PO Q1 2021 - 41

SECTION
TITLE
CONTRACT
RESEARCH

More recently, scientists have turned to qPCR, which delivers results
in one day. However, qPCR is unable to directly quantify Mycoplasma
levels: It is performed by amplifying the target genetic sequence and
measuring the number of cycles it takes to reach a certain threshold,
and this result is then compared to a standard curve, which yields a
relative rather than quantitative answer.
Another limitation of qPCR is its inability to distinguish between
Mycoplasma DNA from living cells versus from Mycoplasma DNA
floating freely in solution.11 To make this distinction, researchers
must measure the ratio of genome copies (GCs) and colony-forming
units (CFUs). Unfortunately, the GC:CFU ratio varies between
cultures because of variable growth rates and the specific culture
conditions. Therefore, it is necessary to obtain an absolute count of
genome copies, which qPCR cannot do. In addition, certain SYBR
based qPCR tests often produce false positives, which can lead to an
overestimation of Mycoplasma concentration.12

Advantages of Using Droplet Digital PCR
for Mycoplasma Detection
One approach that is more ideally suited for quantifying Mycoplasmas
is Droplet Digital PCR (ddPCR) technology. ddPCR technology directly
quantifies target nucleic acids - including Mycoplasma DNA.
This method is based on partitioning (Figure 1): After a researcher
loads a 10 µL sample of DNA, it gets divided into approximately 20,000
Figure 1. Comparison of qPCR and ddPCR technologies.
Quantitative PCR is a bulk measurement with an analog
signal output that is proportional to the concentration of
DNA input. Droplet Digital PCR uses partitioning to provide
a digital signal of 0 or 1 for each partition. The percentage of
empty partitions is used to calculate the copies of input DNA.

uniform 1-nL droplets, each containing one or a few nucleic acid
strands. The nucleic acids in each droplet are amplified using a probe
targeting a genetic sequence that is unique to Mycoplasma species. If
a droplet contains the target sequence, the probe will be cleaved as
the DNA amplifies, and a reporter dye will emit a fluorescent signal. In
contrast, droplets that do not contain the target strand will not emit
a strong signal. Unlike qPCR, ddPCR technology uses probe-based
chemistry and utilizes three primers instead of two, reducing the
chance of non-specific DNA amplification.
The droplets are then streamed in series through a reader that counts
fluorescent versus non-fluorescent droplets. Using Poisson statistics,
software can calculate the concentration of Mycoplasma DNA in the
sample and thereby aid a manufacturer in determining the presence
and level of Mycoplasma contamination in their batch.
ddPCR is already used to detect microbial contamination in other
areas. For example, the technology is more precise than qPCR at
detecting Spiroplasma citri, a pathogenic bacterium found in citrus
fruits.13 In wastewater, it identifies norovirus and poliovirus before
patients present symptoms.14 It can detect E. coli contamination
in food, and it can also detect and quantify SARS-CoV-2 in both
wastewater and nasopharyngeal samples.15,16
ddPCR has also been shown to be more sensitive and specific than
qPCR in the detection of several bacterial species that causes illness
in humans. These include:
L. monocytogenes, a gram-positive bacterium that causes
listeriosis and that is pathogenic at concentrations below the
detection limit of most assays
2.	 F. tularensis, a gram-negative bacterium that causes tularemia and is lethal at small doses
3.	 Mycobacterium avium subsp. Paratuberculosis, which causes
disease in ruminant animals
In one study, researchers directly compared the ability of qPCR and
ddPCR to accurately quantify these three species in suspension and
found that qPCR overestimated the quantity of all three by at least
two-fold.17 In a study looking at the concentration of Mycobacterium
tuberculosis in the blood of rhesus monkeys, researchers using a ddPCR
assay were able to detect the bacterial species only three weeks after
infection, two weeks before it became detectable via qPCR.18
1.	

Recently, researchers examined the ability of ddPCR technology
to detect several Mycoplasma species. In the study, they tested
samples containing three species that are representative of the
range of Mycoplasma found in nature, A. laidlawii, M. pneumoniae,
and M. hyorhinis, and found the limit of detection of A. laidlawii was
4.19GC/well, M. pneumoniae was 6.29 GC/well, and M. hyorhinis was
5.63GC/well.19
The same group wanted to ensure that the technology was not
detecting other species of bacteria, so they tested assay crossreactivity. They tested samples containing the three representative
Mycoplasma species and three control species, C. sporogenes,
L. acidophilus, S. bovis, and confirmed that ddPCR was only
detecting Mycoplasma.19
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PO Q1 2021

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