Method
How a saliva sample becomes a measurement.
Every OraPath result starts as DNA and ends as a Detection Level. Here's everything that happens in between.
Your saliva sample arrives at the laboratory and its DNA is extracted. That DNA is then run through quantitative PCR, once for each target on the panel. PCR copies a specific stretch of DNA repeatedly, roughly doubling it each cycle; the quantitative version watches the copying happen in real time using a fluorescent signal. The cycle at which that signal rises above background is the Ct value, and a sample with more starting DNA crosses it sooner. OraPath reports Ct as a Detection Level: DL = 40 − Ct. Because each cycle doubles, each Detection Level up is roughly twice as much target DNA.
What PCR is
The polymerase chain reaction copies a chosen stretch of DNA over and over. Each cycle heats the sample to separate the double strand, cools it so short synthetic sequences called primers bind to the edges of the target, then lets a polymerase enzyme build a new complementary strand.
One cycle roughly doubles the target. Thirty cycles is a very large multiplication of whatever was there to begin with, which is what makes small amounts of DNA measurable.
What the quantitative version adds
Ordinary PCR tells you at the end whether the target was amplified. Quantitative PCR watches while it happens.
That distinction is the whole basis of the measurement: how soon amplification becomes detectable depends on how much target was present at the start, and you can only see “how soon” if you are watching in real time.
Primers and target specificity
Primers are short DNA sequences designed to bind only to the target. They are what makes the assay specific — they determine what gets copied and therefore what gets measured.
This is also the built-in boundary of the approach. A targeted assay measures the targets it was designed for and nothing else, which is a limitation and a source of precision at the same time.
Watching amplification happen
A fluorescent chemistry in the reaction produces signal in proportion to the amount of product. The instrument reads that signal once per cycle.
Plotted, the result is a curve: nothing distinguishable from background for a while, then a steep rise as doubling compounds, then a plateau as reagents are consumed. The informative part is when the rise begins.
The Ct value
The cycle threshold, Ct, is the cycle number at which signal first rises above background.
It is the assay's actual output. Everything reported downstream is a transformation of it.
Why a lower Ct means more DNA
If a sample starts with more copies of the target, fewer doublings are needed before the signal is detectable, so the threshold is crossed earlier.
The relationship runs backwards from intuition — a smaller number means more — which is one of the reasons the raw value is not what appears on a patient report.
Detection Level
OraPath reports DL = 40 − Ct. The subtraction turns the scale the right way round, so that more organism produces a higher number.
Because each qPCR cycle approximately doubles the DNA present, Detection Level is a log₂ scale: one step up is about twice as much target DNA, not one unit more of something. The bands each category is reported in are set out on the Detection Level page.
What a log scale means when you compare two results
The gap between DL 10 and DL 14 is not “four more”. Four doublings is roughly sixteen times as much.
Microbial abundance spans orders of magnitude, so a log scale is the natural way to report it — but it has to be read as one. A few Detection Levels is a large difference.
Controls
Each run carries controls: material known to contain the target, material known not to, and checks that extraction and amplification behaved as expected.
Controls are what separate a result from a reading. Without them there is no way to distinguish a true negative from a reaction that did not work.
Detection limits
Every assay has a level below which it cannot reliably distinguish target from background. DL 0 means the assay did not detect the target above that limit in that sample.
Several things produce that outcome: the organism is genuinely not present, it is present below the threshold, or the sample did not capture it. Which is why a non-detection is read as what the assay found, not as proof of absence.
Validation and accreditation
The Oral Balance Test is a laboratory-developed test performed by IMMYLabs (CLIA #37D2236199; COLA #32679), validated under CLIA, 42 U.S.C. § 263a. It has not been cleared or approved by the U.S. Food and Drug Administration.
Validation is the work of establishing how an assay behaves before it is used on patient samples — what it detects, how low it goes, how it responds to related sequences, and how consistent it is between runs. That last property is what makes a later sample comparable with an earlier one.
What qPCR is built to answer, and when sequencing answers something else
Both are DNA methods applied to the same kind of sample, and neither measures a clinical outcome. They differ in what question they are shaped for.
Two methods, two different questions. The full comparison, including 16S, is on the method comparison page.
| Targeted qPCR | Shotgun metagenomics | |
|---|---|---|
| Can it find an organism not on the list? | No | Yes |
| Quantity or proportion? | Quantity, against a standard | Proportion of reads |
| Community function | Only for genes explicitly targeted | Across the community |
| Resolution | The targets chosen, measured precisely | Broad, finely resolved |
| Comparing two samples | Directly, on the same scale | Compositional; shifts are relative |
The published methodology
This page describes the method for a general reader. The technical documentation — targets, assay design, validation — is the methodology page, and that is the authoritative version.
Where the research is
Where the relevant findings sit.
qPCR quantifies a target by the cycle at which amplification signal rises above background.
Each amplification cycle approximately doubles the target DNA present, which makes Detection Level a log₂ scale.
Primer design determines what a targeted assay can and cannot measure.
Sequencing output is compositional — it reports proportions of a total rather than quantities.
Targeted and untargeted approaches applied to the same specimens produce correlated but not interchangeable results.
How a sample is collected affects what sequencing and targeted methods recover from the mouth.
How closely DNA-based abundance in saliva reflects abundance at any particular site in the mouth.
In context
The sample
A panel measures what was in the sample. When and how it was collected is part of the result.
The target list
A targeted assay answers precisely about what it targets, and is silent about everything else.
DNA, not activity
The method detects genetic material. Whether an organism was active is a different measurement.
The clinical exam
A Detection Level describes one target in one sample. What is happening in the tissue is established by an examination.
How OraPath measures this
Every target on the Oral Balance Test runs as its own qPCR assay with its own primers, alongside controls that confirm the run behaved as expected.
Results are reported on the Detection Level scale, which is consistent between runs — the property that makes a sample taken months later directly comparable to an earlier one.
See what the method produces.
Curious what your oral microbiome looks like? The Oral Balance Test measures targeted organisms and microbial functions from a saliva sample processed in our Norman, Oklahoma laboratory.
Common questions
How do they test mouth bacteria?
DNA is extracted from a saliva sample and run through quantitative PCR, once per target. Each assay copies a specific DNA sequence and reports how soon amplification became detectable, which reflects how much was there to begin with.
What is a Ct value?
The cycle threshold — the qPCR cycle at which the fluorescent signal rises above background. A lower Ct means more starting DNA.
What is Detection Level?
OraPath's reporting scale, calculated as DL = 40 − Ct. It runs the intuitive direction, and because each cycle doubles, each step up is roughly twice as much target DNA.
Why doesn't the report just give me a Ct?
Ct runs backwards — a smaller number means more organism — and the useful range sits in an awkward stretch of the scale. Detection Level fixes both without changing the measurement.
Does DL 0 mean the organism isn't there?
It means the assay did not detect it above its limit of detection in that sample. The organism may be absent, present below the threshold, or missed by how the sample was collected.
Is qPCR the same as sequencing?
No. qPCR measures a predefined list of targets and quantifies each. Sequencing reads what is present without a predefined list and reports proportions. Neither is the better method in general.
Is the Oral Balance Test FDA approved?
It is a laboratory-developed test performed by IMMYLabs (CLIA #37D2236199; COLA #32679), validated under CLIA, 42 U.S.C. § 263a. It has not been cleared or approved by the U.S. Food and Drug Administration.
References
- Gloor GB, Macklaim JM, Pawlowsky-Glahn V, Egozcue JJ. Microbiome datasets are compositional: and this is not optional. Front Microbiol. 2017;8:2224.
- Wang J, Qi J, Zhao H, et al. Metagenomic sequencing reveals microbiota and its functional potential associated with periodontal disease. Sci Rep. 2013;3:1843.
- Sung CH, Pilla R, Chen CC, et al. Correlation between targeted qPCR assays and untargeted DNA shotgun metagenomic sequencing for assessing the faecal microbiota in dogs. Animals (Basel). 2023;13(16):2597.
- Herrera G, Zouiouich S, Diaz-Mayoral N, et al. Comparison of oral collection methods for 16S rRNA gene and shotgun metagenomic sequencing. Microbiol Spectr. 2026;14(2):e0180625.
- Socransky SS, Haffajee AD. Microbial complexes in subgingival plaque. J Clin Periodontol. 1998;25:134–144.
- Kilian M, Chapple ILC, Hannig M, et al. The oral microbiome — an update for oral healthcare professionals. Clin Microbiol Infect. 2016;22:657–666.
Laboratory developed test · for adjunctive clinical use · not FDA cleared.