1 Introduction

Severe acute respiratory syndrome 2 (SARS-CoV-2) is the viral agent that causes coronavirus disease 2019 (COVID-19). In recent years, the importance of rapid detection of genetic materials of pathogens and finding therapeutic approaches against viruses got more pronounced (Rosenthal 2020; Vandenberg et al. 2021; Zaman et al. 2020; Dastjerdi-Khorzoghi et al. 2019). In the fight against viral pathogens, many techniques have been developed for the detection of the genomic materials of the pathogens to overcome the uncontrolled infections. In this respect, commercial tests for COVID-19 diagnosis comprise three main categories: molecular detection of genetic materials, antigen tests, and serological methods (Machado et al. 2021) from which the former is considered the gold standard method by the World Health Organization (WHO) for detection of SARS-CoV-2. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) is the most applied method; however, this conventional method has limitations such as the long time to results (TTR), up to six hours, as well as its dependence on high-tech instrumentations, e.g., fluorescently equipped thermal cyclers. The development of methods that provide detection of the genetic materials of the virus in less equipped laboratories is necessary for the rapid isolation of asymptomatic and symptomatic patients, especially in low-budget countries. The method would be more beneficial if it could reduce TTR (Song et al. 2021; Afzal 2020).

Isothermal amplification techniques are the method of choice to provide molecular diagnostic tests for the detection of genetic materials that accommodate low TTR with the least instrumentation. These methods have been validated as a point of care (POC) test as well (Bodulev and Sakharov 2020). Methods such as loop-mediated isothermal amplification (LAMP) (Nzelu et al. 2019), helicase-dependent amplification (HDA) (Ma et al. 2017), rolling circle amplification (RCA) (Shen et al. 2019), strand displacement amplification (SDA) (Becker et al. 2019), recombinase polymerase amplification (RPA) (Rostron et al. 2019), and nucleic acid sequence-based amplification (NASBA) (Zhai et al. 2019), unified amplifier-based primer exchange reaction (UniAmPER) (Tavakoli-Koopaei et al. 2022) are examples of isothermal amplification methods that have been developed in recent years for the detection of genetic materials of viruses. Among these methods, LAMP was able to achieve governmental approvals as IVD (Zhao et al. 2016; Zhang et al. 2015; Etchebarne et al. 2017).

LAMP starts with four primers (forward primer, backward primer, forward internal primer, and backward internal primer). After the first round of synthesis, loop primers are attached to the newly synthesized products and continue a highly specific and effective amplification (Nagamine et al. 2002). The reaction products are long double-stranded DNAs.

The LAMP reaction products are produced with high efficiency and can provide colorimetric signals for visual detection with bare eyes. Visual detection methods are the method of choice in many detection systems (Sadeghi et al. 2017; Javani et al. 2017; Anantharaj et al. 2020). The signal could be generated with either pH indicators or magnesium pyrophosphate indicators (Scott et al. 2019; Gachugia et al. 2020; Goto et al. 2009). Phenol red is a pH indicator and malachite green (MG) and hydroxyl naphthol blue (HNB) are magnesium pyrophosphate indicators (Lucchi et al. 2016; Kudyba et al. 2019; Choopara et al. 2017). Real-time monitoring of the reactions is another beneficial aspect that has been addressed in many studies (Dehghanian et al. 2020; Higuchi et al. 1993). Real-time detection of the LAMP reaction is possible through the interaction of the double-stranded long products of LAMP with intercalating dyes (Aglietti et al. 2019; Ristaino et al. 2020).

A clear advantage of the method is that it may get coupled with reverse transcription reactions to target RNA genomes as reverse transcription loop-mediated isothermal amplification (RT-LAMP) in one step (da Silva et al. 2019). RT-LAMP is one of the useful methods for rapid diagnosis of SARS-CoV-2 (Alekseenko et al. 2021; Fowler et al. 2021; Lim et al. 2021).

Primer design is a crucial step in LAMP-based assays. Software such as Primer Explorer (Park et al. 2020; Zhang et al. 2020) and LAMP designer (da Silva Gonçalves et al. 2014; Abdulmawjood et al. 2016; Elvira-González et al. 2017) has been developed to design LAMP primer sets for candidate genomic regions (Jia et al. 2019; Shirshikov et al. 2019). Such software usually results in tens of primer sets that their sensitivity and specificity need to be investigated.

The objective of the study was to develop a validated RT-LAMP primer set that enables accurate and fast visual detection of the SARS-CoV-2 by malachite green. In the first step, the RT-LAMP primer sets were designed for conserved regions of the SARS-CoV-2 genome using both Primer Explorer and LAMP designer software. The top scored designed primer sets of both software were experimentally validated with RT-LAMP assay, and then, the best primer set was chosen for the development of the malachite green-based one-step SARS-CoV-2 diagnostic test (Fig. 1A).

Fig. 1
figure 1

A The steps of reverse transcription loop-mediated isothermal amplification (RT-LAMP). The sample was collected by nasopharyngeal swab at the first step after that the RNA was extracted and applied as a template for RT-LAMP reaction in presence of malachite green. The colorimetric RT-LAMP product signals were visible with the naked eye after incubation at 37 °C for 2 h. B Hot spots region of SARS-CoV-2 genomes that were sequenced from samples of Iranian patients. The gray circles specify the parent nucleotide at the reference genome (MN908947). The colored circle is the mutated nucleotide. The numbers above each circle indicate the position of the mutation at the reference genome

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2 Experimental Details

2.1 Materials

All primers were supplied by Tag Copenhagen. Co. Ltd. (Denmark). RNA isolation kit and one-step RT-qPCR kit were supplied by Behgene, Biotech, Iran, and Sansure Biotech, China, respectively. AddScript cDNA Synthesis Kit was from AddBio, Korea. Bst DNA polymerase large fragment (M0275L) was purchased from New England Biolabs, USA. Mineral oil was from Sinaclone, Iran. Malachite green, agarose, tris (hydroxymethyl)-aminomethane (Tris), boric acid, ethylenediaminetetraacetic acid (EDTA), ethidium bromide solution, and deoxyribonucleotides were purchased from Sigma-Aldrich. The reaction tubes were incubated in LightCycler® 96 instrument (Roche, Germany).

2.2 Clinical Samples and Materials

This study included samples of hospitalized patients with a respiratory infection or suspected patients who were in contact with affected individuals or indicated some clinical signs of COVID-19 and routinely referred to healthcare centers affiliated with Isfahan University of Medical Sciences, Isfahan, Iran. The nasopharyngeal/oropharyngeal swab samples were collected and kept in collection tubes containing 2-ml virus transport medium, delivered to the laboratory, worked with biosafety level 2, and inactivated by mixing to lysis buffer and heating at 65 °C.

2.3 RNA Extraction and RT-qPCR Validation Tests

The genomic RNAs were extracted by RNA isolation kit. A 10-µl aliquot of extracted SARS-CoV-2 RNA was used for RT-qPCR tests by a specific probe-based one-step RT-qPCR kit, targeting ORF1ab, and N genes according to the manufacturer’s protocol. The amplification process was carried out in 25 µl. According to the manufacturer’s protocol, the reverse transcription step was done with incubation for 30 min at 50 °C. Cyclic amplification of synthesized cDNA was carried out as follows: 1 min 95 °C for primary denaturation, followed by 45 cycles at 95 °C (15 s) and 60 °C (30 s). The calculated SARS-CoV-2 cycle threshold (Ct) values were analyzed and applied for quantification of the input RNA sample in the RT-LAMP reaction. Purified RNA samples with the matching number of Ct values were maintained at − 80 °C for further analysis.

2.4 LAMP Primer Design

To define conserved regions of the SARS-CoV-2 genome, Iranian partial and complete genomes which were deposited in both the GISAID and NCBI databases were aligned and compared with the reference genome (MN908947) by Clustal Omega software. The hot spots of nucleotides in Iranian genomes are shown in Fig. 1B. Primer Explorer V5 and LAMP designer were applied to design primers for non-mutated regions of gene N of SARS-CoV-2. The top score primer sets of each software were subjected to cross-reactivity analysis by the NCBI Primer Blast tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). Primers were blasted with similar organism genomes as mentioned in Fig. 2A. Primer details are listed in Table 1 and Fig. 2B and C.

Fig. 2
figure 2

Details for specific binding sites and seqeunces of primers. A Organisms that were analyzed for cross-reactivity check with primer blast. Taxids are mentioned in parentheses. B and C The primers binding sites of Primer Explorer and LAMP designer on the N gene of SARS-CoV-2, respectively

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Table 1 Designed primer sets with both primer explorer and LAMP designer software for N genes of SARS-CoV-2
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2.5 Two-Step RT-LAMP

A reverse transcription reaction was performed with a reverse transcription kit containing 6 µl of extracted RNA, 10 µl of 2X reaction buffer, 1 µM B3 primer, 0.2 mM dNTPs. The reaction volume was 20 µl. LAMP reaction was carried out with a final volume of 25 μl reaction mixture containing 0.2 μM B3 and F3, 1.6 μM of BIP and FIP, 0.8 μM of LF and LB, 1X isothermal amplification buffer [20 mM of Tris–HCl (pH 8.8), 10 mM of KCl, 10 mM of (NH4)2SO4, 2 mM of MgSO4, 0.1% of Triton-X100], 1.2 mM of dNTP mix, supplementary 8 mM of MgSO4, 8 U of Bst DNA polymerase large fragment and 2 μl of cDNA as template. Finally, 25 μl of mineral oil was added on top. The LAMP reactions were carried out at 65 °C for 60 min, and the products were subjected to agarose gel electrophoresis and post-staining with ethidium bromide (0.5 μg/ml) to validate the LAMP reaction. For the colorimetric RT-LAMP assay, the reaction tubes were prepared with the addition of malachite green prior to the reaction.

2.6 One-Step, MG-Assisted RT-LAMP

One-step LAMP reaction was carried out in 25 μl in presence of 10 µl of 2X AddScript reaction buffer, 0.2 μM B3 and F3 each, 1.6 μM BIP and FIP each, 0.8 μM LF and LB each, 1X isothermal amplification buffer, 1.2 mM the dNTPs mix, supplementary 8 mM MgSO4, 8 U Bst DNA polymerase large fragment, 6 μL extracted RNA, and 0.02% malachite green, except mentioned otherwise. Finally, 25 μl mineral oil was added on top of the reaction. Tubes were incubated in a thermal cycler with two-step program: 50 °C for reverse transcription reaction within the first 1 h and 65 °C LAMP amplification within the second 1 h. To confirm the result 5 µl of the reactions was loaded on 2% agarose gel and stained by ethidium bromide.

3 Results

3.1 RT-qPCR for Quantification of RNA Samples

RNA-extracted clinical samples were analyzed by RT-qPCR. Figure 3 depicts the Ct values for the same samples that were applied for RT-LAMP reactions. From this list, two samples were proven to be negative in SARS-CoV-2 RNA and the rest had Ct values from 10 to 33 according to RT-qPCR tests.

Fig. 3
figure 3

Quantification of extracted RNA samples based on one-step RT-qPCR. The corresponding Ct values of each sample are depicted on the side

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3.2 Validation of RT-LAMP Primers Set

Initially, the efficiency and accuracy of the two primer sets were investigated in two-step RT-LAMP reactions with samples 1 and 2, with respected Ct values of 18 and 19. Since the number of primers for amplification via LAMP is higher than other isothermal methods, there are higher chances for false-positive results upon primer dimer extensions and self-amplification (Meagher et al. 2018); hence, control reactions that contained all 6 primers and reaction components except the template were necessarily applied here to indicate if false positives are present (Fig. 4A). Both primer sets were efficient in the detection of SARS-CoV-2 genomic RNA from positive clinical samples. Notably, the primer set which was designed by the Primer Explorer did not show any extension in the no-template control. However, the primer set that was designed by LAMP designer software showed extended bands, apparently due to the presence of primer dimers. Therefore, the first primer set was selected for further experiments in this study.

Fig. 4
figure 4

A LAMP primer sets validation and RT-LAMP reaction in presence of a different concentration of malachite green. A: Two-step RT-LAMP reactions were performed with no-template controls to investigate of primer dimer formation. Primer set 1 which was designed by Primer Explorer software was shown any primer dimer formation neither in the presence nor absent of a template. The top score primer sets of the LAMP designer (primer set 2) were shown primer dimer formation, and it was not suitable for further RT-LAMP reaction. B Primer set 1 was subjected to RT-LAMP reaction in presence of negative control which contains human’s RNA. C RT-LAMP reaction in presence of 0.02% MG (the optimized concentration) with control reaction was loaded into 2% agarose gel. D The two-step RT-LAMP reaction was performed with 0.004, 0.008% MG as mentioned in the previous study. The RT-LAMP assay with MG concentrations of 0.02%, 0.4%, and 0.08% was also done to find an optimized MG concentration

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LAMP primer sets must not show any cross-reactivity with the human genome and transcriptome. Usually, nasopharyngeal samples contain human cells as well as viral particles. Therefore, it is necessary to make sure that the primer set does not bind any RNA from the human transcriptome. To this endeavor, two-step RT-LAMP reactions were performed in presence of negative controls that contained RNA templates, but the RNA samples were from individuals that were negative in SARS-CoV-2 according to RT-qPCR, i.e., samples 3 and 4. Figure 4B shows the results of the RT-LAMP reaction for RNA-negative samples 3 and 4 and RNA-positive samples 5 and 6.

3.3 Two-Step Colorimetric RT-LAMP

RT-LAMP products may be read upon fluorometric or colorimetric signals. Fluorometric signals are usually generated by SYBR green (Naji et al. 2019) or CYTO9 (Tangkanchanapas et al. 2018) which require equipped instruments for detection. On the other hand, colorimetric RT-LAMP products are detectable by the naked eye. As an example, some commercial colorimetric RT-LAMP kits are based on the pH change during RT-LAMP reaction with pH indicators such as phenol red.

Hydroxy naphthol blue (Goto et al. 2009) and malachite green (Nzelu et al. 2016) are other reagents that produce colorimetric signals in RT-LAMP assays. Both reagents are the indicator of magnesium pyrophosphate that is synthesized during the polymerization reaction as a by-product. Here, malachite green-based two-step RT-LAMP (MG-RT-LAMP) assays were performed with positive clinical samples in the presence of 0.004% MG (Lucchi et al. 2016). In addition, the concentration of MG was varied up to 20 fold. Reaction tubes are shown in Fig. 4C. MG in concentrations 0.004% and 0.008 could not pose any obvious difference between positive and negative controls. Here the positive control was sample number 6, and the negative control was sample 3. The color generated in the control tube was detectable from the test when the MG concentration raised above 0.02%. However, the background was also increased, while the concentration of MG went higher. The color produced in the test and control tubes was the same when the malachite green concentration reached 0.04% and above. Increasing the MG concentration enhanced the background signal and prevented the test and control tubes to be distinguished.

Therefore, the best colorimetric signal was produced with 0.02% of the MG concentration. The samples were loaded into an agarose gel to validate the presence of the RT-LAMP reaction products. The result showed that the reaction was efficient in the presence of the optimized concentration of malachite green (Fig. 4D).

3.4 One-Step Colorimetric RT-LAMP

Finally, the reactions were performed with different RNA concentrations as quantified with RT-qPCR. Three tubes of MG-RT-LAMP assays were analyzed with RNA samples with Ct values of 20 (sample 7) and 33 (sample 8) and also a negative RNA (sample 4). The reaction tubes were incubated at 65 °C for 2 h and loaded into agarose gel (Fig. 5). The colorimetric signal of RNA samples with Ct value 20 significantly was above the signal that has been generated in negative RNA samples. However, the RNA samples with Ct value 33 did not show any significant difference to the negative RNA samples. Further experiments validated that SARS-CoV-2 RNA genomes with Ct values below 33 could be detected using one-step MG-RT-LAMP assay with the primer set that was designed by Primer Explorer software, but the system had limitations for detection of lower concentrations of genomic RNAs.

Fig. 5
figure 5

One-step MG-RT-LAMP reactions with different RNA concentrations. A: RT-LAMP reaction was performed in presence of 0.02% malachite green. The test tubes contained extracted RNA with corresponding Ct 20 and 33, respectively. Negative virus samples were also present as the control. B: The same samples of A were loaded at 2% agarose gel as shown above

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4 Discussion and Future Prospect

Rapid diagnosis of SARS-CoV-2 is necessary to prevent the transmission cycle of the disease among people. In response to this urgency, fast and non-expensive methods are required that support detection of the people with various financial and work situations. Isothermal amplification strategies are hence the method of choice in this respect, and many of them have been developed for rapid detection of COVID-19 during pandemic (Huang et al. 2020; Dao Thi et al. 2020; Aoki et al. 2021). LAMP was the most attractive method among other isothermal amplification methods since it applies non-modified primers and allows detection by simple readout systems (Amaral et al. 2021; Alhamid et al. 2022). However, in general, isothermal amplification methods, including LAMP, are not as sensitive as the gold standard detection system (i.e., RT-qPCR) for SARS-CoV-2, which means the limit of detection of the system (LOD) is higher than that of RT-qPCR. Such limitation still allows diagnosis of people with high viral load, which is sufficient to block the infection cycle, since the virus is mostly transmitted by patients with high viral load (Wyllie et al. 2020; Gupta et al. 2020; Wölfel et al. 2020). With an acknowledge to being fast and non-expensive, RT-LAMP would be beneficial for application in low-income countries to avoid the complications of health challenges. RT-LAMP has a simple protocol, and its reaction is performed at an instant temperature. Optimization of the colorimetric RT-LAMP method was investigated in this study by using malachite green, during the amplification process. The results of this study are applicable to further development of visual RT-LAMP systems and being prepared for future pandemics.