19

Most cited references 15

Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China

Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study

A new coronavirus associated with human respiratory disease in China

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Cited by 39

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