Real-Time Sequence-Validated Loop-Mediated Isothermal Amplification Assays for Detection of Middle East Respiratory Syndrome Coronavirus (MERS-CoV)

Abstract

The Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging human coronavirus, causes severe acute respiratory illness with a 35% mortality rate. In light of the recent surge in reported infections we have developed asymmetric five-primer reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for detection of MERS-CoV. Isothermal amplification assays will facilitate the development of portable point-of-care diagnostics that are crucial for management of emerging infections. The RT-LAMP assays are designed to amplify MERS-CoV genomic loci located within the open reading frame (ORF)1a and ORF1b genes and upstream of the E gene. Additionally we applied one-step strand displacement probes (OSD) for real-time sequence-specific verification of LAMP amplicons. Asymmetric amplification effected by incorporating a single loop primer in each assay accelerated the time-to-result of the OSD-RT-LAMP assays. The resulting assays could detect 0.02 to 0.2 plaque forming units (PFU) (5 to 50 PFU/ml) of MERS-CoV in infected cell culture supernatants within 30 to 50 min and did not cross-react with common human respiratory pathogens.

Strand-Exchange Nucleic Acid Circuitry with Enhanced Thermo-and Structure- Buffering Abilities Turns Gene Diagnostics Ultra-Reliable and Environmental Compatible

“…The catalyst (C1) sequence was designed as a selected 29-mer fragment within ORF1A region of MERS-CoV virus gene (MRES-1A, Fig. 2A)3133. Customarily, we describe the DNA reaction components in terms of numbered domains, each of which contains 10–15 mer short sequence.…”

“…LAMP is a most point-of-care promising isothermal gene amplifier32 but has been underused due to its complex products and haunted non-specific false amplicons (Figure S14)3738. Our recent advances have proven one step strand exchange reaction (OSD) could solve this problem by specifically probing single strand loop amplicons among LAMP products303133. Here displacing OSD with CHA circuit may provide further signal enhancement upon anti-false function (Fig.…”

“…As a very important example (Fig. 1C), we employed OHT-CHA as a temperature-independent and structure-irrespective signal enhancer for loop mediated isothermal reaction (LAMP)30313233. LAMP is a most powerful point-of-care (POC) gene amplifier, but its direct detection has been analytically difficult due to structured products and frequent off-target reactions.…”

Initial performance evaluation of a spotted array Mobile Analysis Platform (MAP) for the detection of influenza A/B, RSV, and MERS coronavirus

“…Additionally, given the emergence of MERS coronavirus and subsequent outbreaks thereof in the Middle East and South Korea, it is important that diagnostic assays for respiratory viruses are rapid and deployable at or near the point of care (POC) (Bhadra, 2015; Raj, 2014). Such diagnostic platforms are becoming more common and are capable of detecting a wide array of respiratory pathogens (Zumla, 2014).…”

Amplicon Competition Enables End‐Point Quantitation of Nucleic Acids Following Isothermal Amplification

“…We initially varied the number of T T targets relative to a fixed number of T F targets although the same results can also be achieved by varying T F in the presence of constant T T , as we shall see below. The assay for the open reading frame 1B (ORF1B) gene of the Middle East respiratory syndrome coronavirus (MERS-CoV) [17] was carried out with 60 nM OSD reporters and 10, 10 4 , 10 5 , 10 6 , 10 7 or 10 8 copies of the T T MERS1B plasmid along with 10 6 T F copies (Figure 2A, see Supplementary Tables T1 and T2 for sequences ), while the LAMP-OSD assay for the HF183 sequence, derived from the human feces-specific Bacteroides 16S rRNA gene was carried out with 200 nM OSD reporters and 1, 10, 10 2 , 10 3 , 10 4 , 10 5 or 10 6 copies of T T HF183 plasmid thresholded with 10 5 T F copies (Figure 2B). …”