The oral microbiome, or the microorganisms that live in our mouths, can set us up for good health or disease. Oral microbiome testing helps practitioners and consumers pinpoint the microbes in their mouths and how they might be figuring into oral health symptoms. Thanks to DNA-testing technologies, we can see 50% more of the microbiome than ever before. But what is the best method to test the microbiome? Culturing for microbes was standard practice in the past, but today we have enormous power in DNA-testing methodologies. These include quantitative polymerase chain reaction (qPCR) and metagenomic sequencing. I’ll explain the differences in the two methods here.
Check out the 2nd part of this blog, “The Best Oral Microbiome Tests,” where I outline the most important criteria for picking an oral microbiome test and the pros and cons of each oral microbiome test.
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Testing the Microbiome
Both qPCR and metagenomics testing methods have advantages and disadvantages. It is important for healthcare practitioners to understand the strengths and limitations of these methods when doing microbiome testing.
The pros and cons of either qPCR or metagenomics testing are mainly related to:
(1) Quantitative result versus a qualitative result
(2) The number of organisms measured
(3) The variety of microbes measured
(4) The accuracy with low levels of microbes
(5) Turnaround time
What is quantitative PCR?
Quantitative PCR (qPCR), also known as real-time polymerase chain reaction (RT-PCR), is a targeted method used to quickly find, identify, and measure specific microbes and genes. It makes many duplicates of a specific DNA sequence. It also involves primers, which are short, single strands of nucleic acids, and an enzyme called DNA polymerase, which helps replicate the DNA. In qPCR, specific sections of DNA are copied multiple times. DNA probes that have a colored tag bind to the target gene. This allows scientists to see the amplification process in real-time, which is essentially a process of doubling and then counting the DNA fragments. The result is proportional to the amplified DNA, which helps determine the original amount of DNA present in one milliliter or one gram of the sample (saliva, for example).
qPCR can accurately measure very low quantities of DNA, even down to 10 cells or fewer. However, it can find only microbes that the test specifically looks for. In oral microbiome testing, this means that the test may only measure a handful of targeted pathogens and opportunistic microbes. It doesn’t give a complete view of the microbiome. It may not help patients with more complex dysbiosis. This is an ideal method for measuring disease-causing microbes, but most qPCR panels for the oral microbiome fail to test commensal microbes. This method is also known to be extremely fast for turnaround time, in a matter of days.
When you see results like this, you are seeing quantitative results measured with qPCR:
- 1.0 x 10E6 colony forming units (CFU)/mL or
- 1.0 x 1,000 genomic copies/mL
What is metagenomic sequencing?
Metagenomic sequencing provides a general profile of the microbiome. It is a DNA -sequencing method that determines the order of nucleic acids—adenine, cytosine, guanine, and thymine—in a DNA molecule. Before sequencing, the DNA is copied using PCR to create enough material for analysis. It uses high-throughput sequencing to analyze nucleic acids, followed by computer analysis to identify microbes and their genes. The accuracy of sequencing results depends on the number of reads. A “read” refers to how much of a DNA fragment is sequenced, and sequencing depth is how many times a particular sequence is read.
When a sequencing method applies to RNA, it is called metatranscriptomics. Instead of measuring the microbes themselves, it measures their gene expression. In other words, it measures what they are doing, not who they are.
Sequencing is a broad method that gives a general overview of the microbes or genes in a sample, rather than focusing on specific targets. Metagenomic tests give approximate levels of organisms, not exact measurements. The strength of metagenomics methods is that they can “explore” a microbiome, identifying countless species. It shows relationships in an ecosystem. This method isn’t limited by predetermined targets, the way a qPCR test is.
Qualitative results from metagenomics tests are expressed as:
- % Relative abundance
Metagenomic sequencing has a harder time with measuring low-abundance organisms (those that are found in very small quantities) and it is not quantitative. This can pose a problem for clinical use, because opportunistic and/or pathogens often make up a very low percentage of the microbiome, yet they can cause trouble even at low numbers. This method is also known for having longer turnaround time, in the order of weeks.
When using aggressive clinical treatments like antibiotics, qualitative results are not as strong for comparing before and after treatment. They are percentages of total. The total microbial mass can change. For example, you can think of metagenomics results as expressed as a percentage of a pie. If a pie changes size because of antibiotics or biofilm removal therapy, the percentages of the pie will also change. This makes it hard to directly compare (“apples to apples”) test results before and after treatment.
Picking the Right Method for Your Oral Microbiome Test
Ultimately, both qPCR and metagenomics tests provide tremendous value when assessing the microbiome. That’s why I recommend getting both when possible (see Part 2 of this article, “The Best Oral Microbiome Tests“). The type of test you choose depends on why you are running the test and what you plan to do with the results. It also depends whether you are a practitioner (treating disease) or a consumer (optimizing wellness). Overall, a quantitative qPCR oral microbiome test will likely give you a short list of microbes that usually cause disease, without any commensals or friendly bacteria. It is helpful for managing diseases. But it cannot show you the full picture of the microbiome, only a specific slice. On the other hand, the oral microbiome test via metagenomics gives you a robust list of the oral microbiome- the ecology of the mouth- but it doesn’t quantify those results. It is helpful for balancing the microbiome with less invasive treatments and can be used by consumers or practitioners.
Cass Nelson-Dooley, MS, is a researcher, author, educator, and laboratory consultant. She studied medicinal plants in the rain forests of Panama as a Fulbright Scholar and then launched a career in science and natural medicine. Early on, she studied ethnobotany, ethnopharmacology, and drug discovery at the University of Georgia and AptoTec, Inc. She joined innovators at Metametrix Clinical Laboratory as a medical education consultant helping clinicians use integrative and functional laboratory results in clinical practice. She owns Health First Consulting, LLC, a medical communications company with the mission to improve human health using the written word. Ms. Nelson-Dooley is an oral microbiome expert and author of Heal Your Oral Microbiome. She was a contributing author in Laboratory Evaluations for Integrative and Functional Medicine and Case Studies in Integrative and Functional Medicine. She has published case studies, book chapters, and journal articles about the oral microbiome, natural medicine, nutrition, laboratory testing, obesity, and osteoporosis.
