Detection of Middle East respiratory syndrome coronavirus using reverse transcription loop-mediated isothermal amplification (RT-LAMP)
© Shirato et al.; licensee BioMed Central Ltd. 2014
Received: 25 April 2014
Accepted: 4 August 2014
Published: 8 August 2014
The first documented case of Middle East Respiratory Syndrome coronavirus (MERS-CoV) occurred in 2012, and outbreaks have continued ever since, mainly in Saudi Arabia. MERS-CoV is primarily diagnosed using a real-time RT-PCR assay, with at least two different genomic targets required for a positive diagnosis according to the case definition of The World Health Organization (WHO) as of 3 July 2013. Therefore, it is urgently necessary to develop as many specific genetic diagnostic methods as possible to allow stable diagnosis of MERS-CoV infections.
Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) is a genetic diagnostic method used widely for the detection of viral pathogens, which requires only a single temperature for amplification, and can be completed in less than 1 h. This study developed a novel RT-LAMP assay for detecting MERS-CoV using primer sets targeting a conserved nucleocapsid protein region.
The RT-LAMP assay was capable of detecting as few as 3.4 copies of MERS-CoV RNA, and was highly specific, with no cross-reaction to other respiratory viruses. Pilot experiments to detect MERS-CoV from medium containing pharyngeal swabs inoculated with pre-titrated viruses were also performed. The RT-LAMP assay exhibited sensitivity similar to that of MERS-CoV real-time RT-PCR.
These results suggest that the RT-LAMP assay described here is a useful tool for the diagnosis and epidemiologic surveillance of human MERS-CoV infections.
KeywordsMeddle East respiratory syndrome (MERS) MERS coronavirus (MERS-CoV) RT-LAMP Genetic diagnostic method
Focus forming unit
Middle East respiratory syndrome
Open reading frame
Plaque forming unit
Reverse transcription-loop-mediated isothermal amplification
50% tissue culture infectious dose
Transmembrane protease, serine 2
On 22 September 2012, a novel coronavirus sequence was detected from a 49-year-old patient presenting with severe pneumonia who was initially treated in an intensive care unit in Qatar and then moved to London . The sequence of the PCR amplicon of this isolate was a close match with that of a coronavirus isolated from a 60-year-old patient who had died of severe pneumonia in Jeddah, Saudi Arabia in June 2012 [1, 2]. Together, these two cases marked the beginning of an outbreak of severe respiratory infections caused by a newly identified coronavirus, designated the Middle East Respiratory Syndrome coronavirus (MERS-CoV) . This outbreak is ongoing, with 836 confirmed cases to date that have resulted in 288 deaths in 19 countries (Jordan, Qatar, Saudi Arabia, the United Arab Emirates, Oman, Kuwait, Yemen, Lebanon, Iran, Algeria, Tunisia, France, the Netherlands, Germany, the United Kingdom, Greece, Malaysia, Philippines and the United States of America) as of 14 July, 2014 [The World Health Organization (WHO), Global Alert and Response (GAR), Coronavirus infections, updated on 14 July 2014, http://www.who.int/csr/disease/coronavirus_infections/en/index.html].
Sequence analyses show that MERS-CoV clusters with the group 2c betacoronavirus, and is closely related to the bat coronaviruses HKU4 and HKU5 . Severe acute respiratory syndrome coronavirus (SARS-CoV), which caused severe pneumonia resulting in 8,098 reported infections and 774 deaths between 2002 and 2003 , was also derived from bat coronaviruses [6, 7]. MERS-CoV, with its similar symptoms and phylogeny, is therefore considered a cousin of SARS-CoV. The reservoir for MERS-CoV remains unclear, but recent reports suggest that camels are the most likely candidate, as a form of the virus has been circulating in camels in Saudi Arabia since at least 1992 [8–13].
MERS-CoV is primarily diagnosed using a real-time RT-PCR assay, and at least two different genomic targets are required for a positive diagnosis. The first probe and primer sets for MERS-CoV detection by real-time PT-PCR were developed by Corman et al. shortly following the first reports of the disease [14, 15]. Among them, the probe and primer sets targeting upE and ORF1a exhibit the highest sensitivities, and remain the most widely used targets for MERS-CoV detection. At least two different specific genomic targets are required for a positive diagnosis according to the case definition announced by the WHO as of 3 July 2013 [WHO, GAR, Revised interim case definition for reporting to WHO – Middle East respiratory syndrome coronavirus (MERS-CoV), updated on 3 July 2013, http://www.who.int/csr/disease/coronavirus_infections/case_definition/en/index.html]. A single positive target followed by gene sequencing is also considered positive; however, the current gene sequencing technique requires PCR amplicons, and the ability of conventional RT-PCR to produce a sequencing-quality template is generally lower than that of real-time RT-PCR [16–20]. Therefore, it is urgently necessary to develop as many specific genetic diagnostic methods as possible to allow reliable diagnosis of MERS-CoV infections.
The loop-mediated isothermal amplification (LAMP) method amplifies specific nucleic acid sequences using a set of four or six unique primers [21, 22]. The LAMP procedure is user-friendly, since the reaction mixture is incubated at a single temperature for less than 1 h. Amplification can be detected as the precipitation of magnesium pyrophosphate or by fluorescence under ultra-violet light, and also can be detected in real time by monitoring the turbidity of the pyrophosphate . The LAMP assay can also be used for the detection of RNA by combining reverse transcription with LAMP (RT-LAMP) ; RT-LAMP assays have been developed for a variety of respiratory RNA viruses, including SARS , respiratory syncytial virus [25, 26], and influenza viruses [27, 28]. In this study, a novel RT-LAMP method for the detection of MERS-CoV was developed, with a sensitivity similar to that of real-time RT-PCR targeting upE and ORF1a.
Materials and methods
The MERS-CoV EMC isolate was kindly provided by Ron A. M. Fouchier, Erasmus Medical Center, Rotterdam, The Netherlands. MERS-CoV was propagated and titrated using Vero cells. Human respiratory syncytial viruses (RSV; Long, A2, B WV/14617/85 and 18537) were obtained from the American Tissue Culture Collection (ATCC). Human metapneumovirus (HMPV; Sendai-H/2404/2003) was obtained from the Virus Research Center, Sendai Medical Center, Japan. Human coronavirus (HCoV)-229E isolates ATCC VR-740 and Sendai-H/1121/04  were used. HCoV-NL63 was supplied by Dr. Hoek, University of Amsterdam, Netherlands. Isolate HCoV-OC43 was obtained from ATCC. SARS coronavirus (Frankfurt strain) was supplied by Dr. J. Ziebuhr, University of Würzburg, Germany. Human parainfluenza viruses (PIV) 1 (strain C35) and 3 (strain C243) were obtained from ATCC. Adenoviruses (ADV) (serotype 3, strain G.B.; serotype 4, strain RI-67; and serotype 7, strain Gomen) were obtained from ATCC. Viruses were propagated and titrated using HEp-2, HeLa, RD, Vero cells, or LLC-Mk2 cells . Influenza viruses [Flu; A/California/7/2009 (H1N1pdm), A/Victoria/210/2009 (H3N2), and B/Brisbane/60/2008] were provided by the Influenza Virus Research Center of the National Institute of Infectious Diseases in Japan, and were propagated and titrated using MDCK cells.
Design of primer sets for RT-LAMP
Primer sets for the RT-LAMP assay were designed using the online LAMP primer design software (PrimerExplorer V4; http://primerexplorer.jp/e/) based on the nucleocapsid protein sequence of the EMC isolate of MERS-CoV (GenBank JX869059.2).
Extraction of nucleic acids
RNA was extracted from viral stocks using TRIzol LS or TRIzol reagent (Invitrogen), according to the manufacturer’s instructions. Viral DNA was extracted using Qiagen Genomic-tip (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Total RNA and genomic DNA were quantitated using standard methods to measure the OD value. The MERS-CoV RNA copy number was calculated based upon the standard curve obtained using a TaqMan assay, as described by Corman et al.  (upE probe set and the positive control template). Total RNA was then diluted with ribonuclease-free water containing 10 μg/mL of Ribonucleic Acid from Baker’s Yeast (R6750; Sigma-Aldrich, St. Louis, MO, USA) as carrier RNA.
The RT-LAMP assay was performed using the Loopamp RNA Amplification Kit (RT-LAMP; Eiken, Tokyo, Japan) under the following conditions: 5-μL sample (RNA or DNA) was mixed with 40 pmol each of FIP and BIP primers, 20 pmol each of LF and LB primers, 5 pmol each of F3 and B3 primers, 1-μL Enzyme Mix, and 12.5-μL Reaction Mix; distilled water was added to obtain a final volume of 25 μL. For real-time monitoring of RT-LAMP amplification, the reaction mixture was incubated at 65°C for 30 min in a Loopamp real-time turbidimeter (LA-320C, Eiken). For fluorescence detection, 1-μL Fluorescent Detection Reagent (Eiken) was added to the reaction mixture described above before the start of amplification, and then, fluorescence was detected under ultraviolet light after 30 min of amplification. Negative controls containing only yeast RNA were included in each assay.
To synthesize control RNA for RT-LAMP, the nucleocapsid protein sequence of the EMC strain (28566–29807) was amplified and cloned into the pGEM-T easy vector (Promega, Fitchburg, WI, USA). Point mutations were inserted using a site-direct mutagenesis technique to generate variations of the nucleoprotein sequence. The nucleocapsid protein sequence was amplified by PCR using forward (5′-TAATACGACTCACTATAGGGATGGCATCCCCTGCTGCACC-3′) and reverse (5′-CTAATCAGTGTTAACATCAA-3′) primers with PrimeSTAR Max DNA polymerase (Takara-Bio, Shiga, Japan). The amplicons were gel-purified and were used as templates for RNA transcription using a MEGAscript T7 Transcription Kit (Life Technologies, Carlsbad, CA, USA). The transcribed RNA was quantified using the OD value, the copies number was calculated, and the RNA was diluted with ribonuclease-free water containing 10 μg/mL of yeast RNA.
Real-time RT-PCR assays
Real-time RT-PCR assays using upE and ORF1a sets [14, 15] were also performed for virus detection using a QuantiTect Probe RT-PCR kit (QIAGEN) and LightCycler 480 Instrument (Roche, Basel, Switzerland) following the manufacturers’ protocols. The amplification conditions followed Corman et al. [14, 15].
Virus preparation for spiked samples
For sensitivity assays, Vero cells were infected with MERS-CoV, and incubated for 4 days. Cell supernatants were then collected and centrifuged at 1,500 × g for 30 min at 4°C, and the supernatants were treated with RNaseA (Nippongene, Tokyo Japan) at a concentration of 10 μg/mL for 30 min at 37°C to exclude miscellaneous RNA other than viral RNA.
MERS-CoV RNA was obtained from pre-titrated viral stocks diluted with medium containing pharyngeal swabs obtained from healthy adults using the Universal Viral Transport for Viruses, Chlamydiae, Mycoplasmas and Ureaplasmas (Becton Dickinson and Company, Sparks, MD, USA). Viral isolation was also performed on Vero and Vero/TMPRSS2 cells constitutively expressing type II transmembrane serine protease (TMPRSS2) , which enhances cell entry and fusion formation of MERS-CoV [31, 32]. Diluted viruses were inoculated on Vero and Vero/TMPRSS2 cells, and incubated for 60 min. Cells were then washed with PBS, and incubated in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal calf serum at 37°C. The cytopathic effect was evaluated 5 days after inoculation. Clinical specimens (nasopharyngeal swabs) diagnosed as other respiratory pathogens by RT-PCR assays were used as negative controls. Informed, written consent was obtained at the time of sample collection from all patients. For the specimen diagnosed as human bocavirus, a LAMP assay was performed without the RT reaction.
RT-LAMP primer design
The primer set for MERS-CoV RT-LAMP assay
Position (EMC, JX869059.2)
Sequence (5′ – 3′)
Number of matched MERS sequences*
FIP (F1c + F2)
BIP (B1c + B2)
Sensitivity and specificity of the RT-LAMP assay
The detection limit of the RT-LAMP assay was determined using serially diluted MERS-CoV, and compared with those of real-time RT-PCR using upE and ORF1a assays [14, 15] (Table 2). upE and ORF1a assays were able to detect 1.6 to 3.4 copies of MERS-CoV RNA, consistent with previous reports. The threshold for RT-LAMP within 30 min was also 3.4 copies, indicating a sensitivity equivalent to that of real-time RT-PCR [14, 15].
Sensitivity of the RT-LAMP assay
Specificity of the RT-LAMP assay
4 × 101 TCID50
HCoV 229E (VR-740)
3 × 103 PFU
HCoV 229E (Sendai-H/1121/04)
5 × 103 PFU
2.5 × 102 FFU
HCoV OC43 (VR-1558)
1.3 × 103 TCID50
1 × 105 PFU
Other Respiratory Viruses
RSV A (Long)
1 × 101 PFU
RSV A (A2)
1 × 102 PFU
RSV B (18537)
1 × 102 PFU
RSV B (WV/14617/85)
PIV 1 (C-35)
3 × 104 PFU
PIV 3 (C-243)
5 × 103 PFU
ADV 3 (G.B.)
2.5 × 102 TCID50
ADV 4 (RI-67)
1 × 102 TCID50
ADV 7 (Gomen)
2.5 × 102 TCID50
Flu A/California/7/2009 (H1N1pdm)
8 × 103 TCID50
Flu A/Victoria/210/2009 (H3N2)
2.5 × 106 TCID50
2.5 × 104 TCID50
Detection of MERS-CoV diluted with medium containing pharyngeal swabs
The real-time RT-PCR was highly sensitive for MERS-CoV, with upE and ORF1a assays capable of detecting as few as 1.6 copies of MERS-CoV RNA. The RT-LAMP was also able to detect viral RNA at levels as low as 0.7 copies, showing equivalence with the RT-PCR assay. Viral isolation using Vero/TMPRSS2 cells was more sensitive than that of Vero cells; however, genetic diagnostic assays were consistently more sensitive than culture-based methods. These data suggest that the RT-LAMP assay is capable of detecting MERS-CoV with a sensitivity similar to that of real-time-RT-PCR, even in clinical specimens.
Detection of MERS-CoV using clinical specimens diagnosed as other respiratory viral infections
*MERS-CoV (104 copies)
Other Respiratory viruses
Flu A H1 (Russian)
Flu A H1 (2009 pdm)
Flu A H3
Flu B (Yamagata)
Flu B (Victoria)
RT-LAMP validation for mismatched sequences
Evaluation of RT-LAMP amplification using mismatched sequences
Nucleotide position *
Region in primer
C to T
C to T
T to C
T to C
T to C
G to T
C to T
C to T
As described above, definitive MERS-CoV diagnosis requires amplification of at least two different virus-specific genomic targets according to the case definition reported on 3 July 2013 by the WHO. However, only two real-time RT-PCR targets, upE and ORF1a, are currently available for MERS-CoV detection with high specificity and sensitivity [14, 15]. Indeed, two previous cases reported by the Italian government had to be reclassified as probable MERS-CoV infections, as they were unable to fulfill these criteria (WHO, GAR, MERS-CoV summary and literature update – as of 20 September 2013, http://www.who.int/csr/disease/coronavirus_infections/update_20130920/en/index.html). Additional sensitive and specific genetic diagnostic methods are therefore needed to provide reliable MERS-CoV diagnoses.
This study describes a novel genetic diagnostic method for MERS-CoV based on the RT-LAMP assay, with a sensitivity and specificity equal to that of the upE and ORF1a RT-PCR assays. This assay was also able to detect MERS-CoV RNA in experimentally obtained nasopharyngeal swabs, and never showed cross reactivity to other respiratory viruses, even in the case of clinical specimens.
The RT-LAMP method requires only a single temperature for amplification, with results usually available in less than 1 h by observing magnesium pyrophosphate precipitate or fluorescence signals by the naked eye [21, 22]. Although RT-LAMP amplification can be monitored in real-time using a turbidimeter , the assay can also be performed using basic laboratory equipment, such as a heat block and water bath. The method has been validated using various respiratory viruses, as well as more diverse pathogens, such as bacteria [33–35], protozoa [36–38], and parasites [39, 40]. Furthermore, the reagents necessary to perform RT-LAMP are commercially available. Recently, Abd El Wahed et al., reported a genetic diagnostic method for MERS-CoV based on reverse transcription isothermal recombinase polymerase amplification (RT-PPA) assay. The method is highly sensitive and can detect 10 copies of virus RNA within 3 to 7 minutes. Therefore, it is useful for the diagnosis of MERS-CoV as well. However, RT-RPA requires a specific tubescanner for detection. By contrast, the RT-LAMP assay enables detection of amplicons by observing a magnesium pyrophosphate precipitate or fluorescence signals with the naked eye with no requirement for any specialized instruments. Taken together, the specificity and sensitivity of the RT-LAMP assay described here, in combination with its accessibility and ease of use, make this assay a valuable tool for the diagnosis and epidemiologic surveillance of human MERS-CoV infection, especially for field use.
The primer sets described in this study matched to most of the available sequences. However, several sequences had mismatches with the primers. The effect of these mismatches on the RT-LAMP efficiency was evaluated using a synthesized control RNA template: most of the mismatches did not affect the amplification efficiency of RT-LAMP. However, the substitution in B2 region of the primer, namely, a G to T substitution at position 29018 in the nucleocapsid gene sequence of the EMC isolate, was present in five MERS-CoV sequences and caused a slight decrease in sensitivity. Of the five sequences, KJ156883 (Asir_1_2013) belongs to the Buraidah_1 clade, and is the only sequence in this clade with such a substitution . The other sequences (KJ156944, Riyadh_5_2013; KJ556336, Jeddah_1_2013; KF958702, Jeddah_human1; KF917527, Jeddah_Camel1) belong to the Riyadh_3 clade [42, 43]; none of the other sequences in this clade have a substitution according to the alignment analysis. Therefore, the substitution does not represent a major population in this clade. Although it is important to improve the primers to increase their sensitivity for sequences in the Riyadh_3 clade by using mixed bases, the results of this study suggest that the RT-LAMP primer is useful for detecting the majority of the prevalent MERS-CoV strains.
The success rate of MERS-CoV detection is dependent on the collection of clinical specimens. Drosten et al., reported that up to 106 copies/mL of MERS-CoV RNA are present in lower respiratory tract specimens, such as tracheobronchial secretions, and bronchoalveolar lavage. In contrast, only small amounts of viral RNAs were detected in upper respiratory tract specimens and other tissue . The RT-LAMP primers described here were designed based upon the nucleocapsid protein sequence of MERS-CoV, due to the unique coronaviral replication system. Although coronaviruses generate subgenomic mRNAs to produce each viral protein, all subgenomic mRNAs contain nucleoprotein sequences, as they are located on the 3′end of the coronavirus genome [45–47]. This implies that the RT-LAMP procedure described here will exhibit a high degree of sensitivity for specimens containing cellular components.
This study developed a RT-LAMP assay for the MERS-CoV, which was capable of detecting as few as 3.4 copies of MERS-CoV RNA, and was highly specific, with no cross-reaction with other respiratory viruses. These results suggest that the RT-LAMP assay described here is a useful tool for the diagnosis and epidemiologic surveillance of human MERS-CoV infections.
We thank Dr. Ron A. M. Fouchier, Erasmus Medical Center, Rotterdam, The Netherlands for providing MERS-CoV EMC isolate. We also thank the staff of Mie Prefecture Health and Environment Research Institute, Mie, Japan. This work was supported by a grant-in-aid from the Ministry of Health, Labor, and Welfare, Japan. We thank Dr. Aiko Fukuma and Dr. Shuetsu Fukushi, Department of Virology I, National Institute of Infectious Diseases, Japan, for cloning of the nucleocapsid sequence of MERS-CoV.
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