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. 2018 May 29:9:1101.
doi: 10.3389/fmicb.2018.01101. eCollection 2018.

A Rapid and Specific Assay for the Detection of MERS-CoV

Pei Huang  1   2 , Hualei Wang  2   3   4 , Zengguo Cao  2   3 , Hongli Jin  2   3 , Hang Chi  2 , Jincun Zhao  5   6 , Beibei Yu  7 , Feihu Yan  2 , Xingxing Hu  1   2 , Fangfang Wu  2 , Cuicui Jiao  2 , Pengfei Hou  2   3 , Shengnan Xu  1   2 , Yongkun Zhao  2   4 , Na Feng  2   4 , Jianzhong Wang  1 , Weiyang Sun  2   4 , Tiecheng Wang  2   4 , Yuwei Gao  2   4 , Songtao Yang  2   4 , Xianzhu Xia  2   4
Affiliations

A Rapid and Specific Assay for the Detection of MERS-CoV

Pei Huang et al. Front Microbiol. .

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel human coronavirus that can cause human respiratory disease. The development of a detection method for this virus that can lead to rapid and accurate diagnosis would be significant. In this study, we established a nucleic acid visualization technique that combines the reverse transcription loop-mediated isothermal amplification technique and a vertical flow visualization strip (RT-LAMP-VF) to detect the N gene of MERS-CoV. The RT-LAMP-VF assay was performed in a constant temperature water bath for 30 min, and the result was visible by the naked eye within 5 min. The RT-LAMP-VF assay was capable of detecting 2 × 101 copies/μl of synthesized RNA transcript and 1 × 101 copies/μl of MERS-CoV RNA. The method exhibits no cross-reactivities with multiple CoVs including SARS-related (SARSr)-CoV, HKU4, HKU1, OC43 and 229E, and thus exhibits high specificity. Compared to the real-time RT-PCR (rRT-PCR) method recommended by the World Health Organization (WHO), the RT-LAMP-VF assay is easy to handle, does not require expensive equipment and can rapidly complete detection within 35 min.

Keywords: Middle East respiratory syndrome coronavirus; RT-LAMP-VF; nucleic acid visualization; reverse transcription loop-mediated isothermal amplification; visual detection.

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Figures

FIGURE 1
FIGURE 1
Schematic illustration of the RT-LAMP-VF assay. (A) RT-LAMP was performed in a constant temperature water bath. (B) RT-LAMP products were detected with a vertical flow visualization strip.
FIGURE 2
FIGURE 2
Primer positions of the RT-LAMP-VF assay. The MERS-CoV N gene was retrieved from GenBank and aligned using MEGA7 software.
FIGURE 3
FIGURE 3
Specificity of the RT-LAMP-VF assay as analyzed by extracting RNA from multiple respiratory pathogens and synthesized RNA transcripts.
FIGURE 4
FIGURE 4
Detection of the RT-LAMP-VF assay limit using a series of synthesized RNA transcripts.
FIGURE 5
FIGURE 5
The RNA of MERS-CoV was used to evaluate the RT-LAMP-VF assay. (A) Ten-fold serial dilutions of MERS-CoV RNA were detected with the RT-LAMP-VF assay. (B) The RT-LAMP-VF assay was performed on mixtures in which the dilutions of MERS-CoV RNA were spiked into the total RNA of throat swabs collected from healthy people.
FIGURE 6
FIGURE 6
Sensitivity comparison of conventional RT-LAMP, rRT-PCR, and RT-LAMP-VF. (A) Conventional RT-LAMP assay. (B) The RT-LAMP-VF assay. (C) The rRT-PCR assay for upE. (D) The rRT-PCR assay for N2.
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