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Establishment and Application of a Universal Coronavirus Screening Method Using MALDI-TOF Mass Spectrometry.

ABSTRACT: There are four human coronaviruses (HCoVs), distributed worldwide, that are associated with a range of respiratory symptoms. The discovery of severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV shows that HCoVs pose a significant threat to human health. Our work aims to develop a sensitive method (mCoV-MS) which can not only identify known HCoVs accurately, but also have the ability to provide clues for the emerging HCoVs. The method was performed using a MassARRAY matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) system. We developed a 17-plex analysis to detect six HCoVs in Panel A and another 17-plex analysis to detect Alphacoronavirus and Betacoronavirus in Panel B. All tested primers and probes for the mCoV-MS method were effective, with no cross-reactivity observed with other common respiratory viruses. To confirm the usefulness of the mCoV-MS method we screened 384 pharyngeal and/or anal swab samples collected from bats/rodents, and 131 nasal and throat swabs from human patients. The results showed good concordance with the results of metagenomic analysis or PCR-sequencing. The validation test showed mCoV-MS method can detect potentially pathogenic CoVs in Alphacoronavirus and Betacoronavirus and provide convincingly phylogenetic evidences about unknown CoVs. The mCoV-MS method is a sensitive assay that is relatively simple to carry out. We propose that this method be used to complement next generation sequencing technology for large-scale screening studies.

SUBMITTER: Xiu L

PROVIDER: S-EPMC5552709 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Establishment and Application of a Universal Coronavirus Screening Method Using MALDI-TOF Mass Spectrometry.

Xiu Leshan L Zhang Chi C Wu Zhiqiang Z Peng Junping J

Frontiers in microbiology 20170809

There are four human coronaviruses (HCoVs), distributed worldwide, that are associated with a range of respiratory symptoms. The discovery of severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV shows that HCoVs pose a significant threat to human health. Our work aims to develop a sensitive method (mCoV-MS) which can not only identify known HCoVs accurately, but also have the ability to provide clues for the emerging HCoVs. The method was performed using a ...[more]

PMID: 28848521

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Project description:A method of analysis is presented that utilizes matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to monitor the kinetics and products of RNA cleavage, by use of a program designed to mass-match observed MS peaks with predicted RNA cleavage products. The method is illustrated through application to the study of targeted oxidation of RNA stem loops from HIV-1 Rev Response Element mRNA (RRE RNA) and ribosomal 16S A-site RNA (16S RNA) by metallonucleases. Following incubation of each RNA with catalysts and/or redox co-reactants, reaction mixtures were desalted, and MALDI-TOF MS was used to monitor both time-resolved formation of cleavage products and disappearance of full-length RNA. For each RNA, a unique list was generated that contained the predicted masses of both the full-length, and all of the possible RNA cleavage fragments that resulted from the combination of all possible cleavage sites and each of the six expected overhangs formed at nascent termini adjacent to the cleavage sites. The overhangs corresponded to 2',3'-cyclic phosphate, 3'-phosphate, 3'-phosphoglycolate, 5'- hydroxyl and 5'- phosphate, which corresponded to differing oxidative, hydrolytic, and/or 2'-OH-mediated-endonucleolytic modes of scission. Each mass spectrum was compared with a corresponding list of predicted masses, and peaks were rapidly assigned by use of a Perl script, with a mass-matching tolerance of 200 ppm. Both time-dependent cleavage mediated by metallonucleases and MALDI-TOF-induced fragmentation were observed, and these were distinguished by time-dependent experiments. The resulting data allowed a semi-quantitative assessment of the rate of formation of each overhang at each nucleotide position. Limitations included artifactual skewing of quantification by mass bias, a limited mass range for quantification, and a lack of detection of secondary cleavage products. Nevertheless, the method presented herein provides a rapid, accurate, highly-detailed and semi-quantitative analysis of RNA cleavage that should be widely applicable.

Dataset Information

Establishment and Application of a Universal Coronavirus Screening Method Using MALDI-TOF Mass Spectrometry.

Publications

Establishment and Application of a Universal Coronavirus Screening Method Using MALDI-TOF Mass Spectrometry.

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