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Principle of Emulsion PCR and Its Applications in Biotechnology



Emulsion polymerase chain reaction (PCR) is performed on compartmentalized DNA, allowing a large number of PCR reactions to be carried out in parallel. Emulsion PCR has unique advantages in DNA amplification. It can be applied in many molecular biological assays, especially those requiring highly sensitive and specific DNA amplification. This review discusses the principle of emulsion PCR and its applications in biotechnology. Related technologies are also discussed.
Polymerase chain reaction (PCR) is an
in vitro
cal process that mimics
in vivo
DNA replication, and has
led to breakthroughs in biochemistry, biology, and medi-
cal science. PCR and associated technologies enable early
diagnosis of infectious diseases, rapid prognostic staging
for various cancers, sequencing of DNA and RNA, and
screening and development of antitumor agents (Viljoen
et al., 2005; Hayat, 2008). PCR is expected to continue to
play significant roles in advancing bioscience.
The primary aim of PCR and associated technolo-
gies is to replicate and amplify target DNA sequences on
DNA templates of interest surrounded by nontarget DNA
molecules. PCR is carried out via interactions between
PCR components, including DNA template, polymerase,
primers, nucleotides, and minerals. PCR can identify
fewer than ten target DNA sequences among millions of
nontarget DNA molecules. However, PCR is hampered by
nonspecific reaction between primers and nontarget DNA
and primers, resulting in false negatives in infectious dis-
ease or cancer testing (Viljoen et al., 2005; Pelt-Verkuil et
al., 2008). DNA enrichment, primarily based on PCR, is a
necessary step in metagenomic sequencing and systemat-
ic evolution of ligands by exponential enrichment (SELEX).
Low-frequency DNA variants may be lost during the en-
richment of metagenomic specimens and DNA aptamers
if a method to replicate DNA sequences independently is
not implemented (Fig. 1) (Shanks et al., 2012; Bayat et al.,
2018). The occurrence of nonspecific PCR in early stages
of the PCR process reduces the amplification of target
DNA, which causes false negative results in PCR-based
infectious disease and cancer testing, as well as the loss of
low-frequency DNA variants.
PCR in aqueous droplets emulsified in the oil phase
of water-in-oil emulsions, termed “emulsion PCR,”
has unique attributes for DNA amplification (Kanagal-
Shamanna, 2016). Emulsifying the aqueous PCR phase
in the oil phase can compartmentalize individual DNA
molecules, creating independent PCR environments (Fig.
1). The sensitivity and specificity of PCR can be signifi-
cantly improved by performing PCR in water-in-oil emul-
Principle of Emulsion PCR and Its Applications in
Changhoon Chai*
Department of Applied Animal Science, Kangwon National University, Chuncheon 24341, Korea
Review Article
J Anim Reprod Biotechnol 2019;34:259-266
pISSN: 2671-4639 • eISSN: 2671-4663
Journal of Animal Reproduction and Biotechnology
Received November 20, 2019
Revised December 3, 2019
Accepted December 11, 2019
Changhoon Chai
ABSTRACT Emulsion polymerase chain reaction (PCR) is performed on
compartmentalized DNA, allowing a large number of PCR reactions to be carried out in
parallel. Emulsion PCR has unique advantages in DNA amplification. It can be applied
in many molecular biological assays, especially those requiring highly sensitive and
specific DNA amplification. This review discusses the principle of emulsion PCR and its
applications in biotechnology. Related technologies are also discussed.
Keywords: DNA enrichment, droplet digital PCR, emulsion PCR, polymerase chain
reaction, SELEX
Copyright © The Korean Society of Animal Reproduction and Biotechnology
CC This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (, which permits
unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
J Anim Reprod Biotechnol Vol. 34, No. 4, December 2019
sion (Chai and Oh, 2015; Du et al., 2019). The actual
number of pathogens present in clinical specimens can
be measured using emulsion PCR-based techniques (Mu
et al., 2015). Emulsion PCR-based methods are also used
in next-generation sequencing (NGS), metagenomics se-
quencing, and SELEX to enrich genomic DNA and cDNA
libraries while maintaining the same proportions as the
original libraries (Shanks et al., 2012; Kanagal-Shamanna,
2016; Bayat et al., 2018). The application of emulsion
PCR is expected to support further advances in biological
techniques. However, current emulsion PCR-based tech-
nologies have several issues that remain to be resolved.
For example, emulsion PCR requires the precise opti-
mization of PCR conditions (Kanagal-Shamanna, 2016).
Moreover, PCR emulsions can lose their stabilities during
temperature fluctuations in PCR.
This review addresses the principle of emulsion PCR and
its applications in biotechnology, as well as PCR compo-
nents and procedures. Technical difficulties associated
with emulsion PCR are discussed, and emulsion PCR-
based technologies are reviewed.
DNA extracted from a clinical specimen for the diagno-
sis of infection may include only a few viral or bacterial
DNA molecules among millions of host DNA molecules
(Strain et al., 2013; Kralik and Ricchi, 2017). The size of
genomic DNA and cDNA libraries frequently exceeds 1
× 106 (Ehrlich et al., 2011; Illumina, 2013). The relative
abundance of nontarget DNA can cause false negatives
in conventional PCR-based diagnostic testing and loss of
low-frequency DNA variants in conventional PCR-based
DNA enrichment (Fig. 1). Compartmentalizing individual
DNA molecules in aqueous PCR droplets emulsified in the
oil phase isolates target DNA from nontarget DNA, avoid-
ing some of the challenges associated with conventional
PCR-based assays (Fig. 1).
PCR components such as template DNA, polymerase,
primers, nucleotides and minerals are soluble in water
but not in oil. Water is the continuous phase of the PCR
solution, and has different surface energy properties from
oil. Thus, oil and water are immiscible. Surfactants can
mediate water and oil molecules, allowing water to be
dispersed in oil. Tween 80 (polyoxyethylene sorbitan mo-
nooleate), Span 80 (sorbitan monooleate), Triton X-100
(polyethylene glycol
-octylphenyl ether), ABIL EM 90
(cetyl PEG/PPG-10/1 dimethicone; Evonik Industries,
Essen, Germany), ABIL WE 09 (blend of polyglyceryl-4
isostearate, cetyl PEG/PPG-10/1 dimethicone, and hexyl
laurate; Evonik Industries), and fluorinated silicone are
frequently used as surfactants for emulsion PCR (Williams
et al., 2006; Heredia and Makarewicz, 2016; Witt et al.,
2017). Mineral oil, Tegosoft DEC (diethylhexyl carbon-
ate; Evonik Industries), and silicone oil can be used as the
oil phase of emulsion PCR (Pekin et al., 2010; Heredia
and Makarewicz, 2016; Witt et al., 2017). Surfactants are
generally added to the oil phase. Vortex mixers, magnetic
Polymerase Target DNA Nontarget DNA Primer Surfactant
Emulsion PCR
Conventional PCR
Target specific PCR
Nonspecific PCR
Fig. 1. Schematic illustrates of conven-
tional PCR and emulsion PCR.
Chai. Emulsion PCR in Biotechnology
stirrers, and microfluidic devices can be used to disperse
the aqueous PCR phase in the oil phase (Williams et al.,
2006; Zhu et al., 2012; Kanagal-Shamanna, 2016).
Depending on the specific emulsion PCR method, a
20-200 mL volume of the aqueous PCR phase is added
to the oil phase to prepare a 100-300 mL PCR emulsion
(BioRad.; Chai and Oh, 2015; Witt et al., 2017). Vigorous
mechanical agitation of PCR emulsions may generate 108-
109 irregularly sized aqueous PCR droplets with a mean
diameter of 4-6 mm (Shao et al., 2011; Witt et al., 2017).
Droplet diameter and number can be controlled precisely
using microfluidic devices (Byrnes et al., 2018). Microflu-
idic droplet generators are used in emulsion-based digital
PCR, referred to as droplet digital PCR (Strain et al., 2013).
Typically, microfluidic droplet generators can generate 1
× 105 aqueous PCR droplets with a diameter around 120
mm (BioRad.).
The 100 mL aqueous PCR phase used for emulsion PCR
may include target and nontarget DNA molecules, 1.25
U polymerase, 0.8 mM (80 pmol per reaction) of forward
and reverse primers, 0.2 mM dNTPs (20 nmol per reac-
tion), and other components (Chai and Oh, 2015; Du et
al., 2019). For a 94 kD polymerase, a typical unit of poly-
merase contains about 8 × 1010 polymerase molecules
(Spangler et al., 2009). Thus, 100 mL of the aqueous PCR
phase may contain 1 × 1011 polymerase molecules, 4.82
× 1013 forward and reverse primers, and 1.20 × 1016
dNTPs. Assuming that equally sized aqueous PCR droplets
are dispersed in the oil phase, the frequency (
) of aque-
ous droplets containing
molecules of individual PCR
components follows the Poisson distribution (Equation 1)
(Swami et al., 2008).
; l) = l
! (Equation 1)
Furthermore, the proportion of the sum of individual
PCR components compartmentalized by
units of indi-
vidual PCR components (
) is given by Equation 2:
; l) =
; l) ·
; l) ·
(Equation 2)
where l is calculated by dividing
by the total number
of emulsified aqueous droplets (
is the estimated
number of individual PCR components contained in an
emulsified aqueous droplet, and e is the base of the natu-
ral logarithm.
of polymerase at different
values was calcu-
lated using Equation 2 and plotted in Fig. 2A under the
assumption of
= 1 × 1011 and
= 109. Given that l is
100, the
of polymerase at
= 100 is greater than those
of other components (Fig. 2A). The
values of polymerase
values of 46 and 47 are 6.76 × 10-10 and 1.44 × 10-9,
respectively. As
= 1 × 109, the formation of aqueous
droplets with fewer than 47 polymerases is unlikely. As
seen in the
of polymerase, all aqueous droplets
= 1 × 109 should contain sufficient quantities of
primers and dNTPs for PCR. The
can be varied from 1
× 105 to 1 × 109 according to the emulsification method
(BioRad.; Diehl et al., 2006; Williams et al., 2006). The
of DNA molecules in PCR emulsions is also calculated us-
ing Equation 2. If ≤ 1 × 109 DNA molecules are emulsi-
fied in 1 × 109 aqueous droplets, no aqueous droplet with
more than 11 DNA molecules is likely to be generated (Fig.
2B). The maximum
of DNA molecules at
= 1 × 109
= 1 × 108, 1 × 107, 1 × 106, 1 × 105, 1 × 104, 1
× 103, 1 × 102, and 1 × 101 are 6, 3, 2, 2, 2, 1, 1, and 1,
respectively (Fig. 2B). Assuming that ≤ 105 DNA molecules
are introduced to 105 aqueous droplets, the maximum
of DNA molecules in an aqueous droplet at
= 1 × 105,
1 × 104, 1 × 103, 1 × 102, and 1 × 101 are 8, 3, 2, 1, and
1, respectively (Fig. 2C). Although aqueous droplets in
PCR emulsions may contain more than one DNA molecule
= 1 × 109 and
= 1 × 108, 1 × 107, 1 × 106, 1 ×
105, and 1 × 104, as well as
= 1 × 105 and
= 1 × 105,
1 × 104, and 1 × 103, highly sensitive and specific PCR in
aqueous droplets can be achieved due to the specificity of
PCR primers against their target DNA sequences.
Emulsion PCR is performed via aqueous droplet gen-
eration, PCR amplification inside aqueous droplets, and
detection of PCR inside aqueous droplets. Emulsion PCR
can be categorized into conventional and fluorescence-
based methods. Conventional emulsion PCR includes an
emulsion-breaking step to collect enriched DNA mol-
ecules, and conventional PCR is subsequently performed
with collected DNA molecules from the emulsion. Fluo-
rescence-based emulsion PCR uses fluorescent dyes to
J Anim Reprod Biotechnol Vol. 34, No. 4, December 2019
directly detect DNA amplification inside droplets.
Conventional emulsion PCR methods generally use
mechanical agitation techniques such as stirring and
vortexing to emulsify PCR components. PCR emulsions
can be prepared using two different formulas. Williams
et al. reported an emulsion PCR formulation based on a
mixture of 200 mL of aqueous PCR phase (containing PCR
components) and 400 mL of mineral oil supplemented
with 4.5% Span 80, 0.4% Tween 80, and 0.05% Triton
X-100 (Williams et al., 2006). Diehl et al. and Schütze
Cumulative sum of P
12345678910 11
Cumulative sum of P
Number of DNAs
40 60 80 100 120 140
40 60 80 100 120 140 160
12 345678
12 345678
Cumulative sum of P
Number of DNAs
Fig. 2. The proportion (
) of the sum of Taq polymerase and
DNA molecules compartmentalized by
to their total molecule
number (
) based on the Poisson distribution equation. (A)
of Taq polymerase by
(bottom) and the cumulative sum
(top) under
= 1 × 1011 and number of emulsified aque-
ous droplets in PCR emulsions (
) = 1 × 109. (B) The
of DNA
molecules by
(bottom) and cumulative sum of
(top) under
= 100 – 109 and
= 1 × 109. (C) The
of DNA molecules by
(bottom) and cumulative sum of
(top) under
= 1 × 100 -
1 × 105 and
= 1 × 105.
Chai. Emulsion PCR in Biotechnology
et al. used a formulation composed of 150 mL of aque-
ous PCR phase and 600 mL of oil phase composed of 73%
emollient Tegosoft DEC, 20% mineral oil, and 7% ABIL WE
09 (Diehl et al., 2006; Schütze et al., 2011). In both for-
mulas, bovine serum albumin is included in the aqueous
PCR phase to prevent denaturation of the polymerase at
the water-oil interface (Diehl et al., 2006; Williams et al.,
2006). The
for both conventional emulsion PCR meth-
ods is approximately 1 × 109 (Williams et al., 2006; Witt
et al., 2017). The concentration of dNTP in the aqueous
PCR phase for both methods is 0.2 mM (30 and 40 nmol
per 150 and 200 mL PCR reaction, respectively). Under
these conditions, the average
of dNTPs is 1.81 - 3.13 ×
107. If a single DNA molecule is compartmentalized in an
aqueous droplet with 1.81 - 3.13 × 107 dNTPs and PCR
is designed to amplify a 500-bp fragment, dNTPs should
be used up after the production of about 3.61 - 6.26 ×
104 DNA amplicons, corresponding to approximately 16
PCR cycles. In addition, 3.61 - 6.26 × 104 DNA amplicons
are not sufficient for identification by electrophoresis;
hence, conventional PCR should be used to amplify en-
riched target DNA molecules after emulsion PCR. DNA in
the emulsion may be extracted by adding organic solvents
(diethyl ether, ethyl acetate, and isobutanol), followed by
aqueous PCR phase recovery by centrifugation and DNA
purification using a commercial kit (Witt et al., 2017).
DNA recovery from PCR emulsions is quite tedious and
complicated. Thus, a simple and easy method of DNA re-
covery from PCR emulsions is required for further devel-
opment of conventional emulsion PCR techniques. Since
they generate a large number (~1 × 109) of aqueous PCR
droplets, conventional emulsion PCR methods are suitable
for enriching low-frequency target DNA molecules in up
to 1 × 109 nontarget DNA molecules (Fig. 2B).
Fluorescence-based emulsion PCR methods include flu-
orescent dyes, such as SYBR green and EvaGreen, or fluo-
rescence probes such as TaqMan probes, in the aqueous
PCR phase to generate fluorescence during PCR inside
aqueous droplets (Yang et al., 2014; Martinez-Hernandez
et al., 2019; Zhang et al., 2019). Because fluorescence-
based emulsion PCR methods are capable of visualizing
DNA amplification inside individual aqueous droplets,
they have been applied to digital PCR techniques (Strain
et al., 2013). Fluorescence-based emulsion PCR methods
generally use microfluidic devices to generate aqueous
droplets (BioRad.). Microfluidic and fluorescence-based
emulsion PCR platforms are now commercially available,
such as the QX200 Droplet Digital PCR System (Bio-Rad,
Hercules, CA, USA). This commercialized system uses
silicone oil and fluorinated oil for the oil phase, as well
as surfactants. It is operated with ~1 × 105 aqueous PCR
droplets with 120 mm in diameter that are generated with
20 mL of aqueous PCR phase and 70 mL of oil phase by a
microfluidic droplet generator (BioRad.). The final con-
centration of dNTPs recommended by the droplet digital
PCR system’s manufacturer is 0.8 mM. Under these con-
ditions, the average
of dNTPs at
= 1 × 105 is 9.64 ×
1010. Assuming that the DNA amplicon is 500 bp, the copy
number of DNA amplified with 9.64 × 10 dNTPs may not
exceed 1.93 × 108 and dNTPs may be used up after 28
PCR cycles. However, the manufacturer of the commer-
cial droplet digital PCR system recommends 40 PCR cycles
(BioRad.). Fluorescence-based emulsion PCR methods can
quantify even a single target DNA molecule mixed with
nontarget DNA molecules (Fig. 2C). They may be appro-
priate for early diagnosis of viral and bacterial infection.
Conventional and fluorescence-based emulsion PCR
methods may be performed under regular PCR condi-
tions but with a greater number of cycles. The viscosity of
the oil phase changes with temperature. As temperature
increases and decreases during PCR, aqueous PCR drop-
lets circulate, collide, and are prone to coalesce in PCR
emulsions (Holtze et al., 2008). The coalescence of aque-
ous PCR droplets may compromise the Poisson distribu-
tion and emulsion PCR performance. The coalescence of
aqueous PCR droplets during PCR can be minimized if
PCR is carried out isothermally. The use of loop-mediat-
ed isothermal DNA amplification in fluorescence-based
emulsion PCR methods confers superior amplification
efficiency and specificity compared with previous meth-
ods (Ma et al., 2018). Moreover, since isothermal DNA
amplification-based methods do not require a thermal
cycler, they may support the development of simple, min-
iaturized fluorescence-based emulsion PCR systems.
Metagenomic NGS is used to investigate the taxonomic
composition and genetic content of microorganisms in
foods, as well as biological and environmental speci-
mens (Quince et al., 2017; Chiu and Miller, 2019). DNA
J Anim Reprod Biotechnol Vol. 34, No. 4, December 2019
variants may be present in microbial DNA specimens
at frequencies too low for NGS. Thus, DNA variants in
some specimens must be enriched before sequencing.
DNA enrichment using conventional PCR is biased to-
wards the amplification of short- or high-frequency DNA
variants and DNA sequences that match the polymerase
sequence preference (Blind and Blank, 2015; Yufa et al.,
2015). For example, assuming that a specific DNA variant
is 1.5 times as abundant as another DNA, it will become
1.530 = 191,751.1 times as abundant after 30 PCR cycles.
DNA enrichment for metagenomics sequencing should
amplify DNA molecules in the same proportions as in the
original microbial DNA specimen, which can be achieved
via emulsion PCR (Kihana et al., 2013). Emulsion PCR is
therefore included in many NGS methods for unbiased
DNA enrichment (Linderholm, 2019).
Emulsion PCR is also the standard method for enrich-
ment of DNA or cDNA aptamer candidates during SELEX
(Witt et al., 2017). SELEX is a molecular biological tech-
nique for selecting DNA or cDNA aptamers of interest
from an aptamer library comprising ~1 × 1015 sequences
(Tuerk and Gold, 1990). Due to the enormous size of the
aptamer library, the selection and enrichment of aptamer
candidates should be repeated until the aptamer of inter-
est is identified. In conventional PCR, low-frequency DNA
or cDNA variants can be lost during enrichment. Enrich-
ment of DNA aptamer candidates using emulsion PCR can
prevent the production of PCR byproducts and avoid the
loss of low-frequency aptamer candidates (Shao et al., 2011).
Droplet digital PCR has attracted great interest for the
early diagnosis of infectious diseases and rapid prognos-
tic staging of cancers. For instance, a droplet digital PCR-
based assay capable of quantifying 1 - 104
E. coli
in 1 mL
of blood has been reported (Kang et al., 2014). Droplet
digital PCR-associated diagnostic methods for viruses are
more sensitive than other viral diagnosis methods (Strain
et al., 2013; Myerski et al., 2019), and may be able to
measure a single virus in a clinical specimen (Mu et al.,
The quantity of circulating tumor DNA can be the basis
for cancer prognosis and recurrence prediction (Perkins
et al., 2017), and is also used to investigate the response
and resistance to therapeutic agents (Perkins et al., 2017).
However, it is difficult to measure circulating tumor DNA
using current molecular biological methods, such as
quantitative PCR, because of background DNA from nor-
mal cells (Perkins et al., 2017). A recent study showed that
a droplet digital PCR-based assay exhibited 13% greater
sensitivity than NGS-based assays for KRAS mutations
(Demuth et al., 2018). Droplet digital PCR-based circulat-
ing tumor DNA assays may be a powerful approach for
evaluating cancer patients’ prognosis and response to
No potential conflict of interest relevant to this article
was reported.
This study was supported by a grant from the National
Research Foundation of Korea (NRF-2013R1A1A2060458),
funded by the Korean government.
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Plutella xylostella
using qRT-PCR and
ddPCR. PLoS One. 14:e0220475.
Zhu Z, Jenkins G, Zhang W, Zhang M, Guan Z, Yang CJ. 2012.
Single-molecule emulsion PCR in microfluidic droplets.
Anal Bioanal Chem. 403:2127-2143.
... The use of this technique has been already widely demonstrated to determine geographical origin of food products. Moreover, emulsion PCR has been described as improving amplification gene pattern in related studies (Boers et al., 2015;Chai, 2019;Chai and Oh, 2015). ...
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Greek avgotaracho Mesolonghiou (fish eggs from Flathead Mullet) is a highly valuable food product which holds Protected Destination of Origin status. The aim of this work was to use PCR-DGGE technique to examine whether there is a correlation between bacteria population in fish eggs and geographical origin. Cluster analysis of fish eggs from three geographical locations (Mesolonghi, Australia and Mauritania) discriminated samples according to their provenance. Moreover, we utilized emulsion-PCR amplification in DGGE analysis in order to investigate whether we could obtain further information about food products’ bacteria communities. PCR-DGGE proved to be a suitable method for fish eggs traceability, moreover emulsion PCR-DGGE provides better results. Emulsion-PCR can face up the existing limitations of conventional PCR and thus can be demonstrated as alternative molecular technique for complex and processed matrices, regarding food traceability and authentication.
... The use of this technique has been already widely demonstrated to determine geographical origin of food products. Moreover, emulsion PCR has been described as improving amplification gene pattern in related studies (Boers et al., 2015;Chai, 2019;Chai and Oh, 2015). ...
Full-text available
The establishment of an expression quantification system that can be easily applied for the comparison of microRNAs (miRNAs) from biological samples is an important step toward understanding functional mechanisms in organisms. However, there is lack of attention on the selection of reference genes for miRNA expression profiling in insect herbivores. Here, we explored the candidate reference genes in a notorious pest of cruciferous crops, Plutella xylostella, for normalization of miRNA expression in developmental stages and tissues and in response to a change of food source from artificial diet to host plant Arabidopsis thaliana. We first compared the expression levels and stability of eight small RNAs using qRT-PCR, and found that miR11 was the most suitable reference gene for expression quantification of the miRNAs. We then confirmed this finding using digital droplet PCR and further validated with a well-studied cross-kingdom miRNA derived from A. thaliana (ath-miR159a). However, none of the reference genes was applicable for all experimental conditions, and multiple reference genes were sometimes required within the same experiment. Our work provides a method for the selection of reference genes for quantification of plant-derived miRNAs, which paves the way for unveiling their roles in the insect-plant coevolution.
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Absolute abundances of prokaryotes are typically determined by FISH. Due to the lack of a universal conserved gene among all viruses, metagenomic fragment recruitment is commonly used to estimate the relative viral abundance. However, the paucity of absolute virus abundance data hinders our ability to fully understand how viruses drive global microbial populations. The cosmopolitan marine Pelagibacter ubique is host for the highly widespread HTVC010P pelagiphage isolate and the extremely abundant uncultured virus vSAG 37-F6 recently discovered by single-virus genomics. Here we applied droplet digital PCR (ddPCR) to calculate the absolute abundance of these pelagiphage genotypes in the Mediterranean Sea and the Gulf of Maine. Abundances were between 360 and 8,510 virus mL-1 and 1,270-14,400 virus mL-1 for vSAG 37-F6 and HTVC010P, respectively. Illumina PCR-amplicon sequencing corroborated the absence of ddPCR non-specific amplifications for vSAG 37-F6, but showed an overestimation of 6% for HTVC010P from off-targets, genetically unrelated viruses. Absolute abundances of both pelagiphages, two of the most abundance marine viruses, suggest a large viral pelagiphage diversity in marine environments, and show the efficiency and power of ddPCR to disentangle the structure of marine viral communities. Results also highlight the need for a standardized workflow to obtain accurate quantification that allows cross data comparison.
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Abstract We constructed and validated a novel emulsion PCR method combined with fluorescence spectrophotometry (EPFS) for simultaneous qualitative, quantitative and high-throughput detection of multiple DNA targets. In a single reaction set, each pair of primers was labeled with a specific fluorophore. Through emulsion PCR, a target DNA was amplified in droplets that functioned as micro-reactors. After product purification, different fluorescent-labeled DNA products were qualitatively analyzed by the fluorescent intensity determination. The sensitivity and specificity of the system was examined using four kinds of genetically modified (GM) maize. The qualitative results revealed high specificity and sensitivity of 0.5% (w/w). In addition, the quantitative results revealed that the absolute limit of detection was 103 copies, showing good repeatability. Moreover, the reproducibility assays were further performed using four foodborne pathogenic bacteria to further evaluate the applicability of the system. Consequently, the same qualitative, quantitative and high-throughput results were confirmed with the four GM maize. To sum up, the new EPFS system is the first analytical technology of this kind that enables simultaneous qualitative, quantitative and high-throughput analysis of multiple genes.
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Measuring total cell-free DNA (cfDNA) or cancer-specific mutations herein has presented as new tools in aiding the treatment of cancer patients. Studies show that total cfDNA bears prognostic value in metastatic colorectal cancer (mCRC) and that measuring cancer-specific mutations could supplement biopsies. However, limited information is available on the performance of different methods. Blood samples from 28 patients with mCRC and known KRAS mutation status were included. cfDNA was extracted and quantified with droplet digital polymerase chain reaction (ddPCR) measuring Beta-2 Microglobulin. KRAS mutation detection was performed using ddPCR (Bio-Rad) and next-generation sequencing (NGS, Ion Torrent PGM). Comparing KRAS mutation status in plasma and tissue revealed concordance rates of 79% and 89% for NGS and ddPCR. Strong correlation between the methods was observed. Most KRAS mutations were also detectable in 10-fold diluted samples using the ddPCR. We find that for detection of KRAS mutations in ctDNA ddPCR was superior to NGS both in analysis success rate and concordance to tissue. We further present results indicating that lower amount of plasma may be used for detection of KRAS mutations in mCRC.
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Nucleic acid amplification technology, such as PCR, has enabled highly sensitive and specific disease detection and quantifica-tion, leading to more accurate diagnosis and treatment regimens. Lab-on-a-chip applications have developed methods to parti-tion single biomolecules, such as DNA and RNA, into picoliter-sized droplets. These individual reaction vessels lead to digiti-zation of PCR enabling improved time-to-detection and direct quantification of nucleic acids without a standard curve, therefore simplifying assay analysis. Though impactful, these improvements have generally been restricted to centralized laboratories with trained personnel and expensive equipment. To address these limitations and make this technology more applicable for a variety of settings, we have developed a statistical framework to apply to droplet PCR performed in polydisperse droplets prepared without any specialized equipment. The polydisperse droplet system allows for accurate quantification of ddPCR and RT-ddPCR that is comparable to commercially available systems such as BioRad’s ddPCR. Additionally, this approach is compatible with a range of input sample volumes, extending the assay dynamic range beyond that of commercial ddPCR sys-tems. In this work, we show that these ddPCR assays can reduce overall assay time while still providing quantitative results. We also report a multiplexed ddPCR assay and demonstrate proof-of-concept methods for rapid droplet preparation in multi-ple samples simultaneously. Our simple polydisperse droplet preparation and statistical framework can be extended to a vari-ety of settings for the quantification of nucleic acids in complex samples.
Clinical metagenomic next-generation sequencing (mNGS), the comprehensive analysis of microbial and host genetic material (DNA and RNA) in samples from patients, is rapidly moving from research to clinical laboratories. This emerging approach is changing how physicians diagnose and treat infectious disease, with applications spanning a wide range of areas, including antimicrobial resistance, the microbiome, human host gene expression (transcriptomics) and oncology. Here, we focus on the challenges of implementing mNGS in the clinical laboratory and address potential solutions for maximizing its impact on patient care and public health.
Ex vivo explant models are used to characterize in vitro efficacy of preexposure prophylaxis (PrEP) agents. Tissue is challenged with virus in culture and HIV-1 p24 levels are quantified with enzyme-linked immunosorbent assay (ELISA) on supernatants collected throughout a 14-21-day incubation. Due to the narrow dynamic range of HIV-1 p24 kits, we evaluated whether droplet digital PCR (ddPCR) provides an alternative method to quantify HIV-1 replication in supernatant samples. We used samples from the MWRI-01 study, which evaluated the pharmacokinetic/pharmacodynamic profile of long-acting rilpivirine using the explant model (McGowan et al. Lancet HIV 2016). HIV-1 pol RNA was measured with ddPCR, either directly with a one-step method or reverse transcribed to cDNA before ddPCR (two-step method) on supernatants from the MWRI-01 study. Previously analyzed HIV-1 p24 antigen levels (Alliance; Perkin-Elmer) were available for comparison purposes. Both ddPCR methods strongly correlated with HIV-1 p24 and displayed similar patterns of HIV-1 suppression before and after rilpivirine. Compared to the p24 ELISA, two-step and one-step ddPCR reduced the amount of hands-on time by approximately one-half and two-thirds, respectively. ddPCR also required less sample and based on p24 versus ddPCR correlation, could potentially reduce the explant culture time from 14 to 10 days (r2 = 0.78, p < .001) due to the increased sensitivity of ddPCR. We demonstrate that ddPCR is a suitable alternative to HIV-1 p24 ELISA to quantify HIV-1 infection in the explant model and has the potential to decrease explant culture time.
Systematic evolution of ligand by exponential enrichment (SELEX) is an efficient method used to isolate high-affinity single stranded oligonucleotides from a large random sequence pool. These SELEX-derived oligonucleotides named aptamer, can be selected against a broad spectrum of target molecules including proteins, cells, microorganisms and chemical compounds. Like antibodies, aptamers have a great potential in interacting with and binding to their targets through structural recognition and are therefore called “chemical antibodies”. However, aptamers offer advantages over antibodies including smaller size, better tissue penetration, higher thermal stability, lower immunogenicity, easier production, lower cost of synthesis and facilitated conjugation or modification with different functional moieties. Thus, aptamers represent an attractive substitution for protein antibodies in the fields of biomarker discovery, diagnosis, imaging and targeted therapy. Enormous interest in aptamer technology triggered the development of SELEX that has underwent numerous modifications since its introduction in 1990. This review will discuss the recent advances in SELEX methods and their advantages and limitations. Aptamer applications are also briefly outlined in this review.
PREFACE The Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture is involved in agricultural research and development and assists Member States of FAO and IAEA in improving strategies to ensure food security through the use of nuclear techniques and related biotechnologies, where such techniques have a valuable and often unique role. In particular, molecular diagnostic methods have rapidly evolved in the past twenty years, since the advent of the Polymerase Chain Reaction (PCR). They are used in a wide range of agricultural areas such as, improving soil and water management; producing better crop varieties; diagnosing plant and animal diseases; controlling insect pests and improving food quality and safety. The uses of nucleic acid-directed methods have increased significantly in the past five years and have made important contributions to disease control country programmes for improving national and international trade. These developments include the more routine use of PCR as a diagnostic tool in veterinary diagnostic laboratories. However, there are many problems associated with the transfer and particularly, the application of this technology. These include lack of consideration of: the establishment of quality-assured procedures, the required set-up of the laboratory and the proper training of staff. This can lead to a situation where results are not assured. This book gives a comprehensive account of the practical aspects of PCR and strong consideration is given to ensure its optimal use in a laboratory environment. This includes the setting-up of a PCR laboratory; Good Laboratory Practice and standardised of PCR protocols.