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Clinical Infection & Immunity

Short Communication

Volume 6, Number 2, June 2021, pages 47-50

Abnormal Reverse Transcriptase-Polymerase Chain Reaction Amplification Curve as Possible Pre-Selection Tool for SARS-CoV-2 “English Variant” Sequencing

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new coronavirus that has emerged from December 2019, and has led to a worldwide coronavirus disease 2019 (COVID-19) pandemic [1].

SARS-CoV-2 is a positive sense single-stranded ribonucleic acid (RNA) virus belonging to the Sarbecovirus subgenus. Like coronavirus, SARS-CoV-2 contains four structural proteins (spike (S), membrane (M), envelope (E) and nucleocapsid (N)), and several non-structural proteins (such as RNA-dependent RNA polymerase (RdRp), responsible for viral RNA synthesis). SARS-CoV-2 virus spreads rapidly, infecting over 100 million people and causing over 2.5 million of deaths in nearly 1 year after the World Health Organization (WHO) was alerted by the Chinese health authorities on December 31, 2019 [2].

According to the WHO, diagnosis and confirmation of COVID-19 is based on real-time polymerase chain reaction (PCR) amplification of four genes: E, RdRp/S and N on nasopharyngeal swabs and/or growing the virus on cell culture [3, 4].

Multiple variants of SARS-CoV-2 have been reported from September 2020 [4] arising from the concern for SARS-CoV-2 faster spreading because of a selective advantage and increased transmissibility. Variants include the new lineage called B.1.1.7 emerged in the UK (SARS-CoV-2 variant of concern (VOC) 202012/01 according to Public Health England), which is more infectious than other circulating strains of SARS-CoV-2, becoming the dominant circulating variant in the UK, leading to lockdowns and traveling restrictions in this country [5]. Mutations located in the S gene (A23063T, del21765-770, C23604A, C23271A) might increase the ability of the S protein to bind to its receptor, angiotensin-converting enzyme 2 (ACE2), present on the surface of several cell types, thus increasing the virus infectiveness [6]. The Allplex™ SARS-CoV-2 Assay (Seegene), specifically targeting S gene together with E, RdRp and N genes, allowed us to rapidly sort putative carriers of the B.1.1.7 lineage for further sequencing confirmation by the simple observation of the shape of the amplification curve of the RdRp/S targets. As possible explanation, mutations in the target sequence might moderately affect amplification efficiency, leading to minor changes of the amplification curve slope.

In the present work we aimed to confirm the association of the abnormal amplification curves observed in the reverse transcriptase-PCR (RT-PCR) assay with the presence of B.1.1.7 variant by the gold standard sequencing analysis for the identification of genetic variants of viral RNA.

Approval of the Institutional Review Board is not applicable. All procedures described in the study have been actuated according to ethical principles for medical research involving human subject stated in the Declaration of Helsinki.

We re-analyzed 1,000 SARS-CoV-2 positive samples of oro-nasopharyngeal swabs by the Allplex™ SARS-CoV-2 Assay (Seegene, Seoul, Korea); samples have been archived at -80 °C from October 2020 to March 2021 in the Clinical Pathology and Microbiology Unit of the Vito Fazzi General Hospital of Lecce (Italy) in the context of the diagnostic routine during the COVID-19 pandemic. The assay is an in vitro diagnostic (IVD) real-time RT-PCR test intended for the qualitative detection of SARS-CoV-2 viral nucleic acids in human oro-nasopharyngeal swab. RT-PCR was performed with the CFX96™ Real-time PCR Detection System-IVD (Bio-Rad Laboratories Inc, CA, USA).

Allplex™ SARS-CoV-2 Assay enables simultaneous amplification and detection of target nucleic acids of E, RdRp, S and N genes with four fluorophores: FAM, HEX, Cal Red 610 and QUASAR 670. The amplification of specific gene sequences in the reaction is reported as a sigmoidal amplification curve, the relative fluorescence from each sample was calculated with Bio-Rad CFX Manager software V3.1 (Bio-Rad Laboratories) with the baseline-subtracted curve-fit setting and selecting the fluorescence from FAM (gene E) and Cal Red 610 (RdRp/S gene). The shape of the amplification curves and, specially, of the phase-2 (or logarithmic phase) was carefully inspected to assess the efficiency of the S gene amplification.

Analytical sensibility was 50 copies/reaction for all detected targets. An exogenous gene was used as internal control (IC, highlighted by the fluorophore HEX) to monitor the whole process of nucleic acid extraction and to check for any possible PCR inhibition. Amplification was carried out without RNA extraction, after thermal inactivation (according to the Communication N. 7657/COV19 of October 17, 2020 of the Ministry of Health). Negative and positive controls were included. Allplex™ 2019-nCoV correctly detects SARS-CoV-2 virus RNA even in the presence of the English variant of the virus (B.1.1.7 lineage) and the South African variant (B.1.351 lineage).

A subgroup of 25 samples with non-sigmoidal amplification curve and 25 samples with classical/sigmoidal amplification curve were sent to the Zooprophylactic Institute of Putignano (Italy) for viral RNA sequencing. In brief, targeted sequence of SARS-CoV-2 gene S was performed starting from RNA purification, reverse transcription, multiplex PCR followed by generation of libraries for next-generation sequencing (NGS) on Illumina Platform MiniSeq System.

We found that 130 of 1,000 SARS-CoV-2 positive samples, re-analyzed by RT-PCR Allplex™ 2019-nCoV, showed abnormal amplification curve with non-sigmoidal trend.

In particular, 130 samples positive for the genes E, RdRp/S and N, analyzed by CFX96™ (BIORAD) and CFX Manager™ Software-IVD v1.6 showed abnormal signals for Cal Red 610 fluorophore, which highlights the RdRp/S gene. Table 1 shows the mean of the cycle threshold (Ct) obtained for the RdRp/S genes target of patients subdivided according to the shape of the amplification curve, very low Ct index of high viral load and very infectious patients.

Click to view

Table 1. Cycle Threshold (Ct) of RT-PCR Amplification of SARS-CoV-2 Sequence Targets Obtained for Sigmoidal and Non-Sigmoidal Amplification Curves

Abnormal amplification curves are showed in Figure 1. In particular, the non-sigmoidal curve showed a sequence of the least two inflection points indicating a curve increasing in slope each cycle and then decreasing in slope each cycle; the (classical) sigmoidal curve has an S-shaped form.

Click for large image

Figure 1. Positive SARS-CoV-2 RT-PCR amplification curves. Comparison of the (classical) amplification curve, which appears an S-shaped curve (in bold), and (abnormal) non-sigmoidal amplification curves for RdRp/S target. SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; RT-PCR: reverse transcriptase-polymerase chain reaction; RdRp: RNA-dependent RNA polymerase.

According to the shape of the amplification curves, patients were divided into “classical” and “abnormal” curve groups. Twenty-five of the 130 samples with an “abnormal” trend were sequenced at the Zooprophylactic Institute of Putignano. All of these were identified as the English variant (B.1.1.7 Lineage), while none of 25 patients with classical curve which were sequenced evidenced the presence of the English variant (nor other variants).

From September 2020, the “English variant” (B.1.1.7 lineage) of SARS-CoV-2 began to circulate; becoming dominant in many areas [7].

Allplex™ SARS-CoV-2 Assay was used to test positive samples for SARS-CoV-2 infection archived during the period between October 1, 2020 and March 1, 2021 of the COVID-19 pandemic. The RT-PCR assay’s performance was evaluated on a number of 1,000 nasopharyngeal samples received in UTM transport medium (Copan, Mylan).

We found 130 samples positive for the E, RdRp, S and N genes, with Ct much lower than 35, with an “abnormal” curve for the Cal Red 610 fluorophore identifying the RdRp/S genes (Fig. 1 and Table 1). A group of 25 patients with abnormal amplification curve were sequenced and in all of them the English variant (lineage B.1.1.7) was identified.

In detail, this is the variant VOC 202012/01, lineage B.1.1.7 characterized by the presence of numerous mutations in the S protein of the virus (A23063T, del21765-770, C2360A, C23271A) and by mutations in other regions of the viral genome [1, 4].

The variant was first identified in south-eastern regions of the UK in September 2020 in conjunction with a rapid increase in the number of new confirmed cases of SARS-CoV-2 infection [1]. Almost all the countries of the European Union and the European Economic Area (EU/EEA), including Italy, have notified the presence of cases of SARS-CoV-2 virus infection caused by the VOC variant 202012/01, lineage B. 1.1.7.

The correlation between the abnormal trends of the amplification curve of the Cal Red 610 fluorophore of the RdRp/S genes was suggestive of a mutation in the S gene.

The VOC 202012/01 variant, lineage B.1.1.7 has a higher transmissibility [2, 7, 8], and may be associated with greater virulence [9, 10].

The structural organization of the coronavirus protein S is very similar to that of the protein S of other coronaviruses such as SARS-CoV and Middle East respiratory syndrome (MERS)-CoV. It is a transmembrane trimeric protein formed by three identical units, called protomers. Each CoV-2-S protomer includes two functional subunits: one responsible for binding to the receptor on target cells (the S1 subunit), and the other involved in fusion with the cell membrane (S2 subunit). More in detail, the S1 subunit contains an N-terminal domain (NTD); two subdomains called SD1 and SD2 (subdomains 1 and 2); the receptor binding domain (RBD) responsible for binding to the host cell through interaction with ACE2 and a C-terminal domain that contains the fusion machinery that helps the virus enter cells [11-13].

Interestingly, CoV-2-S possesses an insertion of four amino acids at the boundary between S1 and S2 with respect to the SARS-CoV protein S. These four additional amino acids constitute the cleavage site for a specific human protease called furin [12]. Given the practically ubiquitous expression of furin-like proteases, the presence of this peculiar cleavage site for furin in CoV-2-S has suggested that they may have participated in the acquisition of the broader cell and tissue tropism of CoV-2 compared to SARS-CoV, as well as an increase in its transmissibility and pathogenicity [12]. It has recently been shown that, like TMPRSS2, furin is essential for the entry of CoV-2 into host cells. Furthermore, cathepsin D, a typical protease of lysosomes, is also required for efficient entry of CoV-2 [13].

Considering the greater transmissibility and the rapid diffusion trend in countries earlier affected by the circulation of VOC 202012/01, it is expected that it will become dominant in the Italian and European scenario.

At present, in the European and World scenario characterized by the fast mutation of viral genome, it is necessary to continue to monitor the circulation of the different variants of the SARS-CoV-2, in order to contain and slow down the spreading of the known variant VOC 202012/01 and reinforce measures against their diffusion throughout the country.

The rapid selection of putative SARS-CoV-2 variants by preliminary observation of abnormal RT-PCR curve of the S gene could represent a useful tool for rapid and low-cost identification of suspected variants, with consequent adoption of prevention measures and rapid isolation of supposed very infectious patients, especially because the viral genome sequencing approach or variants target specific amplification is, at the moment, not always easy to be performed in emergency situations, and represents a notable cost for public as well as private health systems. At the moment, more “anomalous” RT-PCR positive samples sequencing are ongoing in order to validate our preliminary results. Anyhow, although the unquestionable convenience in the use of a simple and easy approach as the Allplex RT-PCR for quick population screening, we have to consider that other variants than VOC 202012/01 cannot be detected by this approach and the viral genome sequencing remains the gold standard method for unknown variants identification.

Acknowledgments

None to declare.

Financial Disclosure

None to declare.

Conflict of Interest

The authors declare that they have no conflict of interest.

Informed Consent

Informed consent was not requested because the study was a reanalysis of previous results from archived samples.

Author Contributions

MG and GL conceptualized the study, drafted the initial manuscript, reviewed and revised the article. The data were collected and analyzed by SPF, DC, DM and AS. All authors approved the final article as submitted and agree to be accountable for all aspects of the work.

Data Availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

1. Ciotti M, Ciccozzi M, Terrinoni A, Jiang WC, Wang CB, Bernardini S. The COVID-19 pandemic. Crit Rev Clin Lab Sci. 2020;57(6):365-388.
   doi pubmed
2. Eurosurveillance editorial team. Updated rapid risk assessment from ECDC on the risk related to the spread of new SARS-CoV-2 variants of concern in the EU/EEA - first update. Euro Surveill. 2021;26(3).
   doi pubmed
3. Wang C, Liu Z, Chen Z, Huang X, Xu M, He T, Zhang Z. The establishment of reference sequence for SARS-CoV-2 and variation analysis. J Med Virol. 2020;92(6):667-674.
   doi pubmed
4. Harcourt J, Tamin A, Lu X, Kamili S, Sakthivel SK, Murray J, Queen K, et al. Severe Acute Respiratory Syndrome Coronavirus 2 from patient with coronavirus disease, United States. Emerg Infect Dis. 2020;26(6):1266-1273.
   doi pubmed
5. Pandey S, Yadav B, Pandey A, Tripathi T, Khawary M, Kant S, Tripathi D. Lessons from SARS-CoV-2 pandemic: evolution, disease dynamics and future. Biology (Basel). 2020;9(6).
   doi pubmed
6. Tseng YH, Yang RC, Lu TS. Two hits to the renin-angiotensin system may play a key role in severe COVID-19. Kaohsiung J Med Sci. 2020;36(6):389-392.
   doi pubmed
7. L CC, Mitchell PK, Plocharczyk E, Diel DG. Identification of a SARS-CoV-2 lineage B1.1.7 virus in New York following return travel from the United Kingdom. Microbiol Resour Announc. 2021;10(9).
   doi pubmed
8. Volz E, Hill V, McCrone JT, Price A, Jorgensen D, O'Toole A, Southgate J, et al. Evaluating the effects of SARS-CoV-2 spike mutation D614G on transmissibility and pathogenicity. Cell. 2021;184(1):64-75 e11.
   doi
9. Rahimi F, Talebi Bezmin Abadi A. Implications of the emergence of a new variant of SARS-CoV-2, VUI-202012/01. Arch Med Res. 2021.
   doi pubmed
10. Galloway SE, Paul P, MacCannell DR, Johansson MA, Brooks JT, MacNeil A, Slayton RB, et al. Emergence of SARS-CoV-2 B.1.1.7 Lineage - United States, December 29, 2020-January 12, 2021. MMWR Morb Mortal Wkly Rep. 2021;70(3):95-99.
    doi pubmed
11. Khalid Z, Naveed H. Identification of destabilizing SNPs in SARS-CoV2-ACE2 protein and spike glycoprotein: implications for virus entry mechanisms. J Biomol Struct Dyn. 2020:1-11.
    doi
12. Wrapp D, Wang N, Corbett KS, Goldsmith JA, Hsieh CL, Abiona O, Graham BS, et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science. 2020;367(6483):1260-1263.
    doi pubmed
13. Walls AC, Park YJ, Tortorici MA, Wall A, McGuire AT, Veesler D. Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell. 2020;181(2):281-292 e286.
    doi pubmed

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