A rapid screening assay for L452R and T478K spike mutations in SARS-CoV-2 Delta variant using high-resolution melting analysis

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission has been reported worldwide and novel SARS-CoV-2 variants continue to emerge. A novel SARS-CoV-2 strain, the Delta variant (B.1.617.2), is spreading worldwide. The Delta variant has reportedly high infectivity and immune evasion potency. In June 2021, the World Health Organization categorized it as a variant of concern (VOC). Therefore, it is vital to develop tests that can exclusively identify the Delta variant. Here, we developed a rapid screening assay to detect characteristic mutations observed in the Delta variant using high-resolution melting (HRM) analysis. In this assay, we determined L452R and T478K, among which T478K is an identifier of the Delta variant since L452R is seen in other strains (Kappa and Epsilon variants). Additionally, nested PCR-based HRM analysis, which involved RT-PCR (1st PCR) and HRM analysis (2nd PCR), was developed to improve the specificity and sensitivity. Our method discriminated between the L452R mutant and wild-type L452. In addition, HRM analysis distinguished the T478K mutant from the wild-type T478. Seven clinical samples containing the Delta variant were successfully identified as L452R/T478K mutants. These results indicate that this HRM-based genotyping method can identify the Delta variant. This simple method should contribute to rapid identification of the Delta variant and the prevention of infection spread.

INTRODUCTION

In December 2019, a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged from China and caused coronavirus disease 2019 (COVID-19). Since then, SARS-CoV-2 has spread rapidly worldwide and various variants of it have emerged (Gorbalenya et al., 2020; Wu et al., 2020; Zhou et al., 2020). In February 2021, the World Health Organization (WHO) classified some SARS-CoV-2 variants according to their risks to global public health. Lineage B.1.1.7 (Alpha), B.1.351 (Beta), and P.1 (Gamma) variants were categorized as variants of concern (VOCs), which are the variants that most urgently need monitoring and research (COVID-19 Weekly Epidemiological Update, 25 February, 2021). These three VOCs have the N501Y spike protein mutation in common. This mutation enhances the affinity of viral spike protein to human ACE2 receptors (Starr et al., 2020). Therefore, these N501Y variants rapidly spread across the entire globe.

Since late 2020, novel variants of SARS-CoV-2, the lineage B.1.617, have emerged in India (Cherian et al., 2021). These variants are called “double mutants” because they possess L452R and E484Q spike mutations. The E484 spike amino acid frequently interacts with antibodies. The substitution at E484 may enable the virus to escape the human immune response (Xie et al., 2021; Zhou et al., 2020). The L452R variant is also reported to be a mutant that can evade immunity (Wang et al., 2021; Deng et al., 2021). In Japan, the Ministry of Health, Labour and Welfare launched the L452R screening test to detect the B.1.617 strain. However, there are three sublines belonging to lineage B.1.617 (B.1.167.1, B.1.617.2, and B.1.167.3). Lineages B.1.617.1 (Kappa) and B.1.617.3 have both L452R and E484Q mutations (Table 1). Lineage B.1.617.2 (Delta) also possesses the L452R mutation but has the T478K mutation instead of the L484Q mutation. Since lineage B.1.427/B.1.429 (Epsilon) also has the L452R mutation, the L452R screening test cannot determine which L452R mutant has infected the host (Zhang et al., 2021; Tchesnokova et al., 2021).

Table 1. SARS-CoV-2 variants with L452R spike mutations.

WHO label	Pango lineage	WHO classification	Amino acid substitution
			L452R	T478K	E484Q
Delta	B.1.617.2	VOC	+	+	-
Epsilon	B.1.427/B.1.429	VOI	+	-	-
Kappa	B.1.617.1	VOI	+	-	+
N/A	B.1.617.3	N/A	+	-	+

VOC, variant of concern; VOI, variant of interest; N/A, not applicable

+, mutant; -, wild type

In June 2021, the WHO added the Delta variant (B.1.617.2) as a new VOC (COVID-19 Weekly Epidemiological Update, 1 June, 2021). This variant is suspected to be more infectious and less immunoreactive than its predecessors, enabling it to spread worldwide and become the dominant strain in many countries (Davis et al., 2021). It is thus crucial to identify the Delta variant exclusively with other L452R variants and differentiate it from other L452R variants. On the basis of the mutation spectra shown in Table 1, T478K would be the best screening target for identifying the Delta variant.

We recently developed an assay platform to identify former SARS-CoV-2 variants using high-resolution melting (HRM) analysis (Aoki et al., 2021). This assay can detect subtle differences between two DNA sequences at single-base resolution (Vossen et al., 2009; Zhou et al., 2015). In this study, we improved the assay to increase the sensitivity and specificity using nested PCR and identified the Delta variant using HRM analysis.

MATERIALS AND METHODS

Ethics statement

This project was approved by the Research Ethics Committee of Meijo University (Approval number: 2020-17-1) and Aichi Prefectural Institute of Public Health (Approval number: 20E-4), and it was carried out under the Infectious Diseases Control Law in Japan.

Preparation of standard RNA fragment: in vitro T7 transcription

The SARS-CoV-2 sequence used herein was obtained from NCBI (NCBI Reference Sequence: NC_045512.2) and the GISAID database (www.gisaid.org). Five DNA fragments (wild type, L452R, T478K, E484K, and E484Q mutants; 600–1000 bp in length) with a 5′ T7 upstream promoter sequence were obtained from Eurofins Genomics KK (Tokyo, Japan).

RNA fragments were synthesized by in vitro T7 transcription (CUGA 7 In Vitro Transcription Kit; Nippon Gene Co. Ltd., Tokyo, Japan), in accordance with the manufacturer’s instructions. RNA fragments were purified on spin columns (RNeasy Mini Kit; Qiagen GmbH, Hilden, Germany). Each eluent was treated with DNase I (RNase-Free DNase Set; Qiagen) and repurified on a new spin column. Synthesized single-stranded RNA fragments were used as RT-PCR amplification templates.

Reverse-transcription (RT)-PCR amplification: First PCR

RT-PCR was performed in a single closed tube using a one-step RT-PCR kit (One Step PrimeScript III RT-qPCR Mix, with UNG; Takara Bio Inc., Kusatsu, Shiga, Japan), in accordance with the manufacturer’s instructions. The primer pairs were as follows: outer forward 5′-GATGATTTTACAGGCTGCGTTA-3′ and outer reverse 5′-TGGAAACCATATGATTGTAAAGGA-3′ (Fig. 1). Each reaction mixture (20 μL) contained 2 μL of RNA solution (10⁶ copies/mL), 200 nmol/L of each primer, and 1× master mix. RT-PCR amplifications were performed under the following conditions: RT at 52°C for 5 min, initial denaturation at 95°C for 10 sec, followed by 30 cycles of denaturation at 98°C for 10 sec, annealing at 60°C for 30 sec, and extension at 68°C for 30 sec. After amplification, the reaction mixture was diluted 10,000-fold with water and used as a template for the second PCR and HRM analysis.

Fig. 1

Schematic map of primer annealing sites for RT-PCR and HRM analysis.

HRM analysis: Second PCR

The HRM reaction was performed using an HRM reagent (MeltDoctor HRM Master Mix; Thermo Fisher Scientific, Waltham, MA, USA), in accordance with the manufacturer’s instructions. The second primer pairs (Fig. 1) were as follows: L452 forward 5′-AGGCTGCGTTATAGCTTGGA-3′ and L452 reverse 5′- TCAAAAGGTTTGAGATTAGACTTCC-3′; T478 forward 5′-TTGTTTAGGAAGTCTAATCTCAAACC-3′ and T478 reverse 5′-AAGTAACAATTAAAACCTTCAACACCATTACAAGG-3′. As shown in Fig. 1, the T478 reverse primer was designed based on the E484 coding sequence to avoid the potential influence of E484K/Q mutation. Briefly, each reaction mixture (20 μL) contained 2 μL of diluted RT-PCR reaction mixture, 400 nmol/L of each primer, and 1 × master mix. All reactions were performed in duplicate on a real-time PCR system (LightCycler 96 System; F. Hoffmann-La Roche Ltd., Basel, Switzerland). PCR amplification was carried out under the following conditions: initial denaturation at 98°C for 10 min, followed by 40 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 30 sec, and extension at 72°C for 30 sec. After amplification, HRM was performed with denaturation at 95°C for 60 sec, cooling at 40°C for 60 sec, preheating at 60°C for 1 sec, and melting curve generation from 60°C to 90°C in 1°C/sec increments with 25 acquisitions. HRM curves were analyzed using Gene Scanning Software (F. Hoffmann-La Roche Ltd.) under the default settings. Normalized melting curve and melting peaks (−dF/dT) were acquired by setting pre-melt and post-melt fluorescence as 100% and 0%, respectively.

Clinical samples

From May to June 2021, a total of 14 nasopharyngeal swab or saliva samples were collected from patients suspected of having COVID-19 and those diagnosed with COVID-19 in Aichi Prefecture (except the Ichinomiya, Nagoya, Okazaki, Toyohashi, and Toyota City area). Next-generation sequencing and Sanger sequencing identified seven samples as the Delta variant with L452R and T478K mutations, while the other seven samples had intact L452 and T478. Viral RNA in clinical samples was purified on spin columns (Quick RNA Viral Kit; Zymo Research, Irvine, CA, USA) and stored at −80°C until use.

RESULTS AND DISCUSSION

One-step RT-PCR for template preparation

Our previous study suggested that nested PCR could improve the detection limit of HRM analysis (Aoki et al., 2021). Therefore, we performed nested PCR involving RT-PCR and subsequent HRM analysis. On RT-PCR, the target fragment containing spike protein L452 and T478 was amplified using an outer primer set in a single closed tube (Fig. 1). As a positive control RNA for HRM analysis, the SARS-CoV-2 RNA fragments of wild type and mutants were synthesized by in vitro T7 transcription. After RT-PCR, agarose gel electrophoresis and Sanger sequencing analysis showed that each positive control RNA template was amplified as a specific PCR product (215 bp) (data not shown). These results indicate that our RT-PCR conditions were suitable for the first PCR with an outer primer set.

HRM analysis on standard fragments

After the RT-PCR reaction, HRM analysis as a second PCR was performed using specific primer sets for L452 and T478 sites. First, L452R mutation was determined using diluted RT-PCR reaction mixtures. Normalized melting curves and melting peaks for wild type and L452R mutant are shown in Fig. 2. The L452R mutant plots were dissimilar to that of the wild type. These results indicate that the current HRM analysis can identify L452R variants such as Delta, Kappa, Epsilon, and B.1.617.3 variants. We next investigated the identification of T478K mutation by HRM analysis. As shown in Fig. 1, the T478 reverse primer was designed based on the E484 coding sequence to avoid the potential influence of E484K/Q mutation. The T478K mutant plots were dissimilar to that of the wild type (Fig. 3). No detectable influences of E484 substitution were observed in the HRM analysis (data not shown). The E484 substitute variants have emerged worldwide, such as E484K (Beta, Gamma, Zeta, Eta, Theta, Iota) and E484Q (Kappa). In Japan, the domestic transmission of SARS-CoV-2 with the E484K and E484Q mutations has been confirmed. Therefore, the newly developed HRM analysis has an important advantage in identifying T478K mutation without any interference from the E484 substitution.

Fig. 2

Normalized melting curves and melting peaks of positive control RNAs for the L452 site. Normalized melting curve plots (A) and melting peak plots (B) for the L452 site were acquired using standard fragments of the wild type (blue line) and L452R mutant (red line).

Fig. 3

Normalized melting curves and melting peaks of positive control RNAs for the T478 site. Normalized melting curve plots (A) and melting peak plots (B) for the T478 site were acquired using standard fragments of the wild type (blue line) and T478K mutant (red line).

HRM analysis on clinical samples

Finally, we confirmed the applicability of this HRM analysis to clinical samples. Seven Delta variant samples were analyzed by HRM analysis together with positive control RNAs. After HRM analysis, Gene Scanning Software automatically classified samples into wild type or mutant based on HRM curves. At the L452 site, the melting peaks of seven clinical samples containing the Delta variant were in good agreement with those of L452R positive control RNA and classified as L452R mutants (Fig. 4A). In addition, all Delta variant sample plots of the T478 site differed from the wild-type plots (Fig. 4B). The other seven clinical samples with intact L452 and T478 were in good agreement with wild-type positive control RNAs. These results indicate that our HRM-based genotyping method for L452R and T478K can identify the SARS-CoV-2 Delta variant.

Fig. 4

Melting peaks of clinical samples for the L452 and T478 sites. Melting peak plots for the L452 (A) and T478 (B) sites were acquired using seven clinical samples with the Delta variant (orange line), seven clinical samples with intact L452/T478 (lime line), wild-type positive control RNA (blue line), and mutant positive control RNA (red line).

In conclusion, this study succeeded in developing a rapid screening test for the Delta variant using both L452R and T478K mutations. Because the assay does not require a sequence-specific probe (e.g., TaqMan probe), a cost-effective assay can be constructed rapidly using HRM analysis. Nonetheless, this technique still requires future confirmation using multiple samples to calculate the sensitivity and specificity.

ACKNOWLEDGMENTS

The authors would like to thank all staff of the Laboratory of Virology, Department of Microbiology and Medical Zoology, Aichi Prefectural Institute of Public Health, who performed COVID-19 PCR testing and purified RNA from clinical samples. This work was supported in part by Meijo University Research Project for Countermeasures Against COVID-19. We thank Edanz Group for editing a draft of this manuscript.

Conflict of interest

The authors declare that there is no conflict of interest.

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