Laboratory Testing Methods for SARS-CoV-2 – Key Vocabulary

Introduction

• First reports of COVID-19 made to WHO on 31 Dec 2019; declared

  • Public-health emergency of international concern: 30 Jan 2020

  • Pandemic: 11 Mar 2020

• By 12 May 2020: >4{,}302{,}774 confirmed cases, 289{,}561 deaths across 212 countries/territories.

• SARS-CoV-2 is the 3rd zoonotic, severe, respiratory CoV this century (after SARS-CoV-1 2003, MERS-CoV 2012) and the 7th known human-to-human transmissible CoV.

• Critical need: rapid, accurate diagnostics to enable isolation, quarantine, clinical management, epidemiology, and therapy selection (e.g., convalescent plasma).

Viral Genome & Structure

• Type: positive-sense, single-stranded RNA (ssRNA), Group IV.

• Genome length: \approx 30\,\text{knt} with 14 ORFs → structural, replication, accessory proteins.

  • Key genes: ORF1a/1b (replicase), S, E, M, N, ORF3a/3b, ORF6, ORF7a/7b, ORF8, ORF9b, ORF10, ORF14.

• Virion organization:

  • Lipid bilayer envelope containing E & M proteins.

  • Surface Spike (S) glycoprotein → receptor binding (ACE-2) & “corona” appearance.

  • Nucleocapsid (N) binds RNA genome.

• Mutation surveillance via GISAID reveals early lineage splits; mutations may influence test sensitivity/primer binding.

Why Testing Matters

• Tracks spread, identifies symptomatic & asymptomatic carriers.

• Guides public-health interventions (social distancing, isolation).

• Allows contact tracing, resource allocation, evaluation of immunity & vaccine efficacy.

• Supports selection/donation of convalescent plasma, passive antibody therapies.

Gold-Standard Molecular Test: Real-Time RT-PCR

• Principle: reverse-transcribe viral RNA → cDNA → amplify with sequence-specific primers/probes; fluorescence quantifies target.

• Specimen hierarchy (CDC):

  • High priority: nasopharyngeal swab

  • Lower: oropharyngeal swab, bronchoalveolar lavage, tracheal aspirate, sputum

• Workflow:

  1. RNA isolation via approved kits.

  2. Reverse transcription.

  3. Real-time PCR amplification.

  4. Controls:

  • Positive: synthetic RNA standards (e.g., \text{BetaCoV_Wuhan_WIV04_2019})

  • Internal: human \text{RNAse P} gene

  • Negative: nuclease-free H₂O + negative patient sample.

• Primer sets (WHO consolidated): China CDC (ORF1ab, N), Charité–Berlin (E, RdRp), HKU, NIID Japan, Thailand NIH, US CDC (three N-gene targets + RP), Institut Pasteur, etc.

  • Sensitivity hierarchy (Charité): E gene screen → RdRp confirmation.

• Performance:

  • Sensitivity \approx 95\%; LoD <10 copies/reaction.

  • Throughput: thousands/day; turnaround 2–5 days (depends on supply chain).

• Limitations:

  • False (+) → primer cross-reactivity, lab contamination.

  • False (−) → RNA degradation, poor sampling, mutations in primer sites.

  • Requires expensive equipment & reagents.

Immunoassays (EIA / ELISA)

• Detect host antibodies (IgM, IgG, IgA) or viral antigens.

• Targets: recombinant S protein (rS, esp. RBD) & recombinant N protein (rN).

• Kinetics:

  • IgM: \sim 3!−!7 days post-symptom onset.

  • IgG: \sim 7!−!25 days; persists longer.

• Procedure:

  • Coat microplate with rS/rN.

  • Add serum → bind Abs.

  • Add enzyme-conjugated secondary Ab → add substrate → colorimetric readout.

• Reported metrics:

  • IgG detection: 85.4\% positivity.

  • IgM: 75.6!−!93.1\%; sensitivities peak ≈2 weeks after symptoms.

• Pros:

  • High-throughput, quantitative titers, simpler than RT-PCR once set up.

  • Identifies past exposure; screens donors for convalescent plasma.

• Cons:

  • Window period—early infection may be negative.

  • Cross-reactivity with SARS-CoV-1 or endemic HCoVs → false (+).

  • Unknown correlation of antibody presence with protective immunity.

  • Recombinant protein quality (post-translational modifications) influences accuracy; mammalian expression preferred over E.\ coli.

• Regulation: Many kits fast-tracked under FDA EUA; rigorous validation still required.

Rapid Point-of-Care: Lateral-Flow Immunoassays (LFIA)

• Format: immunochromatographic strip in plastic cassette; read by naked eye.

• Analyte: either antibodies or antigens (S1/S2 or N).

• Sample: finger-stick blood or saliva; \approx 2 drops.

• Time to result: \approx 15 min.

• Mechanism:

  • Pre-coated capture reagents (mAb or recombinant antigen) on test line.

  • Detector Ab conjugated to gold nanoparticles/latex travels via capillary action; binding generates colored line.

• Advantages: rapid, low-cost, portable, suitable for mass screening & epidemiological surveys.

• Disadvantages: lower sensitivity/specificity vs. ELISA and RT-PCR, especially for antigen detection; requires well-characterized mAbs; still needs validation.

Functional Antibody Test: Serum Virus Neutralization (SVN)

• Measures ability of patient serum to block live SARS-CoV-2 infection in cell culture.

• Cells: Vero, Huh-7, or 293T.

• Read-outs: cytopathic effect (5 days), fluorescence, or plaque reduction (24 h).

• Titer: highest serum dilution preventing infection.

• Use-case: qualifying convalescent plasma donors; research on protective immunity.

• Pros: gold-standard for neutralizing Abs; robust & reproducible.

• Cons: labor-intensive, 5-day turnaround, requires BSL-3 & access to live virus; not routine diagnostics.

Emerging Nucleic-Acid Methods

Isothermal Amplification

• No thermal cycling → faster, less equipment.

• Variants:

  • RT-LAMP (loop-mediated): multiple primers; detection in <30 min; LoD 20–200 copies; reported 100\% sensitivity/specificity.

  • RT-RPA (recombinase polymerase): primers to N gene; similar speed & accuracy; amenable to portable devices.

  • Other chemistries: HDA, SDA, NASBA.

CRISPR-Based (SHERLOCK / Cas13a)

• Pre-amplify target (often via RPA) → Cas13a-crRNA binds specific SARS-CoV-2 sequences (S gene, ORF1ab).

• Target recognition activates collateral cleavage of fluorescent/ lateral-flow reporter.

• Sensitivity: 20!−!200\,\text{aM} (attomolar); results in 30–60 min.

• Paper-dipstick adaptation possible; still experimental but promising.

Next-Generation Sequencing (NGS)

• Full viral genome sequencing for surveillance, mutation tracking, environmental sampling.

• Steps: RNA extraction → rRNA depletion → fragmentation → cDNA library → amplification with universal CoV primers → sequencing → submission to GISAID.

• >17{,}000 genomes deposited (June 2020); reveals generally stable genome with occasional deletions (e.g., 81 nt in ORF7a) & spike mutations (e.g., D614G).

• Costly, labor-intensive; not for routine diagnosis.

Biosafety Considerations

• Diagnostic sample handling: BSL-2 with unidirectional airflow and BSL-3 precautions (respiratory PPE, dedicated don/doff areas).

• Viral culture & characterization: BSL-3; regulatory oversight required.

Logistical & Ethical Challenges

• Scale-up limited by reagent shortages, equipment, trained personnel.

• Need for master protocols, shared reference panels, global data sharing.

• Multiple test modalities reduce dependence on single supply chains.

• Ethical collection/use of convalescent plasma; informed consent mandatory.

Comparative Snapshot (Key Metrics)

• RT-PCR: 2!−!5 days; high accuracy; LoD <10 copies.

• ELISA: hours; high accuracy post-seroconversion; informs past exposure.

• LFIA: minutes; moderate accuracy; field screening.

• SVN: \sim5 days; high accuracy; neutralization functionality.

• Isothermal/CRISPR: <30 min; point-of-care; validation pending.

• NGS: \ge 24 h; high resolution; epidemiology & evolution.

Conclusions

• A multiplexed testing ecosystem (molecular, serological, functional, genomic) is essential for comprehensive COVID-19 control.

• Continuous assay validation is required as viral mutations emerge.

• Data sharing, standardized protocols, and diversified supply chains strengthen pandemic response.

• Future directions: integrate rapid POC tests with digital reporting, refine correlates of immunity, and develop biomarkers to predict susceptibility & disease severity.