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Development of a quantitative assay for SARS coronavirus and correlation of GAPDH mRNA with SARS coronavirus in clinical specimens.

ABSTRACT: AIMS: To develop a quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) for severe acute respiratory syndrome coronavirus (SARS-CoV) detection and explore the potential of using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internal control to exclude false negative results. METHODS: SARS-CoV and GAPDH mRNA were both measured in 26 specimens from 16 patients with SARS, 40 follow up specimens from the same batch of patients, and appropriate control subjects. The relation between SARS positivity and GAPDH mRNA concentration was investigated using the chi2 test. Increasing the sensitivity for SARS-CoV and GAPDH mRNA detection was investigated in follow up specimens in which SARS-CoV and GAPDH mRNA were not detected initially. RESULTS: Varying amounts of SARS-CoV were found in the 26 SARS-CoV positive specimens and SARS-CoV was not detected in the 40 follow up specimens and controls. In addition, concentrations of GAPDH mRNA were significantly different between the patients with SARS, follow up specimens, and healthy controls (Kruskal-Wallis test, p<0.05). Moreover, GAPDH mRNA concentrations were highly correlated with SARS-CoV positivity (chi2 = 5.43; p<0.05). Finally, SARS-CoV and GAPDH mRNA were both detected in three follow up urine specimens that were initially negative when the amount of cDNA used was increased from 5 microl to 10 and 15 microl. CONCLUSIONS: This Q-RT-PCR assay can be used to detect SARS-CoV. Moreover, GAPDH mRNA may be useful to rule out false negative results in SARS-CoV detection, and the current extraction method for urine may not be sensitive enough to detect low titres of SARS-CoV.

SUBMITTER: Wong SC

PROVIDER: S-EPMC1770583 | biostudies-literature | 2005 Mar

REPOSITORIES: biostudies-literature

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Development of a quantitative assay for SARS coronavirus and correlation of GAPDH mRNA with SARS coronavirus in clinical specimens.

Wong S C C SC Chan J K C JK Lee K C KC Lo E S F ES Tsang D N C DN

Journal of clinical pathology 20050301 3

<h4>Aims</h4>To develop a quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) for severe acute respiratory syndrome coronavirus (SARS-CoV) detection and explore the potential of using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internal control to exclude false negative results.<h4>Methods</h4>SARS-CoV and GAPDH mRNA were both measured in 26 specimens from 16 patients with SARS, 40 follow up specimens from the same batch of patients, and appropriate control su ...[more]

PMID: 15735160

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Project description:A new human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged at the end of 2019 in Wuhan, China that caused a range of disease severities; including fever, shortness of breath, and coughing. This disease, now known as coronavirus disease 2019 (COVID-19), quickly spread throughout the world, and was declared a pandemic by the World Health Organization in March of 2020. As the disease continues to spread, providing rapid characterization has proven crucial to better inform the design and execution of control measures, such as decontamination methods, diagnostic tests, antiviral drugs, and prophylactic vaccines for long-term control. Our work at the United States Army's Combat Capabilities Development Command Chemical Biological Center (DEVCOM CBC) is focused on engineering workflows to efficiently identify, characterize, and evaluate the threat level of any potential biological threat in the field and more remote, lower resource settings, such as forward operating bases. While we have successfully established untargeted sequencing approaches for detection of pathogens for rapid identification, our current work entails a more in-depth sequencing analysis for use in evolutionary monitoring. We are developing and validating a SARS-CoV-2 nanopore sequencing assay, based on the ARTIC protocol. The standard ARTIC, Illumina, and nanopore sequencing protocols for SARS-CoV-2 are elaborate and time consuming. The new protocol integrates Oxford Nanopore Technology's Rapid Sequencing Kit following targeted RT-PCR of RNA extracted from human clinical specimens. This approach decreases sample manipulations and preparation times. Our current bioinformatics pipeline utilizes Centrifuge as the classifier for quick identification of SARS-CoV-2 and RAMPART software for verification and mapping of reads to the full SARS-CoV-2 genome. ARTIC rapid sequencing results, of previous RT-PCR confirmed patient samples, showed that the modified protocol produces high quality data, with up to 98.9% genome coverage at >1,000x depth for samples with presumably higher viral loads. Furthermore, whole genome assembly and subsequent mutational analysis of six of these sequences identified existing and unique mutations to this cluster, including three in the Spike protein: V308L, P521R, and D614G. This work suggests that an accessible, portable, and relatively fast sample-to-sequence process to characterize viral outbreaks is feasible and effective.

Project description:BackgroundThe COVID-19 pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has overwhelmed health systems worldwide and highlighted limitations of diagnostic testing. Several types of diagnostic tests including RT-PCR-based assays and antigen detection by lateral flow assays, each with their own strengths and weaknesses, have been developed and deployed in a short time.MethodsHere, we describe an immunoaffinity purification approach followed a by high resolution mass spectrometry-based targeted qualitative assay capable of detecting SARS-CoV-2 viral antigen from nasopharyngeal swab samples. Based on our discovery experiments using purified virus, recombinant viral protein and nasopharyngeal swab samples from COVID-19 positive patients, nucleocapsid protein was selected as a target antigen. We then developed an automated antibody capture-based workflow coupled to targeted high-field asymmetric waveform ion mobility spectrometry (FAIMS) - parallel reaction monitoring (PRM) assay on an Orbitrap Exploris 480 mass spectrometer. An ensemble machine learning-based model for determining COVID-19 positive samples was developed using fragment ion intensities from the PRM data.FindingsThe optimized targeted assay, which was used to analyze 88 positive and 88 negative nasopharyngeal swab samples for validation, resulted in 98% (95% CI = 0.922-0.997) (86/88) sensitivity and 100% (95% CI = 0.958-1.000) (88/88) specificity using RT-PCR-based molecular testing as the reference method.InterpretationOur results demonstrate that direct detection of infectious agents from clinical samples by tandem mass spectrometry-based assays have potential to be deployed as diagnostic assays in clinical laboratories, which has hitherto been limited to analysis of pure microbial cultures.FundingThis study was supported by DBT/Wellcome Trust India Alliance Margdarshi Fellowship grant IA/M/15/1/502023 awarded to AP and the generosity of Eric and Wendy Schmidt.

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Development of a quantitative assay for SARS coronavirus and correlation of GAPDH mRNA with SARS coronavirus in clinical specimens.

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Development of a quantitative assay for SARS coronavirus and correlation of GAPDH mRNA with SARS coronavirus in clinical specimens.

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