Development and Evaluation of Novel Real-Time Reverse Transcription-PCR Assays with Locked Nucleic Acid Probes Targeting Leader Sequences of Human-Pathogenic Coronaviruses

Abstract

Based on findings in small RNA-sequencing (Seq) data analysis, we developed highly sensitive and specific real-time reverse transcription (RT)-PCR assays with locked nucleic acid probes targeting the abundantly expressed leader sequences of Middle East respiratory syndrome coronavirus (MERS-CoV) and other human coronaviruses. Analytical and clinical evaluations showed their noninferiority to a commercial multiplex PCR test for the detection of these coronaviruses. C oronaviruses (CoVs) have repeatedly crossed species barriers, and some have emerged as important human pathogens (1, 2). Human coronavirus (HCoV)-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1 predominantly cause mild upper respiratory tract infections, while severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) frequently cause severe pneumonia with extrapulmonary manifestations (3-6). Highly sensitive and specific laboratory diagnostic tests are essential for the control of emerging CoV outbreaks (7). The gold standard for the laboratory diagnosis of CoV infection is the isolation of infectious virus from the respiratory tract and/or other clinical specimens. However, most CoVs are either difficult or dangerous to culture in cell lines (8, 9). The need for convalescent-phase samples and potential false-positive results due to cross-reactivity with other CoVs limit the use of serum antibody detection assays in the acute setting (10). The overall sensitivity of antigen detection assays is inferior to that of molecular assays such as reverse transcription (RT)-PCR (11,12). With the increasing availability of molecular diagnostic facilities and expertise in clinical microbiology laboratories worldwide, RT-PCR has become the test of choice for diagnosing CoV infections (7,(13)(14)(15).Traditionally, the preferred targets of RT-PCR assays are genes that are conserved and/or abundantly expressed from the viral genome (16). For CoVs, the most commonly employed targets include the structural nucleocapsid (N) and spike (S) genes, and the nonstructural RNA-dependent RNA polymerase (RdRp) and replicase ORF1a/b genes (4, 7). Recently, other unique noncoding genome regions not present in related CoVs have also been utilized to develop an RT-PCR for the emerging MERS-CoV (7, 13-15). The World Health Organization (WHO) recommends using the upE assay (regions upstream of the envelope [E] gene) for laboratory screening of suspected MERS cases, followed by confirmation with either the ORF1a or ORF1b assay (7). Notably, a number of single nucleotide mismatches at different positions included in the upE assay forward primer and probe have been detected in recent strains of MERS-CoV and may affect the sensitivity of this assay (17). We hypothesize that additional gene targets may be suitable for design of RT-PCR assays for CoVs and would increase the options of molecular diagnosis for circulating and emerging CoV infections. In this study, we designed and evaluated novel real-time RT-PCR assays with locked nucleic acid (LNA) pro...

Recent advances in the detection of respiratory virus infection in humans

“…Current diagnostic tests for coronavirus include RT‐PCR, real‐time reverse transcription PCR (rRT‐PCR), reverse transcription loop‐mediated isothermal amplification (RT‐LAMP), as well as real‐time RT‐LAMP 51,55–58 …”

Zika Virus Infection in Dexamethasone-immunosuppressed Mice Demonstrating Disseminated Infection with Multi-organ Involvement Including Orchitis Effectively Treated by Recombinant Type I Interferons

“…Total nucleic acid (TNA) was extracted from the blood and necropsied tissues using EZ1 Virus Mini Kit v2.0 and QIAsymphony DSP Virus/Pathogen Mini Kit (QIAGEN, Hilden, Germany), respectively, as we previously described (Zheng et al, 2008;Chan et al, 2016b;Chan et al, 2015a). ZIKV envelope gene was measured by using QuantiNova Probe RT-PCR Kit (QIAGEN) in LightCycler 96 Real-Time PCR System (Roche Diagnostics, Basel, Switzerland).…”

MERS coronavirus outbreak: Implications for emerging viral infections

“…They require minimal instrumentation, thus they can also be used for portable point-of-care testing (Bhadra et al, 2015). Other assays targeting small RNA molecules (leader sequences) have good sensitivity profiles (Chan et al, 2015b).…”