Rapid Genotyping of SARS-CoV-2 Strains Alpha, Delta, Omicron BA.1, Omicron BA.2, Omicron BA.2.12.1, and Omicron BA.4/BA.5 Using ARMS- PCR and Molecular Beacon Technology

doi:10.21203/rs.3.rs-2312263/v1

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Method Article

Rapid Genotyping of SARS-CoV-2 Strains Alpha, Delta, Omicron BA.1, Omicron BA.2, Omicron BA.2.12.1, and Omicron BA.4/BA.5 Using ARMS- PCR and Molecular Beacon Technology

https://doi.org/10.21203/rs.3.rs-2312263/v1

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Background

SARS-CoV-2 subtypes Alpha, Delta, Omicron BA.1, Omicron BA.2, Omicron BA.2.12.1, and Omicron BA.4/BA.5 have significant differences in transmission and immune escape ability. Currently, no effective detection methods are available for these subtypes. Routine detection methods are prone to missed detection.

Methods

In this study, a rapid detection method based on ARMS-PCR and molecular beacon probes was developed for the identification of SARS-CoV-2 subtypes Alpha, Delta, Omicron BA.1, Omicron BA.2, Omicron BA.2.12.1, and Omicron BA.4/5. Specific primers and probes were designed and validated using gel electrophoresis, real-time fluorescence quantitative PCR, and molecular hybridization.

Results

ARMS-PCR and molecular beacon probe-based assays can be applied in RT-PCR and fluorescence assays to differentiate SARS-CoV-2 subtypes Alpha, Delta, Omicron BA.1, Omicron BA.2, Omicron BA.2.12.1, and Omicron BA.4/5.

Conclusions

In the present study, we developed a simple, rapid and accurate detection method based on ARMS-PCR and molecular beacon probes for rapid genotyping of SARS-CoV-2 subtypes Alpha, Delta, Omicron BA.1, Omicron BA.2, Omicron BA.2.12.1, and Omicron BA.4/5. It can be used in real-time fluorescence quantitative PCR and molecular hybridization to identify subtypes of COVID-19, effectively improving the detection rate to provide guidance for disease prevention and treatment.

ARMS-PCR

molecular beacon

COVID-19

Delta

Omicron

The novel coronavirus SARS-CoV-2, which causes COVID-19, is a linear, single-stranded, positive-stranded RNA virus that is susceptible to mutations. The SARS-CoV-2 genome encodes 29 proteins, four of which are structural proteins, including the spike (S) protein, the nucleocapsid (N) protein, the membrane (M) protein, and the envelope (E) protein [1–3]. Because the genomic sequence similarity between SARS-CoV-2 and other coronaviruses can reach 90% or more, the “conserved regions” with low similarity are often selected for specific recognition in clinical tests. Clinically, most detection reagents mainly target the O and N genes, and a small number of kits include the E gene[4].

However, SARS-CoV-2's invasion of cells begins when the S protein binds to the cell surface receptor angiotensin-converting enzyme 2 (ACE2) through its receptor-binding domain (RBD). Therefore, mutations in the S protein often bring great changes to the transmission and immune escape ability of SARS-CoV-2. Korber et al. found that the D614G mutation in the S protein was the most common mutation at that time[5]. Subsequently, Akatsuki et al. mentioned in their study that the Delta strain carrying P681R showed higher pathogenicity than its parent virus [6]. Motozono et al. also found that the L452R mutation in the S protein increased spike stability, viral infectivity, and viral fusion, thereby promoting viral replication[7]. With the currently available detection methods for ORF1ab and the N and E genes, it is difficult to detect the virus and perform genotyping at the same time. In order to distinguish between SARS-CoV-2 subtypes, we need to take into account the sequence of the S protein.

At present, common SARS-CoV-2 nucleic acid detection methods mainly include high-throughput sequencing, RT-PCR, CRISPR, and RT-LAMP. High-throughput sequencing techniques based on sequencing of the entire viral genome can be useful to determine the type of the corresponding strain; however, high-throughput sequencing requires instruments, equipment, and cumbersome operation steps and bioinformatics analysis to process data, so it takes a long time and is associated with high staff requirements. RT-PCR is limited by the channels of the fluorescence quantitative PCR (qPCR) instrument. At present, most qPCR instruments have four to six channels, which cannot detect multiple loci on the basis of the detection of ORF1ab and the N and E genes. The subtype of the virus is usually confirmed using high-throughput sequencing after RT-PCR detection. As an emerging technology, CRISPR has improved sensitivity compared with RT-PCR, but it has not been fully applied to genotyping[8]. Wang et al. applied CRISPR to detect the D614G mutation, but their study did not include the currently emerging varieties[9]. RT-LAMP is a novel nucleic acid amplification method that does not require thermal denaturation, temperature cycling, electrophoresis, or the ultraviolet observation of templates. It is simple, rapid, and highly specific, and has gradually occupied a place in the field of SARS-CoV-2 detection. Unfortunately, this method has not been applied in genotyping. Therefore, the combination of RT-PCR and high-throughput sequencing is still the most common method for genotyping SARS-CoV-2.

The RT-PCR system has been improved to detect different SARS-CoV-2 genotypes. For example, Mohammad et al. used amplification refractory mutation system PCR (ARMS-PCR) to design a genotyping method based on the six genetic lineages of SARS-CoV-2 proposed using GISAID: GH, GR, G, V, L, and S[10]. However, the system established using GISAID, Nextstrain, and Pango to name and track the genetic lineages of SARS-CoV-2 is used primarily by scientists, while the WHO classification method based on the Greek letters α, β, γ, δ, and ο is more widely accepted by the general public[11]. For example, Dikdan et al. established a SARS-CoV-2 detection method based on molecular beacons, focusing on variants of concern (VOCs) [12]. Strain Omicron has gradually replaced other varieties as the world's most widespread SARS-CoV-2 strain. Five Omicron subtypes (BA.1, BA.2, BA.3, BA.4, and BA.5) and their offspring have been described. These subtypes show differences in immune escape. Bruel et al. pointed out that subtypes BA.1 and BA.2 show obvious differences in sensitivity to therapeutic monoclonal antibodies[13]. Li et al. found different hACE2 binding patterns among BA.1.1, BA.2 and BA.3 RBDs [14]. Moreover, subtypes BA.4 and BA.5 could escape the immune effect of antibodies targeting BA.1[15].

The identification of the Omicron strain is a blind area in the current research field. Mutations of SARS-CoV-2 strain Omicron exhibit the following characteristics: dense distribution of mutations, mutations at the same site in different subtypes, and different mutations at the same site. For example, the D405N R408S double mutation occurred simultaneously in BA.2, BA.2.12.1, BA.4, and BA.5. However, at L452, BA.2 did not have a mutation, while BA.2.12.1 harbors an L452Q mutation, while BA.4 and BA.5 harbor an L452R mutation.

In the present study, a method to identify the SARS-CoV-2 variants Alpha, Delta, and Omicron, as well as Omicron subtypes BA.1, BA.2, BA.2.12.1, and BA.4/5, based on the combination of ARMS-PCR and molecular beacons was designed. The subjects were narrowed down to several subtypes by ARMS-PCR-specific primers and then distinguished by molecular beacons. The method has strong specificity; it not only overcomes the high false positive rate of ARMS-PCR, but also reduces the difficulty of probe design. The combination of primers and probes can be used to screen the current Omicron subtypes with strong immune escape ability, which can effectively reduce the probability of missed detection.

Subsection

The 2019 SARS-CoV-2 database (RCoV19) from the National Genomics Data Center (NGDC) contains data for comparison of SARS-CoV-2 subtypes. We obtained the mutation site of each subtype and its mutation frequency. Through screening, we selected mutation sites with mutation frequencies above 0.95 and in close proximity as our screening targets. All mutation sites we used are shown in Fig. 1. The yellow blocks indicate the mutation sites present on the ARMS primers, which were used as our initial screening targets to specifically select subtypes from the majority of the SARS-CoV-2 viruses, while the other blocks indicate the mutations present on the probes of different subtypes, which are able to distinguish between the subtypes that share mutations on the primers.

Plasmid Construction

The sequences of all isoforms were obtained from the open-source database of the New Crown Pneumonia isoform sequences provided by NCBI (www.ncbi.nlm.nih.gov) and GISAID (www.gisaid.org). The sequence numbers are shown in Table 1. After comparison of the published sequences through SnapGene, all sequences contained loci with high mutation rates in agreement with those in RCoV19. We cloned the S gene sequences of all isoforms into the vector pcDNA3.1. Plasmids that served as templates for subsequent experiments were purchased from General Biol (Anhui) Co., Ltd .

The plasmids were centrifuged at 10,000 g for 2 min, and then TE Buffer (Cat. No: BL531A, Biosharp, Beijing, China) was added. The plasmid DNA concentration was measured with a NanoDrop One system (Thermo Scientific) and then diluted with TE buffer. The concentration was confirmed with NanoDrop. These steps were repeated until the concentration of all plasmids was 10 ng/µL. The calibrated plasmids were subsequently diluted 1000× (10 pg/µL) and 10,000× (1 pg/µL) for experiments.

Primer And Probe Design

The sequences of all primers and probes designed using Primer Premier 6, Beacon Designer 8, and SnapGene are shown in Table 1. We aimed to preserve the mutation sites of each isoform while following the basic principles of primer design, so that the mutation sites on the primers are as close to the 3′ end as possible and the mutation sites on the probes are located in the middle of the sequence.

All primers were 18 to 22 nucleotides in length with Tm values of 45–55°C, upstream and downstream consistency as high as possible, a GC content of 40–60%, G/C within the last five bases, no four or more replicates of any base, and an amplicon size of 100–200 bp. The specificity of SARS-CoV‐2 and other organisms was examined using BLAST in NCBI. Primers were purchased from Zhejiang Sunya Biotechnology Co., Ltd.

To enhance the specificity of stem sequence recognition, probe size is 30–40 bp, with a loop of usually 20–30 bp in length which is complementary to the target sequence and a stem of usually 5–7 bp in length with high GC content. They are paired with each other to form the stem structure. The fluorescent groups are labeled at one end of the probe, while quenchers are labeled at the other end. A 5′ FAM fluorescent group and a 3′ Dabcyl quenching group were used to adapt to our molecular hybridization method. Other fluorescent groups can be used according to the research needs. Probes were purchased from General Biol (Anhui) Co., Ltd .

TABLE 1 Template, primer and probe sequences for COVID-19 subtypes

The letters in bold represent hairpins of molecular beacons and the red letters represent point mutated nucleotides A wide center space indicates a missing position.

Pcr And Gel Electrophoresis

To test the amplification using ARMS-PCR primers, we used a Bio-Rad T 100 thermocycler for PCR amplification. Each reaction system contained 4 µL 5× Taq Pro Buffer (#PN101, Vazyme Biotech Co. Ltd., Beijing, China), 0.5 µL forward primer (10 µM), 0.5 µL reverse primer (10 µM), 1 µL template (10 pg/µl), 0.2 µL Taq Pro HS DNA polymerase (Vazyme Biotech Co. Ltd., Beijing, China), and 12.8 µL ddH₂O.

The PCR program was as follows: 95°C for 3 min, followed by 30 cycles of 95°C for 10 s and 50°C for 15 s. The PCR products were analyzed using 1.5% agarose electrophoresis and visualized using an Azure Imaging Systems 280 (USA).

Primer Optimization

To optimize primer specificity, we mismatched the base pair at the penultimate or third position at the 3′ end and then detected the specificity of individual primer pairs using a real-time fluorescence qPCR instrument (Bio-Rad CFX Connect) and Vazyme ChamQ SYBR qPCR Master Mix (without Rox). Each reaction system contained 10 µL 2× ChamQ SYBR qPCR Master Mix (Vazyme Biotech Co. Ltd., Beijing, China), 0.4 µL forward primer F (10 µM), 0.4 µL reverse primer (10 µM), 1 µL template (1 pg/µL), and 8.2 µL ddH₂O. The PCR program was as follows: 95°C for 3 min, followed by 40 cycles of 95°C for 10 s and 50°C for 15 s. The fluorescence signal was collected during the reaction. Finally, the primers with non-specific samples and no amplification or large Ct value differences were selected for subsequent experiments, and for the primers that did not meet the requirements, the specificity was enhanced by adjusting the sequence or introducing a new mismatch.

Probe Specificity Detection

The probe assay was used to detect samples of different templates by matching the screened primers with the corresponding probes to confirm whether the corresponding probes can specifically distinguish between different target mutants that have the same primers.

qPCR amplifications were carried out in a 20 µL reaction volume containing 4 µL 5× Taq Pro Buffer (Vazyme Biotech Co. Ltd., Beijing, China), 1 µL (1 pg/µL) DNA template, 0.4 µL Taq DNA polymerase (5 U/µL, Vazyme Biotech Co. Ltd., Beijing, China), 0.4 µL (10 µM) each of the forward and reverse primers, 0.2 µL Beacon (10 µM), and 13.6 µL ddH₂O.

For real-time fluorescence PCR detection using Vazyme Taq Pro HS DNA polymerase reagent in a Bio-Rad CFX Connect system, the PCR program was as follows: 95°C for 3 min, followed by 40 cycles of 95°C for 10 s and 55°C for 15 s. The fluorescence signal was collected during the reaction. The specificity of the probe was demonstrated by the fact that the amplification curve was only detected when using the corresponding template.

Molecular Hybridization

Molecular hybridization is a method for detecting specific products at time nodes after amplification. After amplification, a fluorescent probe was added to detect the background signal value. Due to the presence of the molecular beacon probe stem–loop structure, the probe did not produce fluorescence in the normal state. After denaturation at 95°C, the probe specifically combined with the amplification product at 55°C, and the emitted fluorescence signal was captured using a fluorescence receiver. The signal value at this time was multiplied by the difference from the background.

First, a preliminary amplification was carried out on that fragment. In this case, we formed multiplex PCR by combining multiple primer pairs, such as A570D-Primer + G339D-Primer + P681R-Primer or T547K-primer + LPPA24-27S-primer. The feasibility of this method was tested using a multiplex PCR protocol. Each reaction system contained 10 µL 5× Taq Pro Buffer (Vazyme Biotech Co. Ltd., Beijing, China), 2.5 µL forward primer (10 µM), 2.5 µL reverse primer (10 µM), 10 µL template (1 pg/µL), 1 µL Taq Pro HS DNA polymerase (Vazyme Biotech Co. Ltd., Beijing, China), and (29 − 5 × N) µL ddH₂O, where N = 1–3. The PCR program was as follows: 95°C for 3 min, followed by 35 cycles of 95°C for 10 s and 50°C for 15 s.

The amplification product was added to a black 384-well plate (#w/lid Cell Culture Sterile PS, Thermo Fisher Scientific) with 1 µL of beacon probe (10 µM). The probe with the FAM fluorophore was then detected using a SpectraMax iD5 multi-mode microplate reader at 55°C (excitation at 484 nm, emission at 525 nm). The average value from three replicates was taken as the background, i.e., the fluorescence signal of the reagent itself when the probe was not attached to the template.

After denaturation at 95°C for 3 min, molecular hybridization of the probe to the amplified product was performed at 55°C for 30 s. At this point the products were read again on a SpectraMax iD5 multi-mode microplate reader. The average value from the three repeats was taken. The signal change at this point reflects the detection of the template by the probe. Specific templates will produce a more pronounced increase in signal.

Sensitivity Test

To verify the detection limit of this assay, both RT-PCR and molecular hybridization were used. The real-time fluorescence quantification system and the molecular hybridization system performed as expected based on previous tests. First, the plasmid copy number of 9100 bp at 1 ng/µL = (6.02 × 10²³ × 10^− 6)/(9100 × 660) = 10¹¹ copies/mL. Real-time fluorescence qPCR was performed using concentrations of 10¹² copies/mL, 10¹⁰ copies/mL, 10⁸ copies/mL, 10⁶ copies/mL, 10⁴ copies/mL, and 10² copies/mL. Plasmid positive controls were used, and deionized water was used as the negative control as the detection limit reference. Molecular hybridization was performed using concentrations of 10¹⁰ copies/mL, 10⁸ copies/mL, 10⁶ copies/mL, 10⁴ copies/mL, and 10² copies/mL, with deionized water as a negative control as the assay-limiting reference.

ARMS-PCR primer screening gel electrophoresis results

ARMS-PCR primers enhanced the specificity by the difference in bases between mutants and the introduction of mismatches. Finally, primer pairs that could specifically amplify the corresponding mutants were screened. ARMS-PCR primers were specific. Primer specificity is improved by base differences between mutant strains and the introduction of mismatches. Ultimately, pairs of primers that amplify only specific mutant strains were screened for. As shown in Fig. 2, the P681R primer can only produce a PCR product when the Delta subtype with mutation is used as template. Similarly, the A570D primer can only produce a PCR product when the Alpha subtype is used, the G339D primer amplifies the Omicron subtype, and the T547K primer amplifies the BA.1 subtype. This is all due to the specificity of the ARMS-PCR primers. BA.2 and BA.4/5 subtypes, which both harbor the LPPA24-27S mutation, and BA.2, BA.2.12.1, and BA.4/5, which all harbor the D405N R408S double mutation, were amplified using the same primer pairs, and genotyping of these subtypes is based on different mutations present in the molecular beacons.

Beacon Specificity Test Results

In the detection by agarose gel electrophoresis, the ARMS-PCR primers showed specificity, but the primers alone could not distinguish between different subtypes harboring the same mutation. Therefore, we added the probes to the qPCR detection system. As shown in Fig. 3, subtypes with mutations at the same site can be amplified using ARMS-PCR primers, but the probes designed at sites with different mutations can detect single subtypes with specificity, thus achieving the purpose of specific genotyping.

Molecular Hybridization Results

The molecular hybridization results are shown in Fig. 4, where the horizontal axis indicates the different SARS-CoV-2 subtype S protein sequences and the vertical axis indicates the value of the detected fluorescence signal. In the triple PCR primer combination consisting of Delta-P681R, Alpha-A570D, and Omicron-G339D, the signal of P681R-Beacon is significantly higher than that of the other templates in the presence of the Delta template. Similarly, A570D-Beacon and G339D-Beacon were able to distinguish between different subtype templates in the triple amplification products. The double primer combinations consisting of T547K-BA.1, LPPA24-27S-BA.2 + BA.4/5, and D405NR408S-BA.2 + BA.2.12.1 + BA.4/5 further illustrate that the molecular beacons can achieve specific differentiation of SARS-CoV-2 subtypes based on amplification products screened using ARMS-PCR primers.

Sensitivity Detection Results

The results of the sensitivity test are shown in Fig. 5. In the assay on the real-time fluorescence PCR instrument, templates with concentrations of 10¹² copies/mL, 10¹⁰ copies/mL, 10⁸ copies/mL, and 10⁶ copies/mL were able to detect the amplification curve before 40 cycles, and the linear standard curve consisting of copy number and CT value R2 = 0.9981 > 0.99. Also, in the fluorescence assay of the enzyme standard, samples above 10⁴ copies/mL showed a significant increase in the background signal compared to the negative samples. The above results indicate that the method is suitable for samples with copy number above 10⁶ copies/mL in the real-time fluorescence PCR, and the detection limit can be reduced to 10⁴ copies/mL in the enzyme-labeled assay.

According to the US Centers for Disease Control (CDC) (accessed on June 21, 2022), the new Omicron subtype BA.4/5 accounted for 34.9% of all new cases reported in the week of June 18 in the United States. In the present study, we developed a method for the rapid detection of SARS-CoV-2 strains Delta, Alpha, Omicron BA.1, Omicron BA.2, Omicron BA.2.12.1, and Omicron BA.4/5 based on the combination of ARMS-PCR and molecular beacons, which can effectively improve the detection efficiency of immune escape strains, reduce detection costs, and provide a new technical means for the prevention of viruses and the treatment of disease.

In the present study, Delta-P681R, Alpha-A570D, and Omicron- G339D formed a triple PCR primer combination, T547K-BA.1 and LPPA24-27S-BA.2 + BA.4/5 formed a double primer combination, and D405NR408S-BA.2 + BA.2.12.1 + BA.4/5 formed a single primer pair. Each combination distinguished three templates by three probes each. Such a combination can also be applied to the real-time fluorescence qPCR system by changing the fluorescent group type, because commonly used RT-PCR instruments generally have four to six channels. We can add the general primer probe of the N gene or ORF1ab outside the triple as the positive internal reference for detecting SARS-CoV-2 for the simultaneous detection and genotyping of SARS-CoV-2.

The detection limit of the real-time fluorescence PCR system was about 10⁶ copies/mL, while in the fluorescence signal assay of the zymograph, the detection limit was 10⁴ copies/mL. This difference may be due to the fact that in the zymograph assay, in contrast to the RT-PCR assay, the probe is added to the system at the end of amplification, so it does not need to undergo a dozen or even dozens of cycles. This retains the sensitivity of the probe.

Based on this, we believe that more than 10 types of typing probes can be used in the microfluidic hybridization plate; the samples can be amplified in 35 cycles and then evenly placed into each well, and the fluorescence signal can be detected in a fixed position while the microfluidic plate is rotating while being read. The increase in signal in each well is used to determine the type of sample. Based on the results of the specificity test, we can assume that the increase in signal when the droplet binds to the probe is indicative of one or more of the subtypes included in the test.

The main limitation of this study is the lack of positive clinical samples. To ensure the accuracy of detection of subtypes BA.2 and BA.4/5, two protocols were designed for the detection of BA.2 and BA.4/5 (Fig. 1 and Table 1).

The advantage of this method is that multiple subtypes can be genotyped with only one fluorescence assay, overcoming the limitations of the RT-PCR caused by the limited number of channels. With the adjustment of primer probe design, the complete point of care testing (POCT) molecular hybridizer coupled with multiple PCR protocols can be applied to a wider range of fields, such as respiratory disease typing, human papillomavirus typing, thalassemia detection, and EGFR tumor-targeting drug screening.

Herein, we describe a method based on ARMS-PCR and molecular beacon analysis to distinguish between SARS-CoV-2 subtypes by identifying functional mutations, facilitating SARS-CoV-2 subtype identification and variant tracking.

Ethics approval and consent to participate：

Not applicable.

Consent for publication：

Not applicable.

Availability of data and materials：

The sequences of all isoforms were obtained from the open-source database of the

New Crown Pneumonia isoform sequences provided by NCBI (www.ncbi.nlm.nih.gov) and GISAID (www.gisaid.org).

Competing interests：

The authors declare that they have no competing interests

Funding：

This research received no external funding.

Authors' contributions：

J.L. and L.C. have made substantial contributions to the design of the work; X.H. and Q.F. have analysis data; F.Y.have drafted the work ; F.K. have substantively revised it. L.C., Y.Z., Y.L. conducted part of the lab work. All authors have read and agreed to the published version of the manuscript.

Acknowledgements：

We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.

Dermaku-Sopjani, M.; Sopjani, M., Molecular Characterization of SARS-CoV-2. Curr Mol Med 2021, 21 (7), 589-595.Author 1, A.; Author 2, B. Title of the chapter. In Book Title, 2nd ed.; Editor 1, A., Editor 2, B., Eds.; Publisher: Publisher Location, Country, 2007; Volume 3, pp. 154–196.
Kirtipal, N.; Bharadwaj, S.; Kang, S. G., From SARS to SARS-CoV-2, insights on structure, pathogenicity and immunity aspects of pandemic human coronaviruses. Infect Genet Evol 2020, 85, 104502.Author 1, A.; Author 2, B. Book Title, 3rd ed.; Publisher: Publisher Location, Country, 2008; pp. 154–196.
Yang, H.; Rao, Z., Structural biology of SARS-CoV-2 and implications for therapeutic development. Nat Rev Microbiol 2021, 19 (11), 685-700.Author 1, A.B.; Author 2, C. Title of Unpublished Work. Abbreviated Journal Name year, phrase indicating stage of publication (submitted; accepted; in press).
Rai, P.; Kumar, B. K.; Deekshit, V. K.; Karunasagar, I.; Karunasagar, I., Detection technologies and recent developments in the diagnosis of COVID-19 infection. Appl Microbiol Biotechnol 2021, 105 (2), 441-455.Author 1, A.B. (University, City, State, Country); Author 2, C. (Institute, City, State, Country). Personal communication, 2012.
Korber, B.; Fischer, W. M.; Gnanakaran, S.; Yoon, H.; Theiler, J.; Abfalterer, W.; Hengartner, N.; Giorgi, E. E.; Bhattacharya, T.; Foley, B.; Hastie, K. M.; Parker, M. D.; Partridge, D. G.; Evans, C. M.; Freeman, T. M.; de Silva, T. I.; Sheffield, C.-G. G.; McDanal, C.; Perez, L. G.; Tang, H.; Moon-Walker, A.; Whelan, S. P.; LaBranche, C. C.; Saphire, E. O.; Montefiori, D. C., Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus. Cell 2020, 182 (4), 812-827 e19.Author 1, A.B.; Author 2, C.D.; Author 3, E.F. Title of Presentation. In Proceedings of the Name of the Conference, Location of Conference, Country, Date of Conference (Day Month Year).
Saito, A.; Irie, T.; Suzuki, R.; Maemura, T.; Nasser, H.; Uriu, K.; Kosugi, Y.; Shirakawa, K.; Sadamasu, K.; Kimura, I.; Ito, J.; Wu, J.; Iwatsuki-Horimoto, K.; Ito, M.; Yamayoshi, S.; Loeber, S.; Tsuda, M.; Wang, L.; Ozono, S.; Butlertanaka, E. P.; Tanaka, Y. L.; Shimizu, R.; Shimizu, K.; Yoshimatsu, K.; Kawabata, R.; Sakaguchi, T.; Tokunaga, K.; Yoshida, I.; Asakura, H.; Nagashima, M.; Kazuma, Y.; Nomura, R.; Horisawa, Y.; Yoshimura, K.; Takaori-Kondo, A.; Imai, M.; Genotype to Phenotype Japan, C.; Tanaka, S.; Nakagawa, S.; Ikeda, T.; Fukuhara, T.; Kawaoka, Y.; Sato, K., Enhanced fusogenicity and pathogenicity of SARS-CoV-2 Delta P681R mutation. Nature 2022, 602 (7896), 300-306.Author 1, A.B. Title of Thesis. Level of Thesis, Degree-Granting University, Location of University, Date of Completion.
Motozono, C.; Toyoda, M.; Zahradnik, J.; Saito, A.; Nasser, H.; Tan, T. S.; Ngare, I.; Kimura, I.; Uriu, K.; Kosugi, Y.; Yue, Y.; Shimizu, R.; Ito, J.; Torii, S.; Yonekawa, A.; Shimono, N.; Nagasaki, Y.; Minami, R.; Toya, T.; Sekiya, N.; Fukuhara, T.; Matsuura, Y.; Schreiber, G.; Genotype to Phenotype Japan, C.; Ikeda, T.; Nakagawa, S.; Ueno, T.; Sato, K., SARS-CoV-2 spike L452R variant evades cellular immunity and increases infectivity. Cell Host Microbe 2021, 29 (7), 1124-1136 e11.Title of Site. Available online: URL (accessed on Day Month Year).
Broughton, J. P.; Deng, X.; Yu, G.; Fasching, C. L.; Servellita, V.; Singh, J.; Miao, X.; Streithorst, J. A.; Granados, A.; Sotomayor-Gonzalez, A.; Zorn, K.; Gopez, A.; Hsu, E.; Gu, W.; Miller, S.; Pan, C. Y.; Guevara, H.; Wadford, D. A.; Chen, J. S.; Chiu, C. Y., CRISPR-Cas12-based detection of SARS-CoV-2. Nat Biotechnol 2020, 38 (7), 870-874.
Wang, Y.; Zhang, Y.; Chen, J.; Wang, M.; Zhang, T.; Luo, W.; Li, Y.; Wu, Y.; Zeng, B.; Zhang, K.; Deng, R.; Li, W., Detection of SARS-CoV-2 and Its Mutated Variants via CRISPR-Cas13-Based Transcription Amplification. Anal Chem 2021, 93 (7), 3393-3402.
Islam, M. T.; Alam, A. R. U.; Sakib, N.; Hasan, M. S.; Chakrovarty, T.; Tawyabur, M.; Islam, O. K.; Al-Emran, H. M.; Jahid, M. I. K.; Anwar Hossain, M., A rapid and cost-effective multiplex ARMS-PCR method for the simultaneous genotyping of the circulating SARS-CoV-2 phylogenetic clades. J Med Virol 2021, 93 (5), 2962-2970.
Aleem, A.; Akbar Samad, A. B.; Slenker, A. K., Emerging Variants of SARS-CoV-2 And Novel Therapeutics Against Coronavirus (COVID-19). In StatPearls, Treasure Island (FL), 2022.
Dikdan, R. J.; Marras, S. A. E.; Field, A. P.; Brownlee, A.; Cironi, A.; Hill, D. A.; Tyagi, S., Multiplex PCR Assays for Identifying all Major Severe Acute Respiratory Syndrome Coronavirus 2 Variants. J Mol Diagn 2022, 24 (4), 309-319.
Bruel, T.; Hadjadj, J.; Maes, P.; Planas, D.; Seve, A.; Staropoli, I.; Guivel-Benhassine, F.; Porrot, F.; Bolland, W. H.; Nguyen, Y.; Casadevall, M.; Charre, C.; Pere, H.; Veyer, D.; Prot, M.; Baidaliuk, A.; Cuypers, L.; Planchais, C.; Mouquet, H.; Baele, G.; Mouthon, L.; Hocqueloux, L.; Simon-Loriere, E.; Andre, E.; Terrier, B.; Prazuck, T.; Schwartz, O., Serum neutralization of SARS-CoV-2 Omicron sublineages BA.1 and BA.2 in patients receiving monoclonal antibodies. Nat Med 2022, 28 (6), 1297-1302.
Li, L.; Liao, H.; Meng, Y.; Li, W.; Han, P.; Liu, K.; Wang, Q.; Li, D.; Zhang, Y.; Wang, L.; Fan, Z.; Zhang, Y.; Wang, Q.; Zhao, X.; Sun, Y.; Huang, N.; Qi, J.; Gao, G. F., Structural basis of human ACE2 higher binding affinity to currently circulating Omicron SARS-CoV-2 sub-variants BA.2 and BA.1.1. Cell 2022, 185 (16), 2952-2960 e10.
Science(2022), New versions of Omicron are masters of immune evasion. https://www.science.org/content/article/new-versions-omicron-are-masters-immune-evasion.

No competing interests reported.

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Rapid Genotyping of SARS-CoV-2 Strains Alpha, Delta, Omicron BA.1, Omicron BA.2, Omicron BA.2.12.1, and Omicron BA.4/BA.5 Using ARMS- PCR and Molecular Beacon Technology

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Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Subsection

Plasmid Construction

Primer And Probe Design

Pcr And Gel Electrophoresis

Primer Optimization

Probe Specificity Detection

Molecular Hybridization

Sensitivity Test

Results

ARMS-PCR primer screening gel electrophoresis results

Beacon Specificity Test Results

Discussion

Conclusions

Declarations

References

Additional Declarations

Status:

Version 1