Molecular detection of SARS‐CoV‐2 strains and differentiation of Delta variant strains.

AN0159326416;[6moc]01sep.22;2022Sep28.05:24;v2.2.500

Molecular detection of SARS‐CoV‐2 strains and differentiation of Delta variant strains 

The Delta variant of SARS‐CoV‐2 has now become the predominant strain in the global COVID‐19 pandemic. Strain coverage of some detection assays developed during the early pandemic stages has declined due to periodic mutations in the viral genome. We have developed a real‐time RT‐PCR (RT‐qPCR) for SARS‐CoV‐2 detection that provides nearly 100% strain coverage, and differentiation of highly transmissible Delta variant strains. All full or nearly full (≥28 kb) SARS‐CoV‐2 genomes (n = 403,812), including 6422 Delta and 280 Omicron variant strains, were collected from public databases at the time of analysis and used for assay design. The two amino acid deletions in the spike gene (S‐gene, Δ156‐157) that is characteristic of the Delta variant were targeted during the assay design. Although strain coverage for the Delta variant was very high (99.7%), detection coverage for non‐Delta wild‐type strains was 93.9%, mainly due to the confined region of design. To increase strain coverage of the assay, the design for CDC N1 target was added to the assay. In silico analysis of 403,812 genomes indicated a 95.4% strain coverage for the CDC N1 target, however, in combination with our new non‐Delta S‐gene target, total coverage for non‐Delta wild‐type strains increased to 99.8%. A human 18S rRNA gene was also analyzed and used as an internal control. The final four‐plex RT‐qPCR assay generated PCR amplification efficiencies between 95.4% and 102.0% with correlation coefficients (R2) of >0.99 for cloned positive controls; Delta and non‐Delta human clinical samples generated PCR efficiencies of 93.4%–97.0% and R2 > 0.99. The assay also detects 98.6% of 280 Omicron sequences. Assay primers and probes have no match to other closely related human coronaviruses, and did not produce a signal from samples positive to selected animal coronaviruses. Genotypes of selected clinical samples identified by the RT‐qPCR were confirmed by Sanger sequencing.

Keywords: assay; COVID‐19; Delta variant; diagnosis; Omicron variant; PCR; SARS‐CoV‐2

INTRODUCTION

Since the COVID‐19 pandemic began, there have been over 235 million confirmed human cases globally, and over 4.8 million deaths (https://covid19.who.int/, October 2021). In October 2021, a total of 6.2 million vaccines doses have been administered worldwide, which have contributed to slowing the spread of SARS‐CoV‐2, yet, it remains unclear when the global pandemic will end. Multiple variants of concern (VOC) strains have emerged, some with increased transmission rates and potentially increased pathogenicity (Cherian et al., 2021; Dougherty et al., 2021; Farinholt et al., 2021; Focosi et al., 2021; Kumar et al., 2021). The transmission rate of the Delta variant (B.1.617.2) is twice as high as previous variants and is also the predominant strain currently circulating in the United States and in many other countries or regions (Dougherty et al., 2021; Duong, 2021; Teyssou et al., 2021; https://www.cdc.gov/; https://www.gisaid.org/hcov19‐variants/). Nearly 398,000 vaccine doses have been administered in the United States, (https://www.cdc.gov/), and current data indicate that vaccines do offer robust protection against severe infections and death. However, it is still possible for vaccinated people to become infected and serve as source of transmission (Farinholt et al., 2021; Rotondo et al., 2021; Subbarao, 2021; Tenforde et al., 2021; The Lancet Infectious D, 2021). Due to the mutations in the SARS‐CoV‐2 strains, suspected false negative results from some PCR assays have been predicted (Zitek, 2020). A strain coverage analysis of current SARS‐CoV‐2 sequences in this study has also indicated that continued mutations in the viral genomes have resulted in reducing strain coverage of certain assays, including the CDC N1 target and a target developed in this lab in the early stage of the pandemic.

According to US Centers for Disease Control and Prevention (CDC), the Delta variant is currently the only circulating VOC in the United States. A combination of point mutations and deletions has been used to characterize the variant. Major mutations in the spike gene (S‐gene) include T19R, Δ156‐157, L452R, T478K, D614G, P681R and D950N (ECDC, 2021). Efforts have been made to sequence SARS‐CoV‐2 genomes from test‐positive samples, primarily to monitor for and identify the Delta variant strains (Herlihy et al., 2021; Lam‐Hine et al., 2021; Teyssou et al., 2021). However, sequencing procedures are labour intensive and can require several days or sometimes up to 2 weeks to obtain results. We have developed and validated a multiplex reverse transcription real‐time PCR (RT‐qPCR) for the detection of >99% of all SARS‐CoV‐2 strains that can concurrently detect and differentiate >99% of known Delta variant strains.

MATERIALS AND METHODS

Clinical sample and positive amplification controls

One hundred thirty SARS‐CoV‐2 nasal swab clinical samples were collected by Kansas State University Lafene Health Center, including 40 wild‐type, 54 Delta variant strains and 36 negative samples, and submitted to Kansas State Veterinary Diagnostic Laboratory (KSVDL) Public Health section for COVID‐19 diagnosis using ThermoFisher Scientific TaqPath™ COVID‐19 Combo Kit (Waltham, MA, USA). Selected SARS‐CoV‐2 positive samples were PCR tested and genotyped followed by Sanger sequencing confirmation. Viral RNA of a confirmed Delta variant and a wild‐type strain were extracted by MagMax™ Viral/Pathogen Nucleic Acid Isolation Kit (Applied Biosystems/ThermoFisher, Foster City, CA, USA) according to the manufacturer's recommendations. A pair of sequencing primers amplifying a 446 base pair (bp) of wild‐type and 440 bp of Delta variant strains (Table 1) that encompassed the 6 nucleotide (nt) deletion region in the Delta variant was used for RT‐PCR amplification. Amplicon size was verified on Qiagen QIAxcel (Valencia, CA, USA), and purified using the Qiagen QIAquick PCR Purification Kit. Purified PCR products were ligated into the pCR™4‐TOPO™ vector using the TOPO™ TA Cloning™ Kit (Invitrogen/ThermoFisher, Carlsbad, CA), according to the manufacturer's instructions. They were then transformed into Mix & Go competent cells (Zymo Research, Irvine, CA 92614, U.S.A.), and blue‐and‐white colonies were screened on isopropyl β‐d‐1‐thiogalactopyranoside (IPTG), 5‐bromo‐3‐chloro‐3‐indolyl β‐d‐Galactopyranoside (X‐Gal) and carbenicillin‐containing LB plates for overnight culturing. The selected white colonies were cultured in carbenicillin‐containing LB broth, and plasmid DNA was extracted using Qiagen QIAquick Spin Miniprep Kit. The presence of the cloned insert was confirmed through the prototype of this newly designed RT‐qPCR assay and Sanger sequencing.

1 TABLEPrimers and probes used in this study

Primer and probe design

The first 840 nt in the S‐gene of SARS‐CoV‐2 from 100 Delta variant and 100 non‐Delta strains were randomly selected and downloaded from public databases. The downloaded sequences that encompassed the Δ156‐157 deletion region were aligned in CLC Main Workbench 7 (Qiagen, Valencia, CA, USA). A common primer pair that amplifies both the Delta variant and the wild‐type strains was designed. Minor groove binder (MGB) probes for both the Delta variant and non‐Delta strains were designed in the same region (Table 1) to maximize for competitive binding affinity of a probe to its corresponding genotype. Primers and probes were synthesized from Applied Biosystems/ThermoFisher. The SARS‐CoV‐2 viral genome is roughly 30,000 nt. To check the comprehensive strain coverage of the new assay, all genome sequences with at least 28,000 nt were downloaded for in silico analysis. Following this criterion, 403,812 full or near full genomes of SARS‐CoV‐2 (including 6422 Delta and 280 Omicron variant strains) were obtained, and analyzed for strain coverages of the newly designed primers and probes, together with the CDC N1 primers and probe.

Optimization of PCR conditions

A 20 μl RT‐qPCR reaction composed of 5 μl of template RNA, 0.5 μM of each forward and reverse qPCR primers, 0.25 μM of SARS2‐delta probe (SARS2‐dPr), 0.5 μM of SARS2‐wild type (SARS2‐wPr) and 18S probes, 0.125 μM of nCoV‐N1 probe, and 5 μl of 4× TaqPath™ 1‐Step RT‐qPCR Master Mix (Applied Biosystems/ThermoFisher) was used. Annealing temperatures for the primers and both wild‐type and Delta probes were tested with a thermal gradient ranging from 50 to 65°C, which resulted in an optimum annealing temperature of 59.5°C. Therefore, the thermocycling parameters used for the following experiments were set with an RT reaction at 48°C for 10 min, then an inactivation and denaturation at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 20 s and annealing/extension at 60°C for 40 s. All RT‐qPCR reactions were run on a Bio‐Rad CFX96 Touch Real‐Time PCR Detection System, and cycle threshold value (Ct) of different clinical samples and cloned plasmids were generated and analysed with the Bio‐Rad CFX Maestro 2.2 software.

Analytical analysis using cloned positive control plasmids and clinical samples

The analytical sensitivity of the assay was first studied by generating standard curves with triplicates of 10‐fold serial dilutions of cloned plasmids. The analytical sensitivity was then assayed with high‐concentration clinical samples of a Delta variant and a wild type strain for standard curve analysis. PCR amplification efficiencies and correlation coefficients were calculated with the Bio‐Rad CFX Maestro 2.2 software.

Analysis of assay's specificity

The specificity was first analyzed in silico by comparing closely related coronaviruses including SARS‐CoV‐1, MERS, HKU1, 229E, NL63 and OC43 human coronavirus strains (Figure 1). As current animal coronavirus strains are distantly related to SARS‐CoV‐2, they were not analyzed in silico, but selected animal coronavirus positive samples were tested with the new SARS‐CoV‐2 assay (Table 3). The animal samples used were positive to bovine coronaviruses, canine coronavirus, porcine epidemic diarrhea virus (PEDV); porcine deltacoronavirus (PDCoV), and porcine transmissible gastroenteritis virus (TGEV).

Limit of detection determination

The concentrations of cloned plasmids were measured by a Nanodrop spectrophotometer (ThermoFisher). The limit of detection (LOD) Ct values were determined by generating 10‐fold serial dilutions of the plasmids, and LOD Ct values were refined with twofold serial dilutions from the end‐point 10‐fold diluted sample. The target copy numbers corresponding to LOD Ct values were calculated using formula below:

<semantics>Plasmid<mspace />copies/μl=6.02×1023×Xng/μl×10−9Plasmid<mspace />length<mspace />bp×660

X: Concentration in ng/μl measured by Nanodrop spectrophotometer.

Diagnostic validation using clinical samples

In total, 94 positive and 36 negative clinical human nasal swab samples submitted to KSVDL were selected for SARS‐CoV‐2 testing and differentiation of Delta variant and wild‐type strains. All clinical samples were previously tested by KSVDL Public Health section with the commercial TaqPath™ COVID‐19 Combo Kit (ThermoFisher).

Sequencing confirmation of selected Delta variant and wild‐type strains

The cloning primers encompassing the 6‐nt deletion region (Δ156‐157 aa) in the S‐gene (Table 1) and amplifying a 446 bp (wild type) or 440 bp (Delta variant) fragment were also used for Sanger sequencing confirmation. Out of the 94 positive clinical human samples, 10 wild‐type and 10 Delta variant samples were randomly selected and subjected to RNA extraction and RT‐PCR amplification using TaqPath™ 1‐Step RT‐qPCR Master Mix (Applied Biosystems/ThermoFisher). The amplified fragments were purified through Qiagen QIAquick PCR Purification Kit; concentrations were measured by ThermoFisher Nanodrop, and adjusted according to the sequencing facility's guidelines. The concentration‐adjusted amplicons were sent to GeneWiz (South Plainfield, NJ, US) for sequencing. The raw sequencing data were trimmed and assembled using Qiagen CLC Main Workbench. The identity of assembled sequences was confirmed by comparing to the annotated sequences in the NCBI GenBank database.

RESULTS

In silico sequence analysis and primer and probe design

A common pair of primers that amplifies from both the Delta variant and non‐Delta wild‐type strains was designed using a pilot dataset of 100 Delta variant and 100 non‐Delta strains. The Delta variant probe was designed to envelope the 6‐nt deletion in the S‐gene (Δ156‐157 aa deletion) that is characteristic of Delta strains, while the wild‐type probe design targeted the corresponding region of the deletion site (Table 1). In order to utilize additional nucleotides to increase distance between the probes and primer on the same strand, both probes were placed on the antisense strand. To ensure the strain coverage of the primers and probes, a collection of 403,812 full or nearly full SARS‐CoV‐2 sequences, including 6422 Delta and 280 Omicron variant sequences, were collected from public databases and used to map the primers and probes. The coverage was calculated by determining the co‐presence of two primers and each probe in their respective datasets. Primers and probes covered 99.7% and 93.9%, respectively, of the Delta (6422 sequences) and non‐Delta strains (397,110 sequences) examined (Table 2). Analysis of the CDC N1 primer and the probe set indicated a strain coverage of 95.4% of the 397,110 sequences. However, the newly designed non‐Delta target together with the CDC N1 target had a combined strain coverage of 99.8% (Table 2). Although the assay will not differentiate Omicron variant, the non‐Delta wild‐type assay can detect 98.6% of Omicron strains. The CDC N1 probe has a single base mismatch close to the 5′ end of most Omicron sequences, it is unlikely to prevent the probe from binding to these templates. If that is true, the CDC N1 assay would detect 99.6% (279/280) of Omicron strains (Table 2).

2 TABLEStrain coverage analysis of three molecular targets used in this assay

1 * : The SARS2 wild‐type assay matched 276 Omicron sequences; The CDC N1 probe has a single base mismatch close to the 5′ end to the Omicron sequences; If the mismatch does not prevent binding to the template, the number of matching sequences would be 279 (99.6%).

Standard curve analysis using genotype‐confirmed plasmids

The genotypes of cloned plasmids containing the Delta variant or the non‐Delta wild‐type segments were confirmed by the prototype of this RT‐qPCR assay and Sanger sequencing (data not shown). Standard curves generated by 10‐fold dilutions of the plasmids using the multiplexed assay indicated a broad detection range (Figure 2). PCR amplification efficiency of the Delta variant genotype was 99.9% with a correlation coefficient (R2) of 0.998. The S‐gene wild‐type target indicated the PCR amplification efficiency of 102.4% with an R2 of 0.998. The CDC nCoV‐N1 target indicated the PCR efficiency of 95.4% with an R2 of 0.997 (Figure 2).

Limit of detection determination

The standard curves using cloned plasmids indicated that the assay can consistently amplify all three targets in the presence of the 18S rRNA gene up to an endpoint Ct of ∼35. Based on the Nanodrop measured concentrations of the plasmids and the dilution factor to reach Ct 35, the calculated copy number per PCR reaction at this limit of detection Ct was between 14 and 24 copies.

Standard curve analysis using clinical samples

Standard curve generated with a Delta variant sample

Standard curve using the human Delta variant sample, verified by sequencing, is shown in Figure 3. The sample was selected because its high viral concentration could offer a wide dynamic range of dilutions to be tested for. From the figure, PCR amplification efficiency for the Delta variant target was 93.7% with a correlation coefficient (R2) of 0.998, and PCR amplification efficiency of the nCoV‐N1 wildtype target was 93.4% with an R2 of 0.989. Because the non‐Delta target in the S‐gene was also designed in the Δ156‐157 aa deletion region, no signal was generated from this probe.

Standard curve generated with a non‐Delta wildtype sample

A standard curve was generated with a human non‐Delta wild‐type sample (Figure 4). Results of the S‐gene wild‐type target indicated a PCR amplification efficiency of 97.0% with a correlation coefficient (R2) of 0.996. The PCR amplification efficiency for the CDC N‐gene target, nCoV‐N1, was 94.8% with an R2 of 0.998. Similarly, no signal was observed for the Delta variant probe.

Specificity of the assay

In silico analysis of closely related human coronaviruses

Results from an in silico analysis of five sequences each from the Delta variant and non‐Delta wild‐type of SARS‐CoV‐2, SARS‐CoV‐1, MERS, and one sequence each for HKU1, 229E, NL63 and OC43 human coronavirus strains indicated that the newly designed SARS‐CoV‐2 assay does not match to any of the above‐mentioned strains. The two newly designed SARS‐CoV‐2 targets are highly specific to their respective genotypes as well (Figure 1).

Animal coronavirus testing

All species of coronavirus positive animal samples that were available in our collection tested negative by the new SARS‐CoV‐2 RT‐qPCR assay indicating that the new assay does not detect these animal coronaviruses (Table 3)

3 TABLETesting results of animal coronavirus positives specimens

Diagnostic validation using human clinical samples

SARS‐Cov‐2 positive clinical sample testing

All 94 human clinical samples used in this study that previously tested positive to SARS‐CoV‐2 by the ThermoFisher TaqPath™ COVID‐19 Combo Kit also tested positive by our newly developed RT‐qPCR assay. Out of the 94 clinical samples, 40 were identified as non‐Delta wild‐type strains, while 54 tested positive for the Delta variant (Table 4).

4 TABLEHuman Delta variant and non‐Delta wild‐type sample testing results

4 Abbreviations: SARS2‐d, Delta variant target; SARS2‐w, Wild‐type target; nCoV‐N1, CDC 2019‐nCoV_N1 target; 18S‐3, Human 18S rRNA gene target as internal control.

SARS‐CoV‐2 negative clinical sample testing

In addition to the 94 positive clinical samples, we also tested 36 clinical samples that were previously determined as negative for SARS‐CoV‐2. All 36 samples also tested negative by the new RT‐qPCR differential assay (Table 5).

5 TABLEHuman negative clinical sample testing results

5 Abbreviations: SARS2‐d, Delta variant target; SARS2‐w, Wild‐type target; nCoV‐N1, CDC 2019‐nCoV_N1 target; 18S‐3, Human 18S rRNA gene target as internal control.

Sequencing confirmation of selected Delta variant and wild type strains

Sequence identities of 10 Delta and 10 non‐Delta strains to the top matches in NCBI are shown in Table 6. The genotypes of all 20 sequences were identified to be the same as those in the NCBI GenBank database. Figure 5 also provides a visual representation of the probe region of the 10 Delta variant and 10 wild‐type strains in the beginning of the S gene.

6 TABLESanger sequencing confirmation of selected Delta and non‐Delta clinical samples

DISCUSSION

The emergence of multiple VOC strains has introduced additional challenges to SARS‐CoV‐2 diagnostics and disease management (Safarchi et al., 2021; Zimmerman et al., 2021). Currently, the most prevalent VOC circulating in the United States and globally remains the Delta variant (Lee, 2021; Li et al., 2021; https://www.cdc.gov/), which has a higher transmission rate, and potentially increased pathogenicity and evasion of the vaccinated immune system compared to other strains (Dougherty et al., 2021; Farinholt et al., 2021; Focosi et al., 2021; Kumar et al., 2021). The detection of Delta variant strain is mainly through Sanger sequencing or next‐generation sequencing (Cherian et al., 2021; Zimmerman et al., 2021), which are labour intensive and require a long turn‐around time to results. Although the Delta variant has become the predominant strain in some countries, like in the United States, in other countries and regions, multiple VOC strains are still co‐circulating (Campbell et al., 2021; https://www.gisaid.org/hcov19‐variants/). Therefore, a fast PCR‐based detection of the more transmissible Delta variant can serve as an important public health tool for variant tracing and epidemiological investigations.

One characteristic of the Delta variant is a two amino acid deletion in the S gene in position 156‐157 (ECDC, 2021). These two amino acid deletions are conferred by six nucleotide deletions, and serves as a better diagnostic target for differential detection compared to other tests that target on single‐nucleotide mutations. Both our analytical and diagnostic validation data have indicated that the multiplex RT‐qPCR is highly specific, and can differentiate the Delta variant from other SARS‐CoV‐2 strains. Both specific amplification conferred by primers (Graber et al., 2021) and specific detection by probes (Heijnen et al., 2021) have been used for SARS‐CoV‐2 variant detections. Each strategy has its own advantages. In this study, we chose to use specific probes for Delta and non‐Delta strain differentiations, mainly because the genotype‐specific probes can be labelled with different dyes, and the genotype of the sample is readily interpreted. To ensure the accuracy of the PCR genotyping results, selected Delta variant and wild‐type strains were subjected to Sanger sequencing for further genotype confirmation.

A main goal driving the development of this assay was to identify as many SARS‐CoV‐2 strains as possible, whether they were Delta variants or otherwise. To ascertain the coverage of our assay, 403,812 SARS‐CoV‐2 full genomes, including 6422 Delta and 280 Omicron variant strains, were downloaded and analyzed for their in silico binding potential to the primer and probe designs from this current study. Our full‐genome data analysis indicated that our newly designed wild‐type target, in combination with the CDC N1 target, should detect greater than 99% of all SARS‐CoV‐2 strains, including 98.6% of Omicron strains (Table 2). The coverage to the Omicron strains would be 99.6% if the CDC N1 probe that has a single‐base mismatch close to the 5′ end can still bind to the template and generate signal. Our data analysis also indicated that the 156‐157 aa deletion in the S‐gene is unique only to B.1.617.2 (Delta) and B.1.617.3 variant strains. As circulation of the B.1.617.3 strains have greatly decreased, our newly developed test should just detect the Delta variant strains. The assay designed for the mutant target should also detect greater than 99% of Delta variant strains (Table 2). Thus, our newly developed SARS‐CoV‐2 assay should detect more than 99% of all Delta and non‐Delta SARS‐CoV‐2 strains.

The genomes of viruses are continuously mutating, as evidenced during this COVID‐19 pandemic. Monitoring for the changes and modifying detection assays accordingly will likely be required through the duration of the pandemic. We will continue to monitor for these changes, and keep our assay up‐to‐date in order to detect the majority of contemporary VOC strains.

ETHICAL STATEMENT

All human nasal swab samples were collected by Lafene Health Center, Kansas State University, and submitted to Kansas State Veterinary Diagnostic Laboratory for diagnosis under CLIA certification # 17D0648239. The research activities of this study is also authorized under Kansas State University IBC # 1322.9.

CONFLICT OF INTEREST

All authors declare no conflict of interest in this study.

ACKNOWLEDGEMENT

This study was supported by Kansas State Veterinary Diagnostic Laboratory, Kansas State University.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available in National Center for Biotechnology Information (GenBank) at https://www.ncbi.nlm.nih.gov/. These data were derived from the following resources available in the public domain:‐ Global initiative on sharing all influenza data, https://www.gisaid.org/

By Vaughn Hamill; Lance Noll; Nanyan Lu; Wai Ning Tiffany Tsui; Elizabeth Poulsen Porter; Mark Gray; Tesfaalem Sebhatu; Kyle Goerl; Susan Brown; Rachel Palinski; Sasha Thomason; Kelli Almes; Jamie Retallick and Jianfa Bai

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