SARS-CoV-2 Diagnostics: RT-PCR, Antigen Testing, and Serology — Principles and Interpretation

The COVID-19 pandemic put three categories of viral diagnostic testing under public scrutiny like never before: molecular testing (RT-PCR), antigen testing, and serology (antibody testing). While SARS-CoV-2 is no longer the diagnostic emergency it once was, the underlying principles of these three testing approaches are foundational to all of clinical virology — the same RT-PCR, antigen, and serology principles apply to influenza, RSV, HIV, hepatitis, and virtually every viral pathogen you will encounter clinically.

Figure: Structure of Coronavirus

This article uses SARS-CoV-2 diagnostics as a teaching case study to build a durable understanding of how these three testing modalities work, what each one actually measures, and — critically — how to interpret results correctly.

Background — Why SARS-CoV-2 Needed Three Different Testing Approaches

SARS-CoV-2 is an enveloped, positive-sense, single-stranded RNA virus of the genus Betacoronavirus, with a genome of approximately 30 kb — the largest known RNA virus genome. Its surface is studded with characteristic club-shaped spike (S) proteins that bind to ACE2 (angiotensin-converting enzyme 2) receptors on human respiratory epithelial cells, mediating viral entry.

No single test type can answer every clinical question about a viral infection:

Is the virus present right now, and how much of it? → Molecular testing (RT-PCR)
Is the virus present right now, but I need a fast answer without lab infrastructure? → Antigen testing
Was this person infected at some point in the past, even if they're not infected now? → Serology (antibody testing)

Understanding why each test answers a different question — rather than treating them as interchangeable — is the single most important diagnostic concept this article teaches.

Part 1 — RT-PCR (Molecular Testing) and Ct Value Interpretation

Figure: Genome of SARS-CoV-2 with the most common RT-PCR targets highlighted

How RT-PCR Works

Reverse transcription polymerase chain reaction (RT-PCR) detects the presence of viral genetic material in a sample. It is important to understand a key limitation from the outset: RT-PCR detects viral RNA, not infectious virus — it cannot distinguish between live, infectious virus and non-infectious viral RNA fragments remaining after the infectious virus has already been cleared.

The process:

Viral RNA is extracted from the patient sample, purified, and concentrated
The purified extract is combined with primers, nucleotide bases, reverse transcriptase enzyme, and fluorescently labelled probes
Primers attach to specific target regions of the viral genome, and the enzyme synthesizes a complementary DNA (cDNA) strand
The reaction undergoes repeated thermal cycles (typically up to 40 cycles), with the target sequence doubling exponentially with each cycle
Fluorescently labelled probes emit a signal proportional to the amount of newly synthesized target — the earlier this exponential signal rises above background, the more virus was present in the original sample

RT-PCR assays may target one or multiple viral genes (commonly the N, E, S, and RdRP genes for SARS-CoV-2) — multi-target assays generally offer better specificity and resilience against viral mutation in any single target region.

Understanding the Cycle Threshold (Ct) Value

The cycle threshold (Ct) is the thermal cycle number at which the fluorescent signal first exceeds the background threshold for positivity.

The core relationship to remember:

Low Ct = high viral concentration in the sample (signal crossed threshold early)
High Ct = low viral concentration in the sample (signal crossed threshold late, near the 40-cycle limit)
A 3-point increase in Ct value corresponds to approximately a 10-fold decrease in the quantity of viral genetic material — this logarithmic relationship is the basis for using Ct as a semi-quantitative (not just qualitative) measure

Why Ct value matters clinically: Low Ct values are generally associated with higher infectivity risk. However, this relationship is not absolute — a high Ct can also occur very early in infection (before viral replication has peaked) or from an inadequately collected or partially degraded sample, not just from declining infectivity late in disease. A single Ct value, without clinical context, should never be the sole basis for an infectivity decision.

### Why Ct Values Cannot Be Compared Across Laboratories

This is a critical and frequently misunderstood point: Ct values from different laboratories, or different RT-PCR assay platforms, are not directly comparable. Multiple factors affect the final Ct value independent of true viral load:

Pre-analytical (sample-related) factors:

Adequacy and technique of sample collection
Anatomical site and timing relative to infection course
Presence of PCR inhibitors in the sample

Analytical (laboratory/assay-related) factors:

Total sample collection buffer/transport medium volume
Sample preparation method (heat inactivation vs lysis-based extraction)
Reagent volumes used at each step
The specific RT-PCR assay and platform used — different assays have different limits of detection (LoD), gene targets, and cycling parameters

Each RT-PCR assay has its own validated limit of detection (LoD) — the lowest viral concentration the assay can reliably and consistently detect. This is why a "positive" or "negative" result, and any reported Ct value, must always be interpreted in the context of the specific assay used to generate it.

Figure: commercial testing methods for covid-19 (SARS-CoV-2)

Part 2 — Antigen Testing

Principle

Antigen tests detect specific viral proteins directly, most commonly the nucleocapsid protein of SARS-CoV-2 (some assays instead target the receptor-binding domain of the spike protein). Unlike RT-PCR, which amplifies genetic material to detectable levels, antigen tests rely on the antigen already being present in sufficient quantity in the sample to be directly detected — there is no amplification step.

Mechanism (lateral flow immunoassay format, the most common):

The patient sample disrupts viral particles, exposing internal nucleocapsid protein
The extracted sample is applied to the test device's sample well and migrates along the test strip by capillary action
Viral antigen present in the sample binds to anti-nucleocapsid antibodies conjugated to a visible indicator (commonly colloidal gold or a fluorescent label), forming an antigen-antibody-conjugate complex
This complex migrates further and is captured by immobilized antibodies at the test line, producing a visible (or fluorescent) signal proportional to antigen concentration
Excess conjugate continues to the control line, where it is captured regardless of test outcome — confirming the test ran correctly

Result Interpretation

Positive: Indicates presence of viral antigen (and therefore likely virus), but must be correlated with clinical presentation — does not rule out co-infection with another pathogen
Negative: Presumptive only. Does not rule out infection and should never be the sole basis for a clinical management decision, particularly in a symptomatic patient with high pre-test probability — confirmatory molecular testing should be considered

Advantages and Limitations

Advantage	Limitation
Fast — results typically in 10–30 minutes	Lower sensitivity than RT-PCR — the principal trade-off
Inexpensive relative to molecular testing	No amplification step means a minimum viral load threshold must be present to detect
Point-of-care compatible — no laboratory infrastructure required	Generally most sensitive during peak viral shedding (early-to-mid symptomatic phase); less reliable very early or late in infection

The core teaching point: antigen tests trade sensitivity for speed and accessibility. This sensitivity-speed trade-off is a recurring theme across nearly all rapid point-of-care diagnostics in clinical microbiology, not just for SARS-CoV-2.

→ Immunochromatography/Lateral Flow Immunoassay: Principle and Uses

Part 3 — Serology (IgG/IgM Antibody Testing)

Principle and Purpose

Unlike RT-PCR and antigen testing, which detect the virus itself, serology detects the host immune response to infection — specifically IgM and IgG antibodies directed against viral antigens (commonly spike or nucleocapsid protein). This fundamentally changes what the test can tell you: serology does not diagnose a current infection reliably — it indicates the immune system has encountered the virus, recently or in the past.

Clinical uses of serology:

Estimating the true extent of past infection in a population, including asymptomatic cases missed by symptom-triggered molecular testing
Assessing immune status of high-risk individuals (e.g. healthcare workers) following suspected past exposure
Evaluating immune response during vaccine clinical trials and post-vaccination antibody studies

Antibody Kinetics — Why Timing Matters

Figure: Variation of the Levels of SARS-CoV-2 RNA and Antigen, IgM and IgG after infection.

Antibody	Typical detection timeline	What it indicates
IgM	Detectable several days after infection onset	Marker of recent/acute infection
IgG	Detectable later than IgM, following class-switching	Marker of more established or past infection; typically more durable

A test showing both IgM and IgG positive suggests a relatively recent infection where class-switching has already occurred. IgM-only positive suggests a very recent infection. IgG-only positive suggests prior infection, with the immune system having already transitioned to longer-lived antibody production.

Figure: Schematic diagram for SARS-CoV-2 IgG/IgM assay

Lateral Flow Immunoassay Cassette Design (Mechanism)

A typical SARS-CoV-2 IgG/IgM rapid test cassette contains:

Sample well — absorbent pad where the specimen (serum/plasma) is applied
Conjugate pad — contains SARS-CoV-2 recombinant antigen conjugated to a visible label (e.g. colloidal gold), plus a separate control conjugate
Nitrocellulose membrane — containing the IgG test line (immobilized anti-human IgG), the IgM test line (immobilized anti-human IgM), and the control line (captures excess conjugate regardless of result)
Wick/absorbent pad — draws sample across the membrane by capillary action

Figure: SARS-CoV-2 IgM/IgG Assay

As the specimen migrates, any anti-SARS-CoV-2 antibodies present bind to the labelled viral antigen conjugate, forming a complex. This complex is captured at the corresponding IgG or IgM test line, producing a visible coloured line whose intensity correlates with antibody concentration. The control line must always appear for a result to be considered valid — its absence indicates a failed/invalid test regardless of what appears (or doesn't) at the test lines.

Figure: SARS-CoV-2-IgG/IgM assay procedure and interpretation

Result Interpretation Table

IgM line	IgG line	Control line	Interpretation
Positive	Positive	Positive	Both IgM and IgG present — infection with recent onset (IgM) and established class-switching (IgG)
Positive	Negative	Positive	IgM only — recent infection
Negative	Positive	Positive	IgG only — prior infection (and likely some degree of immunity)
Any	Any	Negative	Test invalid, regardless of test line results — must be repeated

Important Limitations of Serology

Negative results do not rule out infection — particularly early in illness, before antibodies have had time to develop. The sensitivity of antibody tests in the first few days after infection is generally poor.
Cross-reactivity can produce false positives — pre-existing antibodies from infection with other related coronaviruses (including endemic common-cold coronaviruses) can cross-react with assay antigens if the target antigen is not sufficiently unique to the virus being tested for.
Antibody persistence is variable and depends on the individual, the antigen targeted, infection severity, and time since infection or vaccination — a single antibody test is a snapshot, not a permanent immunity certificate.
Antibody tests should never be used as the sole basis for diagnosing acute infection — they complement, but do not replace, molecular or antigen testing in the acute setting.

Comparison Table — RT-PCR vs Antigen vs Serology

Feature	RT-PCR (Molecular)	Antigen Test	Serology (IgG/IgM)
What it detects	Viral RNA (amplified)	Viral protein (nucleocapsid, directly)	Host antibody response
Detects current infection?	Yes — most sensitive	Yes — but less sensitive than RT-PCR	Generally no — reflects past/recent exposure
Detects past infection?	No (RNA clears with infection resolution, generally)	No	Yes — this is its primary value
Turnaround time	Hours (lab-based); ~45 min (point-of-care cartridge systems)	10–30 minutes	15–20 minutes
Relative sensitivity	Highest	Lower than RT-PCR	Not applicable in the same sense — depends on time since infection
Requires lab infrastructure?	Traditionally yes (point-of-care cartridge systems now exist)	No — point-of-care	No — point-of-care
Best clinical use	Confirming active infection; quantifying viral burden via Ct value	Rapid screening when speed/access matters more than maximum sensitivity	Population/epidemiological surveys; past exposure assessment; vaccine trials
Key limitation	Cannot distinguish infectious virus from residual RNA fragments	Lower sensitivity — false negatives possible, especially outside peak shedding window	Window period before seroconversion; cross-reactivity; not for acute diagnosis

References and Further Reading

Public Health England. Understanding cycle threshold (Ct) in SARS-CoV-2 RT-PCR.
Li, Z., Yi, Y., Luo, X., et al. (2020). Development and clinical application of a rapid IgM-IgG combined antibody test for SARS-CoV-2 infection diagnosis. Journal of Medical Virology. https://doi.org/10.1002/jmv.25727
Zhao, J., Yuan, Q., Wang, H., et al. (2020). Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019. Clinical Infectious Diseases. https://doi.org/10.1093/cid/ciaa344
Petherick, A. (2020). Developing antibody tests for SARS-CoV-2. The Lancet, 395(10230), 1101–1102. https://doi.org/10.1016/S0140-6736(20)30788-1
American Society for Microbiology. COVID-19 Testing FAQs. https://asm.org/Articles/2020/April/COVID-19-Testing-FAQs
U.S. Food & Drug Administration. In Vitro Diagnostics EUAs.
Tille, P. M. (2017). Bailey & Scott's Diagnostic Microbiology (14th ed.). Mosby Elsevier.