Development of a multiplex Loop-Mediated Isothermal Amplification (LAMP) assay for on-site diagnosis of SARS CoV-2

A newly identified coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), has spread rapidly from its epicenter in China to more than 150 countries across six continents. In this study, we have designed three reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primer sets to detect the RNA-dependent RNA polymerase (RdRP), Envelope (E) and Nucleocapsid protein (N) genes of SARS CoV-2. For one tube reaction, the detection limits for five combination SARS CoV-2 LAMP primer sets (RdRP/E, RdRP/N, E/N, RdRP/E/N and RdRP/N/Internal control (actin beta)) were evaluated with a clinical nasopharyngeal swab sample. Among the five combination, the RdRP/E and RdRP/N/IC multiplex LAMP assays showed low detection limits. The sensitivity and specificity of the RT-LAMP assay were evaluated and compared to that of the widely used Allplex™ 2019-nCoV Assay (Seegene, Inc., Seoul, South Korea) and PowerChek™ 2019-nCoV Real-time PCR kit (Kogenebiotech, Seoul, South Korea) for 130 clinical samples from 91 SARS CoV-2 patients and 162 NP specimens from individuals with (72) and without (90) viral respiratory infections. The multiplex RdRP (FAM)/N (CY5)/IC (Hex) RT-LAMP assay showed comparable sensitivities (RdRP: 93.85%, N: 94.62% and RdRP/N: 96.92%) to that of the Allplex™ 2019-nCoV Assay (100%) and superior to those of PowerChek™ 2019-nCoV Real-time PCR kit (RdRP: 92.31%, E: 93.85% and RdRP/E: 95.38%).


Introduction
In December 2019, an outbreak in Wuhan, China of a severe respiratory illness was caused by a previously unrecognized coronavirus, which has since been named severe acute respiratory a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 2019-nCoV Assay (Seegene, Inc., Seoul, South Korea). We suspected the sensitivity and specificity of the multiplex RT-LAMP assay to be 95% with a desired margin of error of 0.04%. Under these conditions, the number of required samples is 114.0475 (rounded up to 115) per group. In this experiment, we have tested total 292 samples (130 positive and 162 negative). A total of 130 clinical samples, including nasopharyngeal (NP) swabs, oropharyngeal (OP) swabs, sputum, saliva and urine, were collected from 91 patients suspected of being infected with SARS CoV-2 in the Republic of Korea. All clinical samples were confirmed using the Allplex TM 2019-nCoV Assay (Seegene, Inc., Seoul, South Korea) and PowerChek™ 2019-nCoV Real-time PCR kit (Kogenebiotech, Seoul, South Korea). To assess the specificity of the multiplex SARS CoV-2 RT-LAMP assay, 162 NP swab specimens were tested from individuals with (72) and without (90) viral respiratory infections. Respiratory viral infections, as confirmed by PCR using the Anyplex TM II RV16 detection kit, included 39 coronavirus (KHU1, NL63, 229E), 3 influenza virus A/H1N1, 3 influenza virus A/H3N2, 3 influenza virus B, 3 respiratory syncytial virus (RSV) A, 3 RSV B, 3 adenovirus, 3 parainfluenzavirus (PIV) types 1 to 4, 3 human bocavirus (HboV), 3 human enterovirus (HEV), 3 human rhinovirus (HRV) and 3 metapneumovirus (MPV). RNA was extracted from 200 μL of SARS CoV-2 clinical samples using an InviMag Universal RNA Mini Kit (Stratec Molecular, Berlin, Germany), according to the manufacturer's manual. RNA extraction from the 162 NP swab controls was performed using the QIAamp Viral RNA Mini kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. RNA was stored at -50˚C. The SARS CoV-2 RT-LAMP was performed blindly with the operator unaware of any previous test results.

Primer design
The RT-LAMP primer sets for SARS CoV-2 were designed from conserved regions of the RdRP, E and N genes ( Table 1). All LAMP primers including two outer primers (forward primer F3 and backward primer B3), two inner primers (forward inner primer FIP and backward inner primer BIP), and two loop primers (forward loop primer LF and backward loop primer LB) were designed using the Primer Explorer version 4 software (Eiken Chemical Co., Tokyo, Japan). A 32-oligomer or 35-oligomer fluorophore strand-displaceable probes was designed at the 5' end of the LB primer, and the 30-oligonucleotide or 35-oligonucleotide quencher was complementary to the probe. Strand-displaceable probes were 5 0 -labeled with FAM, Hex and Cy5 for RdRP, E and N, respectively. Before use in LAMP, all primers were assessed for specificity by performing a BLAST search. All LAMP primers and probes were synthesized by Macrogen, Inc (Seoul, South Korea).

Limits of detection
pTOP Blunt V2 plasmids, including partial RdRP, E or N gene sequences of SARS CoV-2, were used to test the limit of detection of the RT-LAMP assay. All plasmids were constructed by Macrogen, Inc. (Seoul, South Korea). The plasmids were serially diluted 10-fold from 1 × 10 8 copies/μL to 1 × 10 0 copies/μL to determine the detection of limit of the multiplex SARS CoV-2 RdRP/N/IC RT-LAMP assay. In addition, the detection limit of the multiplex SARS CoV-2 RdRP/N/IC RT-LAMP was tested on 10-fold serially diluted clinical samples from SARS CoV-2 patients.

Optimization of the multiplex SARS CoV-2 RT-LAMP assay
The sensitivity of the SARS CoV-2 RdRP, E and N gene RT-LAMP was evaluated by testing synthetic plasmid standards, including synthetic partial RdRP, E and N genes ranging from 10 8 to 10 0 copies/μL, respectively (Fig 1). The limits of detection for the RdRP gene E gene and N gene were 1x10 1 copies/μL, 1x10 1 copies/μL and 1x10 2 copies/μL, respectively.  (Table 2). Among the three ratios of both the RdRP/N primer set and E/ N primer set, a ratio of 1:0.5 showed faster Ct values (12.64/12.03 and 14.04/11.6, respectively) and the most stable graph. In the case of the RdRP/E primer set and RdRP/E/N primer set, the ratio of 1:1 and 0

Comparison of detection limits of the multiplex SARS CoV-2 RT-LAMP assay with two commercial RT-qPCR assays for SARS CoV-2 clinical samples
The detection limits of monoplex SARS CoV-2 LAMP primer sets were compared to those of two commercial RT-qPCR kits (Allplex™ 2019-nCoV Assay and PowerChek™ 2019-nCoV Real-time PCR kit) for 10-fold serial dilutions of SARS CoV-2 NP samples (range of 10 −3 -10 −7 ) ( Table 4 and  (Table 5). For the SARS CoV-2 clinical samples

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(Kogenebiotech, Seoul, Korea) ( Table 6). As a result, all three molecular diagnostic tests showed no cross-reactivity with other infectious viruses. Particularly, the multiplex SARS CoV-2 RdRP/ N/IC RT-LAMP assay do not cross-react with human coronavirus 229E, NL63 and OC43.

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(MERS CoV) and severe acute respiratory syndrome coronavirus (SARS CoV) [18,19]. The genome of SARS CoV-2 consists of approximately 30,000 bases [20,21]. A phylogenetic analysis revealed that genome sequences of SARS CoV-2 from different patients were extremely similar (with 99.98% identity) and that SARS CoV-2 was closely related (with 88% identity) to two bat-derived SARS-like coronaviruses, bat-SL-CoVZC45 and bat-SL-CoVZXC21, collected in 2018 in Zhoushan, in eastern China [22]. Commercial SARS CoV-2 diagnostic RT-PCR kits detect 2-3 genes to produce a more accurate diagnosis of SARS CoV-2. Since the sensitivity of each primer set in clinical samples may be different, it is diagnosed as a positive sample when 2-3 genes are all positive, and if only one is identified, it is re-tested with another kit. Therefore, the multiplex primer set to detect two more genes is important in developing the SARS CoV-2 LAMP kit. Currently, several SARS CoV-2 LAMP primer sets were reported [23][24][25][26]. They were mostly developed with fast colorimetric detection of one or two genes suitable for on-site diagnosis [27][28][29][30]. However, it has disadvantages in not producing diagnose with multiplex testing and having to test each primer set individually. In particular, the LAMP assay has been reported to be highly susceptible to contamination [31,32], and the recently reported SARS CoV-2 RT-LAMP assay has also pointed out such a problem [33]. Therefore, if an RT-LAMP test for one clinical sample is performed with three or four LAMP primer sets (including internal control) individually, the degree of contamination may also increase. In addition, when conducting clinical tests in large quantities, the number of clinical trials more than doubles, and the advantage of a rapid diagnosis of the LAMP assay may be diluted. Therefore, the multiplex SARS CoV-2 RdRP/N/IC RT-LAMP assay developed in this study has an advantage in minimizing the contamination and enabling a mass diagnosis.
Finally, the multiplex SARS CoV-2 RdRP/N/IC RT-LAMP assay showed 100% specificity, with no cross reactivity for NP samples from patients infected with other respiratory viruses (including Coronavirus 229E, NL63 and OC43) and from uninfected healthy controls. Unfortunately, the multiplex SARS CoV-2 RdRP/N/IC RT-LAMP assay was not tested for cross reactivity against SARS CoV or other bat-derived SARS-like coronaviruses.
While the two types of RT-qPCR kit take 2 hours and 30 minutes of assay time, the multiplex SARS CoV-2 RdRP/N/IC RT-LAMP assay is very fast and produces results within 40 minutes, so if we use a 15-minute nucleic acid auto-extractor, it is possible to finish an assay within 1 hour. Therefore, if used with multi-channel isothermal equipment, such as a T16-ISO Instrument (Axxin, Australia), it will be useful for airports, ports, emergency rooms, and drive thru type SARS CoV-2 testing systems.
Here, we have developed a multiplex SARS CoV-2 RdRP/N/IC RT-LAMP assay capable of detecting RdRP, N genes and IC (actin beta, IC) in a single tube. Since the multiplex SARS CoV-2 RdRP/N/IC RT-LAMP assay takes less time (approximately 40 min), compared to the commercial Allplex™ 2019-nCoV Assay and PowerChek™ 2019-nCoV Real-time PCR kit (usually 2-3 hours), it shows promise for deployment as an on-site molecular diagnostic test.