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. 2023 May 23;262:124711. doi: 10.1016/j.talanta.2023.124711

A polyethylene glycol enhanced ligation-triggered self-priming isothermal amplification for the detection of SARS-CoV-2 D614G mutation

Luxin Yu a,b,1, Zibin Tang a,b,1, Yuanzhong Sun a,b,1, Hai Yi a,b, Yuebiao Tang a,b, Yangqing Zhong a, Dongchun Dian a, Yanguang Cong a, Houqi Wang b, Zhaoyang Xie a,b,∗∗∗, Suhui He a,b,∗∗, Zhangquan Chen a,b,∗
PMCID: PMC10205128  PMID: 37244245

Abstract

We presented a polyethylene glycol (PEG) enhanced ligation-triggered self-priming isothermal amplification (PEG-LSPA) for the detection D614G mutation in S-glycoprotein of SARS-CoV-2. PEG was employed to improve the ligation efficiency of this assay by constructing a molecular crowding environment. Two hairpin probes (H1 and H2) were designed to contain 18 nt and 20 nt target binding site at their 3′ end and 5′ end, respectively. In presence of target sequence, it complemented with H1 and H2 to trigger ligation by ligase under molecular crowding condition to form ligated H1–H2 duplex. Then 3′ terminus of the H2 would be extended by DNA polymerase under isothermal conditions to form a longer extended hairpin (EHP1). 5′ terminus of EHP1 with phosphorothioate (PS) modification could form hairpin structure due to the lower Tm value. The resulting 3’ end overhang would also fold back as a new primer to initiate the next round of polymerization, resulting in the formation of a longer extended hairpin (EHP2) containing two target sequence domains. In the circle of LSPA, long extended hairpin (EHPx) containing numerous target sequence domains was produced. The resulting DNA products can be monitored in real-time fluorescence signaling. Our proposed assay owns an excellent linear range from 10 fM to 10 nM with a detection limit down to 4 fM. Thus, this work provides a potential isothermal amplification method for monitoring mutations in SARS-CoV-2 variants.

Keywords: SARS-CoV-2, D614G mutation, Polyethylene glycol, Isothermal amplification

Graphical abstract

A polyethylene glycol enhanced ligation-triggered self-priming isothermal amplification for the detection of SARS-CoV-2 D614G mutation with high sensitivity and selectivity.

Image 1

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1. Introduction

During the past three years, the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a severe threat to global public health. Up to February 08, 2023, the World Health Organization (WHO) Situation Report has recorded over 754 million confirmed cases of COVID-19 and over 6.8 million deaths all over the world [1]. As the COVID-19 pandemic spread rapidly, mutations of spike glycoprotein have occurred in the SARS-CoV-2 during virus evolution. D614G mutation which caused by the A23403G substitution in the spike protein resulting in replacement of aspartic acid with glycine at position 614 of the spike protein (D614G) is one of the SARS-CoV-2 mutations in all variants of concern (VOCs). The D614G variant was reported to increase the infectivity and influence the immunity and partial vaccine escape of the COVID-19 virus [[2], [3], [4]]. Additionally, the mutations such as K417 N, E484K, N501Y were verified to cause antibody resistance of SARS-CoV-2 variants [5]. The continuous emergence of novel SARS-CoV-2 variants call for direct and rapid detection approach for monitoring of the transmission of the virus all over the world.

To date, antigen and antibody detections have been used for early diagnostics [6,7]. Point-of-care antigen tests are cost-effective and easy-to-use to provide results in short analysis time [8,9]. However, antigen assay cannot provide the mutation information directly. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) is considered as the gold standard for the SARS-CoV-2 detection. However, qRT-PCR is not suitable for direct mutation detection [[10], [11], [12], [13]]. There are many PCR-based methods including amplification refractory mutation system-PCR (ARMS-PCR) [14,15], variant-specific RT-qPCR [16], and high-resolution melting analysis [17] which have been developed for SARS-CoV-2 mutation detection. Unfortunately, these methods are limited to the problems of complicated procedures, easy contamination and requirement for professional personnel which hinder their application outside central laboratories [18]. Recently, isothermal amplification-based approaches have been developed as potential alternatives to conventional PCR-based approaches due to their convenience, rapidity, and low-cost. For example, recombinase polymerase amplification (RPA) [19,20] and loop-mediated isothermal amplification (LAMP) [[21], [22], [23]] have been applied to detect SARS-CoV-2. Unfortunately, these methods also suffer from the limitations of nonspecific amplification and false-positive results. Furthermore, RPA or LAMP associated detection methods are difficult to be employed in direct nucleic acid point mutation analysis. Notably, Changjun Hou and his colleagues cleverly took advantage of the mutated gene-PIK3CAH1047R with the recognition site of endonuclease FSpI to initiate strand displacement amplification and successfully detect the target [24]. However, this strategy requires specific recognition site so that it’s hard to be used as a universal single-nucleotide polymorphisms (SNP) detection method. Recently, nucleic acid sequencing-based methods are standard tests for analyzing SARS-CoV-2 variant with the advantages of accuracy. However, these methods are often complex, labor intensive and costly. Therefore, universal detection technologies for rapidly and directly monitoring of SARS-CoV-2 mutation are urgently needed.

Self-priming isothermal amplification has been developed for nucleic acid assay due to its cost-effectiveness and portability [[25], [26], [27], [28]]. Ellington and co-workers proposed a 5′ end phosphorothioate (PS) modification self-priming isothermal amplification system for SNP assay with a detection limit of 5 pM [29]. Ligation based isothermal amplification is one of widely used techniques in mutation or single nucleotide polymorphism detection [[30], [31], [32]]. However, the amplification efficiency of these techniques is greatly affected by the ligation rate, especially at the low target concentration. Several literatures reported that the ligation rate would be increased sharply by conducting the ligation reaction in a molecular crowding environment created by polyethylene glycol (PEG) [[33], [34], [35], [36]]. PEG as one of macromolecules can cause large changes in equilibria or rates of chemical reactions. Several works claimed that in vitro ligation at which DNA ligase joins DNA sequences with blunt or short cohesive ends increased ligation rate over 1000-fold by PEG. High concentrations of PEG 6000 increase the ligation rate by increasing the effective concentration of DNA probes to be joined in a molecular crowding environment created by PEG [37,38]. Kae Sato's group has developed sensitive DNA detection strategies through rolling circle amplification assisted by PEG [39,40].

Upon this background, we herein propose a polyethylene glycol (PEG) enhanced ligation-triggered self-priming isothermal amplification (PEG-LSPA) for fluorescent detection of SARS-CoV-2 mutation at gene level. In this work, we firstly employed the high specific ligase reaction to discriminate mutant target and wild type target. Isothermal amplification was initiated by destabilizing base stacking in the double helical structure with phosphorothioate (PS)-modification which result in the reduction of the melting temperature (Tm) of PS-DNA/DNA duplex [41], and formation of self-primer structure due to unstable PS-DNA/DNA double strands. Then we exploited PEG 6000 to enhance ligation efficiency and sensitivity of this method by constructing a molecular crowding environment. Thus, this work provides a promising universal isothermal amplification method for directly and rapidly detecting SARS-CoV-2 mutations.

2. Material and methods

2.1. Reagents and chemicals

The Bst 2.0 WarmStart DNA polymerase, HiFi Taq ligase and deoxynucleotide triphosphates (dNTPs) solution were purchased from New England Biolabs (New England, USA). SYBR Green Ⅰ, betaine and PEG 6000 was purchased from Solarbio (Guangzhou, China), NuSeive GTG agarose was obtained from Lonza (Basel, Switzerland). Other chemicals were purchased from standard commercial sources and were of analytical grade. The 0.22 μm MILLEX®GP filter unit was purchased from Merck Millipore (Merck Millipore, USA). Ultrapure water (18.2 MΩ/cm) was used to prepare the buffer solution. Phosphorothioate and phosphorylation modified hairpin probes and other oligonucleotide sequences used in this study were synthesized and purified through HPLC by Sangon Biotech (Shanghai, China). The detailed oligonucletide sequences were listed in Table S1 of the supplementary data.

2.2. Preparation of hairpin probes (H1 and H2)

All of the hairpin probes (H1 and H2) were heated to 95 °C for 5 min and cool down to room temperature and incubated at room temperature for 2 h, respectively. Finally, the treated H1 and H2 were stored at 4 °C until use.

2.3. Preparation of PEG solution

Stock solution of 40% PEG 6000 (weight/volume) was made in deionized water and filtered by 0.22 μm filter (Merck Millipore, USA).

2.4. Experimental procedures of PEG-LSPA reaction

The analytical procedures basically consisted of two steps: ligation and amplification. Briefly, for ligation: 10 μL of ligation reactions were prepared with 1 × HiFi Taq DNA ligase buffer (20 mM Tris-HCl, 25 mM KAc, 10 mM MgAc, 1 mM NAD, 10 mM DTT, 0.1% Triton X-100, pH 7.6), 10 nM different concentrations of target DNA, 10 nM H1, 10 nM H2, 15% PEG 6000, 10 U of HiFi Taq DNA ligase. Subsequently, above-mentioned ligation reactions were heated up to 95 °C for 5 min and gradually cooled down to 65 °C for 20 min. For amplification: 10 μL of amplification reactions were prepared with 2 μL of 10 × isothermal amplification buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 50 mM KCl, 2 mM MgSO4, 0.1% Tween 20, pH = 8.8), 4 U of Bst 2.0 WarmStart DNA polymerase, 0.5 μL of 25 mM dNTPs mixture, 2 μL of 10 × SYBR Green Ⅰ and 25 μM betaine. Then the above amplification reaction solutions were added to ligation reaction tubes and then incubated at 65 °C for 60 min. Meanwhile, the fluorescence signal from SYBR Green Ⅰ staining was monitored at an interval of 2 min at 65 °C by a QuantstudioTM 6 Flex System (ABI, USA).

The detection procedure of LSPA reaction without PEG was conducted almost the same as abovementioned PEG-LSPA without adding 15% PEG 6000.

2.5. Analysis of real samples

Human serum and saliva were firstly filtered with 0.22 μm microfiltration membrane to remove potential bacterium or insoluble particles. A series of samples were prepared by adding target DNA of different concentrations into 10% serum and saliva samples. The spiked recovery tests were conducted through the standard addition assay. The detection procedure was the same as described in experimental procedures of PEG-LSPA reaction.

2.6. Agarose gel electrophoresis analysis

In this assay, two kinds of agarose gel electrophoresis were employed to analyze small and large amplification fragments, respectively. For the small fragment amplification products analysis, 4% GTG agarose gel (3% NuSeive GTG agarose + 1% agarose) were prepared in 1 × TBE buffer (89 mmol/L Tris; 89 mmol/L Boric acid; 2 mmol/L EDTA pH 8.3) at 65 V constant voltage for about 75 min. After staining for 30 min in a 4SGelred solution, the gel was photographed by FluorChem FC3 Chemiluminescent imaging system (ProteinSimple, USA). To analyze large fragments amplification products, 2% agarose gel prepared in 1 × TAE buffer (40 mmol/L Tris-acetate; 1 mmol/L EDTA; pH 8.0–8.6) at 90 V constant voltage for about 30 min. After staining for 30 min in a 4SGelred solution, the gel was photographed by FluorChem FC3 Chemiluminescent imaging system (ProteinSimple, USA).

3. Results and discussion

3.1. Principle of PEG-LSPA reaction

The working principle of the PEG-LSPA reaction is illustrated in Scheme 1 . SARSCoV-2 D614G mutation (A23403G) (MT) nucleic acid sequence is chosen as detection target. The main DNA probes of the nucleic acid amplification system are hairpin probe 1 (H1) and hairpin probe 2 (H2). As Scheme 1 illustrated, H1 is designed to contain a 20 nt phosphorothioate (PS)-modified self-priming domain at its 5'end and an 18 nt target binding domain overhung at its 3′ end, which complements with SARS CoV-2 D614G mutation sequence. The last base (C) of H1 at 3′ end is complementary with the mutant sit (G). H2 is synthesized to possess an 8 nt hairpin structure at its 3'end and a 22 nt target recognition site overhung at its 5′ terminus which complements with SARS CoV-2 D614G mutation sequence. In presence of target sequence, it complements with H1 and H2 to form a double-strand structure. Subsequently, ligation is efficiently triggered by HiFi Taq ligase under molecular crowding condition which is created by PEG 6000. Then 3′ terminus of the H2 can be extended by DNA polymerase under isothermal conditions, leading to the formation of a longer extended hairpin (EHP1) with one target sequence domain (yellow region). The Tm value is reduced as a consequence of phosphorothioate modification of 20 bases at 5′ terminus. 20 bases on the 5′ end modified with phosphorothioate are unstable and can form a short stem-loop hairpin at low temperature due to the lower Tm value. The resulting overhanging 3′ end can also fold back as a new primer to initiate the next round of polymerization, resulting in the formation of a longer extended hairpin (EHP2) containing two target sequence domains (yellow region). In the circle of spontaneously self-primer formation and polymerization, the ligated H1–H2 chain can spontaneously be extended to be a long hairpin-structure concatemer (more than 10,000 bp) containing a great number of target sequence domain (EHPx). The resulting long hairpin concatemers can be monitored in real-time through duplex-specific fluorescent signaling (SYBR Green Ⅰ). In the presence of the wild type (WT) SARS-CoV-2 sequence (D614) or in the absence of MT target sequence, the ligation between H1 and H2 can not happen due to the mismatch of the last base on the 3′ end of H1. No long-extended hairpin products are generated.

Scheme 1.

Scheme 1

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Schematic illustration of the PEG enhanced ligation-triggered self-priming isothermal amplification for fluorometric detection of SARS-CoV-2 D614G mutation.

3.2. Feasibility of PEG-LSPA reaction

In order to validate that the isothermal amplification does take place as expected for amplification of nucleic acid detection signal. We firstly monitored real-time fluorescence signals produced from SYBR Green Ⅰ staining for the reaction products. As shown in Fig. 1 A, an obviously rapid enhancement of fluorescence signal was only observed in the D614G sample (red curve), indicating a dramatic isothermal amplification. In contrast, the limited fluorescence signal was observed in the wild type (WT) sample (blue curve) and negative control (NC) (black curve) due to the partially complementary of hairpin probe H1 and extended H2.

Fig. 1.

Fig. 1

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Feasibility evaluation of the isothermal amplification for the detection of SARS-CoV-2 D614G Mutation with 10 nM target. (A) Real-time fluorescence curves during isothermal amplification with 10 nM D614G Mutation, ancestral WT and NC. (B) Fluorescence images took by scanner and smartphone under UV light. (C). Agarose gel electrophoresis analysis of isothermal reaction products. 1: DNA marker (25 bp – 500 bp), 2: H1 (10 μM), 3: H2 (10 μM), 4: Target D614G (10 μM), 5: Target WT (10 μM), 6: H1(10 μM) + H2(10 μM), 7: H1(10 μM) + H2(10 μM) + Target D614G(10 μM), 8: H1(10 μM) + H2(10 μM) + Target WT(10 μM), 9: H1(10 μM) + H2(10 μM) + Target D614G (10 μM) + ligase, 10: H1(10 μM) + H2(10 μM) + Target WT(10 μM) + ligase, 11: H1(10 nM) + H2(10 nM) + Target D614G(10 nM) + ligase + polymerase (60 min), 12: H1(10 nM) + H2(10 nM) + Target WT(10 nM) + ligase + polymerase (60 min). 13: NC (60 min). 14: DNA marker (100 bp – 5000 bp).

Importantly, the fluorescence signal of D614G sample was about 9 times higher than ancestral WT sample and NC at 60 min, which manifested that mutant nucleic acid was very effectively identified against the wild type nucleic acid and negative control based on PEG-LSPA reaction. The fluorescence images also verify this point (Fig. 1B), only the tube with D614G target produced super-bright fluorescence signal, which could be directly visualized under a UV light illuminator by scanner and smartphone. No visible fluorescence signal was observed in the WT tube or NC tube.

To support the above fluorescence results, we next conducted gel electrophoresis analysis for the products from the isothermal amplification. Only lane 11 containing 10 nM D614G target showed an obvious band in the sample well (Lane 11) as seen in Fig. 1C, whereas none of the band was observed in 10 nM WT target (Lane 12). Meanwhile, no clear band was seen in the absence of target nucleic acid (Lane 13). Lane 6 to lane 10 displayed the reaction procedure of LSPA. These experimental results demonstrated that our constructed PEG enhanced ligation-triggered self-priming isothermal amplification assay can work for SARS-CoV-2 D614G mutation.

3.3. Optimization of key reaction parameters

To obtain the best performance for SARS-CoV-2 mutation detection, various reaction parameters including ligation and polymerization temperature, amplification time, the concentration of H1 and H2 probes were optimized by comparing real-time fluorescence signals produced from the reaction for SARS-CoV-2 D614G mutation to SARS -CoV-2 wild type target and those without target (NC).

Firstly, the most important factor that affects the sensitivity and specificity of this approach is the ligation temperature. As displayed in Fig. 2 A, the fluorescence intensity of MT at 65 °C or 62 °C was almost the same. However, when the ligation temperature was 62 °C, the fluorescence signal of WT increased sharply due to the non-specific ligation reaction. The gel analysis results in Fig. 2B also verifide this point, when the ligation and polymerization temperature was 62 °C, the amplification product of the WT target (lane 2) was almost the same as the MT target, indicating the non-specific ligation of H1 and H2 in the presence of WT target. When the temperature was elevated to 65 °C, no obvious bands were observed in the WT target (lane 10) and NC (lane 11). Meanwhile, an obvious band was observed in the sample well (lane 9). Therefore, 65 °C was selected as the optimal ligation temperature in the following experiments.

Fig. 2.

Fig. 2

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Optimization of the experiment conditions. (A) Real-time fluorescence curves during isothermal amplification with different ligation temperature in the presence of 10 nM MT, 10 nM WT target and NC. (B) Agarose gel electrophoresis analysis of isothermal reaction products with different ligation temperature. M: DNA marker (100 bp – 5000 bp) 1: SARS-CoV-2 D614G Mutation (MT) target, 2: SARS-CoV-2 wild type (WT) target, 3: negative control (NC), 4–8 non-complementary target, 9: MT target, 10: WT target, 11: NC. (C) Real-time fluorescence curves during isothermal amplification with different PEG-LSPA reaction time ranging from 0 min to 120 min. (D) Agarose gel electrophoresis analysis of PEG-LSPA reaction products with different amplification time ranging from 0 min to 70 min. 1: 0 min, 2: 10 min, 3: 20 min, 4: 30 min, 5: DNA marker (100 bp – 5000 bp), 6: 40 min, 7: 50 min, 8: 60 min, 9: 70 min. (F) Real-time fluorescence curves during isothermal amplification with different concentrations of H1 and H2 in the presence of 10 nM MT target and NC.

Another factor that affects the sensitivity and specificity of the approach is the PEG-LSPA reaction time. As shown in Fig. 2C, the real-time fluorescence curve sharply elevated from 0 min to 60 min and finally reached the plateau over 60 min. However, further increasing of the amplification time led to the increasing of back-ground signal. The gel analysis results further clarified this view. As displayed in Fig. 2D, more obvious bands were displayed in the sample well from 0 min to 60 min. These results indicated more and more large molecular products were produced as the amplification time increasing. Therefore, 60 min was selected as the PEG-LSPA reaction time in the following experiments to obtain best performance.

In this work, the concentrations of H1 and H2 were the key factors that affect the analytical performance of the biosensor because partially complemented hairpin probe H1 and the extended H2 could produce background fluorescence signals. The concentrations of H1 and H2 were studied on basis of the fluorescence response toward the detection of 10 nM SARS-CoV-2 D614G mutation (MT) target and 10 nM wild type (WT) target. As displayed in Fig. 2E, the fluorescence response of 20 nM H1 and H2 at 60 min was almost the same as 10 nM H1 and H2. However, the fluorescence response of WT with 20 nM H1 and H2 is higher than WT with 10 nM H1 and H2 due to increasing the background signal. The fluorescence response of 5 nM H1 and H2 at 60 min is only 1/2 of the 10 nM H1 and H2 because of fewer templates for isothermal amplification. Based on these results, 10 nM of H1 and H2 was employed as the optimal concentration for this assay.

3.4. Sensitivity of the PEG-LSPA reaction

To determine the sensitivity of the PEG-LSPA reaction, SARS- CoV-2 mutation sequences was used at different concentrations ranging from 1 fM to 10 nM for measuring the real-time fluorescence signals from the reaction products under optimal reaction conditions. As shown in Fig. 3 A, the fluorescence intensity increased as the concentration of the target increased in the range from 1 fM to 10 nM. A good linear relationship between the fluorescence and the logarithm of target concentration was from 10 fM to 10 nM. The correlation coefficient is 0.9855 for the linear calibration curve as shown in Fig. 3B. Based on three times the standard deviation corresponding to three measurements of negative control samples (NC) [[42], [43], [44]], the detection limit (LOD) was calculated to be 4 fM, which is superior to previous proposed self-priming approaches [29] (Table S2). In order to further verify that PEG improved the ligation efficiency and sensitivity of this method by constructing a molecular crowding environment, we conducted the ligation-triggered self-priming isothermal amplification for target nucleic acid at series of concentrations in the range from 10 fM to 10 nM without PEG. The fluorescence signals were monitored in a real-time manner. The fluorescence results in Fig. S1 showed that the LOD of the assay without PEG for increasing the ligation efficiency is only 10 pM, which is inferior to the PEG-LSPA reaction by 3 orders of magnitudes. These results strongly prove that the proposed PEG-LSPA reaction could yield higher sensitivity than that without PEG owing to increasing the ligation efficiency of this method in a molecular crowding environment.

Fig. 3.

Fig. 3

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Sensitivity of the proposed assay for SARS-CoV-2 D614G mutation. (A) Real-time fluorescence curve detected from the isothermal reaction for target at different concentrations in the range from 1 fM to 10 nM. (B) Calibration curve of the target assay with different concentrations of target. Error bars represent standard deviation, n = 3.

3.5. Specificity of the PEG-LSPA reaction

The specificity of this PEG-LSPA strategy was next assessed by testing perfectly matched target (D614G), a single base mismatched target (M1), two-base mismatched target (M2), three-base mismatched target (M3), WT target, as well as NC. As presented in Fig. 4 A, a distinct enhancement of fluorescence signal was detected only in the D614G target. In contrast, when the target M1, M2, M3 and WT were tested, the gently increased fluorescence signals were observed. Then the fluorescence signals of each target were quantified for analysis. Fig. 4B displayed that the fluorescence intensity from WT target was approximately 15% of the signal from the perfectly matched D614G target and the fluorescence intensity from one to three base mismatched targets was almost the same as the negative control sample. These results showed that this assay has good specificity to distinguish SARS-CoV-2 D614G mutation sequence from wild type sequence or 1–3 mismatched targets. As shown in Fig. S2, the mutation sites in HCoV-229E sequence, HCoV-HKU1 sequence, HCov-NL63 sequence, HCoV-OC43 sequence, MERS-CoV sequence, and SARS-CoV sequence are more than WT and M3. Therefore, our proposed assay can also employ to distinguish different beta noroviruses.

Fig. 4.

Fig. 4

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Specificity of the proposed assay. (A) Real-time fluorescence curve detected from the isothermal reaction for D614G mutation, WT, M1, M2, M3 and NC. (B) Fluorescence intensity of D614G mutation, WT, M1, M2 M3 and NC.

3.6. Real sample performance of the PEG-LSPA reaction

To demonstrated whether our developed approach could be applied to real sample analysis, a spiking test was carried out in buffer, saliva and 10% human serum. Aliquots of the serum and saliva samples were spiked with different concentrations of target DNA. As displayed in Fig. 5 , for PEG-LSPA analysis in buffer, saliva and 10% human serum samples, as the concentrations of D614G target increase from 0 p.m. to 10 nM, the fluorescence increase gradually. The fluorescence intensities of the same concentration in three types of samples are almost the same. These results indicated that this assay is potential to be applied to detect real sample.

Fig. 5.

Fig. 5

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Real sample performance of the PEG-LSPA reaction in buffer, saliva and 10% human serum. Error bars represented the SD of at least 3 independent experiments. All measurements were performed in triplicate.

4. Conclusions

In this study, we have demonstrated a direct and rapid nucleic acid sensing system with high sensitivity and selectivity by isothermal amplification protocol. PEG was employed to create a molecular crowding condition by increasing the effective concentration of H1, H2 and target. Phosphorothioate (PS)-modified at 5′ end destabilize DNA duplex to form self-primer at 3’ end at isothermal condition. The sensing system is capable to sensitively and directly detect SARS-CoV-2 D614G mutation even down to 4 fM with excellent specificity to discriminate a single-nucleotide mutation between SARS-CoV-2 variants and wild type sequence or different beta noroviruses. By substituting the ligase, this assay can be used to detect RNA sample without reverse transcription directly. In addition, this assay can be employed to monitor various SARS-CoV-2 mutations by substituting the sequences of corresponding hairpin probes. Based on these advantages, this new nucleic acid detection approach is expected to serve a great potential universal platform for the direct detection of mutations in SARS-CoV-2 variants. We anticipate that the fabricated PEG-LSPA strategy can ultimately be further developed to be a great potential platform for the direct detection of other respiratory viruses.

Credit author statement

Luxin Yu: Conceptualization, methodology, resources, supervision, formal analysis, project administration, writing – original draft and writing – review and editing. Zibin Tang: Methodology, formal analysis, investigation, validation and writing – original draft. Yuanzhong Sun: Methodology, formal analysis, investigation and validation. Hai Yi: Methodology, formal analysis, investigation and validation. Yuebiao Tang: Investigation and validation. Yangqing Zhong, Dongchun Dian Yanguang Cong, and Houqi Wang: Investigation. Zhaoyang Xie and Suhui He: supervision and Investigation. Zhangquan Chen: Conceptualization, methodology, resources, supervision, Methodology, project administration and writing – review and editing. All the authors read and corrected the manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by Discipline Construction Project of Guangdong Medical University (4SG22023G), and the Dongguan Science and Technology of Social Development Program (202050715032212).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.talanta.2023.124711.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Multimedia component 1
mmc1.docx (922.5KB, docx)

Data availability

No data was used for the research described in the article.

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