Journal of Molecular Medicine

https://doi.org/10.1007/s00109-020-01992-x

ORIGINAL ARTICLE

Diagnosing the novel SARS-CoV-2 by quantitative RT-PCR:

variations and opportunities
Horllys Gomes Barreto 1 & Flávio Augusto de Pádua Milagres 1 & Gessi Carvalho de Araújo 1 &
Matheus Martins Daúde 1 & Vagner Augusto Benedito 2

Received: 9 July 2020 / Revised: 5 October 2020 / Accepted: 7 October 2020

# Springer-Verlag GmbH Germany, part of Springer Nature 2020

Abstract
The world is currently facing a novel viral pandemic (SARS-CoV-2), and large-scale testing is central to decision-making for the
design of effective policies and control strategies to minimize its impact on the global population. However, testing for the
presence of the virus is a major bottleneck in tracking the spreading of the disease. Given its adaptability regarding the nucleotide
sequence of target regions, RT-qPCR is a strong ally to reveal the rapid geographical spreading of novel viruses. We assessed
PCR variations in the SARS-CoV-2 diagnosis taking into account public genome sequences and diagnosis kits used by different
countries. We analyzed 226 SARS-CoV-2 genome sequences from samples collected by March 22, 2020. Our work utilizes a
phylogenetic approach that reveals the early evolution of the virus sequence as it spreads around the globe and informs the design
of RT-qPCR primers and probes. The quick expansion of testing capabilities of a country during a pandemic is largely impaired
by the availability of adequately trained personnel on RNA isolation and PCR analysis, as well as the availability of hardware
(thermocyclers). We propose that rapid capacity development can circumvent these bottlenecks by training medical and non-
medical personnel with some laboratory experience, such as biology-related graduate students. Furthermore, the use of
thermocyclers available in academic and commercial labs can be promptly calibrated and certified to properly conduct testing
during a pandemic. A decentralized, fast-acting training and testing certification pipeline will better prepare us to manage future
pandemics.

Keywords COVID-19 . Coronavirus . Infrastructure . Polymerase chain reaction . Testing

Introduction sequencing and the public data deposition on January 12 to

help with the development of diagnostic kits [1]. WHO de-
The World Health Organization (WHO) received notification clared on March 11, 2020, a pandemic the coronavirus disease
on December 31, 2019 of pneumonia cases of unknown eti- 2019 (COVID-19), which is caused by SARS-CoV-2. In
ology in Wuhan, China. On January 11, 2020, the Chinese March 21, after nearly 3 months of the first notification, over
authorities identified a new type of coronavirus, which was 291,000 thousand cases were confirmed, and 12,776 deaths
isolated on January 7, 2020, which allowed its rapid were reported worldwide [2]. As of May 1, 2020, over 3.2
million cases have been confirmed globally, with a death toll
surpassing 234,000 people [3].
Electronic supplementary material The online version of this article In addition to mitigation approaches (e.g., promotion of
(https://doi.org/10.1007/s00109-020-01992-x) contains supplementary hygiene, social distancing, isolation of infected people, and
material, which is available to authorized users. restricting traveling), comprehensive testing of the infec-
tion in the population is central to track the disease spread-
* Vagner Augusto Benedito ing as well as inform public policies. It has been suggested
vagner.benedito@mail.wvu.edu
that the demand for health services can only be maintained
1 at manageable levels through a prompt adoption of public
Molecular Analysis Laboratory, Department of Health Sciences,
Faculty of Medicine, Federal University of Tocantins (UFT), health measures to suppress virus spreading [4]. Indeed,
Palmas, TO, Brazil countries that have adopted broad testing strategies early
2
Genetics and Developmental Biology Program, West Virginia have better succeeded in limiting the spread of the disease,
University, Morgantown, WV, USA such as South Korea, Vietnam, and New Zealand [5].
 J Mol Med

Ideally, tests should be easy to sample and analyze, genome sequences that were deposited at the NCBI
quick to return results, accurate and precise, scalable, and GenBank as it spread throughout the world as well as to
inexpensive. Often, antibody-based point-of-care tests study the various testing approaches recommended by offi-
(POCT) fit this description. However, rapidly evolving ep- cial government authorities from several countries in order
idemics due to novel viruses do not allow the timely devel- to assess methodological testing differences and to indicate
opment of antibody-based tests. Thus, viral load tests based prospects of effective diagnosis via RT-PCR. Our approach
on real-time, quantitative RT-PCR (referred herein as RT- underscores the importance of analyzing the evolution of
qPCR) are an ideal platform for the rapid development of genome sequences of novel viruses as it spreads around the
test kits due to the easy adaptability to the nucleotide se- globe, allows for the revision of adopted testing reagents,
quence of the target. and contributes to avoiding the development of imprecise
Currently, RT-qPCR is a reliable test widely used for the molecular tests based on mutated regions, as already ob-
detection of symptomatic and asymptomatic patients infect- served [11, 12].
ed with SARS-CoV-2 [6, 7] with a technical limit of detec-
tion (LOD) <10 copies/reaction [8], and a detection thresh-
old of 3.8 RNA molecules per reaction, depending on the
amplified region and the primers and probes used in the Methods
analysis [9]. Indeed, although RT-qPCR requires special
equipment, it allows for a relatively simple and rapid diag- Phylogenetic analysis
nosis by amplifying segments of the coronavirus genetic
material with high specificity and reliability. Multiple re- The keyword “SARS-CoV-2” was used to search for com-
search and clinical institutions around the world have de- plete genomes of the novel coronavirus deposited to the
veloped molecular assays to diagnose SARS-CoV-2 and National Center for Biotechnology (NCBI) nucleotide da-
made the sets of RT-qPCR primers and probes publicly tabase (https://www.ncbi.nlm.nih.gov/genbank/sars-cov-
available (Table 1). 2-seqs/). We analyzed 226 SARS-CoV-2 genome se-
Extensive testing of the population (and the subsequent quences deposited at the NCBI GenBank from samples
isolation of those infected) is essential to containing a pan- collected by March 22, 2020 (Online Resource 1).
demic and avoiding a premature collapse of entire national Following, we selected the complete genome sequences
health systems. For example, Li et al. [10] used infection data and sorted them according to the collection dates
to model the spreading of the novel coronavirus in China (Table 2). We only considered the first complete genome
before travel restrictions were imposed. They concluded that deposited from each country for further analyses, since
undiagnosed SARS-CoV-2 carriers accounted for 79% of the this study did not intend to propose new molecular assays
cases and the primary source of infection, which rapidly for the diagnosis of SARS-CoV-2 but rather demonstrate
spread the disease across the globe [10]. However, as the the importance of genomic analysis of the virus, especial-
global demand for RT-qPCR testing kits surges abruptly dur- ly regarding disease dissemination and validation of as-
ing a fast-spreading pandemic, commercial suppliers are un- says already developed via RT-PCR.
able to deliver kit components in a timely manner, which Nucleotide sequences were utilized to calculate the phylo-
prevents the effective assessment of the disease spreading in genetic distances through the tool ClustalW [13] using default
the population in order to identify and isolate those infected parameters. The tree was generated through the MEGA 7.0
and avoid further spreading. software [14], with branches inferred using the Maximum
The present work aimed to contribute to the study of Likelihood method based on the Hasegawa-Kishino-Yano
SARS-CoV-2 diagnosis through the analysis of the virus model [15].

Table 1 Summary of available

protocols (adapted from WHO– Country Institute Target genes
House Assays, 2020)
China Center for Disease Control and Prevention (CDC) ORF1ab, N
France Institut Pasteur RdRP (2 targets)
Germany Charité - Universitätsmedizin Berlin RdRP, E, N
Hong Kong SAR The University of Hong Kong (HKU) ORF1b-nsp14, N
Japan National Institute of Infectious Diseases (NIID) Pancorona, multiple targets,
spike protein
Thailand National Institute of Health (Thai NIH) N
USA Centers for Disease Control and Prevention (CDC) N (3 target regions)
J Mol Med

Table 2 Complete genome sequences of the novel coronavirus (SARS- Results

CoV-2) deposited at the NCBI GenBank by March 22, 2020

GenBank ID Collection date Location Genome size (bp) Phylogenetics

MN908947 2019-12 China: Wuhan 29,903 Through the analysis of the 14 complete genomes available
LC529905 2020-01 Japan 29,903 (Fig. 1) available during the early stages of the pandemic, we
MT039890 2020-01 South Korea 29,903 could trace the geographical spreading of SARS-CoV-2 since
MT072688 2020-01-13 Nepal 29,811 the beginning of the pandemic [1]. Accordingly, the phyloge-
MN985325 2020-01-19 USA: WA 29,882 netic classification of complete genome sequences can be used
MT192772 2020-01-22 Vietnam 29,891 to trace sources of infection and inform clinical and outcomes
MT007544 2020-01-25 Australia: Victoria 29,893 of novel viruses and should be considered during the design of
MT192759 2020-01-25 Taiwan 29,862 treatments and, eventually, vaccines [12]. While several ther-
MT066156 2020-01-30 Italy 29,867 apeutic approaches to treat COVID-19 are being developed in
MT050493 2020-01-31 India: Kerala State 29,851 the course of the pandemic [16], tracking the evolution of the
MT093571 2020-02-07 Sweden 29,886 virus may also be helpful to determine the impact on the host,
MT233519 2020-02-27 Spain: Valencia 29,782 including high-risk populations and genetic variations associ-
MT126808 2020-02-28 Brazil 29,876 ated with different responses, as well as to inform vaccine
MT240479 2020-03-04 Pakistan: Gilgit 29,836 development and interventions (cf. [17]).
Our phylogenetic analysis (Fig. 2) shows the formation of
three distinct clades. In line with our findings, a recent study
that analyzed 160 complete SARS-CoV-2 genomes also
Virus mutations and molecular assays developed in- found three central variants named A, B, and C. The A variant
house was the ancestral type and closest to a coronavirus from bats
and pangolins. The B variant had a higher incidence in East
For the analysis of amplified regions used in diagnostics, we Asia, while the C variant was found first in patients from
used the tool ClustalW [13] to align all regions of the 14 France, Italy, and Sweden and is the prevalent version in
SARS-CoV-2 complete genome sequences against the primer Europe [12]. This analysis also revealed the main origins of
and probe sequences recommended for diagnosis by various the virus in other countries, such as Brazil, which reported its
research and clinical institutions around the world (Table 1). first sequenced genome from a patient who had returned from
The images displaying nucleotide sequences alignments were Italy. Indeed, in our phylogenetic reconstruction, Brazil and
generated by GeneDoc (http://www.nrbsc.org/gfx/genedoc/; Italy fell in the same clade (variant C), alongside Australia,
Online Resource 2). Additionally, we searched the Web of Sweden, and South Korea, which in line with the report by
Science database for studies on PCR variations used to Forster et al. [12]. In a recent study, 54.8% of the SARV-CoV-
diagnose viruses and compared the methods. 2 cases in Brazil by March 5, 2020, were estimated to be

Fig. 1 Timeline of complete

SARS-CoV-2 genome sequences
deposited to GenBank, according
to their collection dates. The viral
structure image was designed
using resources at Freepik.com
 J Mol Med

[27] developed an analysis pipeline to facilitate real-time

mutation tracking of SARS-CoV-2. This analysis initially
focused on the spike (S) protein because it mediates cell
infection, and it is the focus of most vaccine strategies
and antibody-based therapeutics. Using this method, the
authors identified fourteen mutations that are accumulat-
ing in S [27]. In the host, a major focus of study is the
gene ACE, which is directly involved with SARS-CoV-2
infection and which genetic variations may be involved
with the severity of the disease and may help explain
why the virus is hitting southern Europe so hard [28]. In
our study, however, we focused our analysis on the re-
gions used for diagnosis via PCR.
We examined the regions used for the design of SARS-
CoV-2 primer and probe sets by leading official institutions
around the world (Fig. 3). We noticed that the most common
genomic region of the virus used for diagnosis comprises the
gene “N”, followed by ORF1b, ORF1a, and the gene “E”
(Fig. 3). Five institutions from four countries (Thailand,
China (The University of Hong Kong (HKU) and Center for
Disease Control and Prevention (CDC), the United States, and
Japan) recommended the “N” gene region for diagnosis, with
at least one set of primers and probe. The US Centers for
Disease Control and Prevention (CDC) developed a SARS-
Fig. 2 Phylogenetic analysis of 14 complete SARS-CoV-2 genome se-
quences published by several countries. The molecular phylogenetic anal- CoV-2 Detection Panel composed of three sets of primers and
ysis was conducted in MEGA7 and inferred by the maximum likelihood probes for that region, which comprise a universal set for
method on the Hasegawa-Kishino-Yano model. For more complete stud- SARS beta-coronaviruses, and two other sets specific for
ies on the evolution of SARS-CoV-2 during the pandemic and follow-up SARS-CoV-2 [29]. Chu et al. [8] developed two quantitative
discussions on proper methodologies of phylogenetic analyses, please
refer to [18–21] assays via RT-qPCR to detect two different regions of the viral
genome (ORF1b and gene ‘N’—HKU-Hong Kong/China)
originated from contaminated travelers arriving from Italy and demonstrated that could detect SARS-CoV-2 < 10
[22]. copies/reaction [8]. On the other hand, Corman et al. [9] de-
Phylogenetic analyses assessing the origin of the novel veloped an assay targeting the genes N, E and ORF1b (RNA-
coronavirus have already been published. These studies re- dependent RNA polymerase/RdRP) (Charité/Germany) and
vealed that bats might be the primary reservoir of SARS- obtained the best sensitivity with the respective limits of de-
CoV-2 [23–25]. Therefore, phylogeny is an essential tool to tection (LOD): 8.3, 5.2 and 3.8 copies/reaction, at the 95%
monitor the evolution of pandemic pathogens and can help, detection probability [9].
considering its limitations, in tracing the spreading of viruses. Our SARS-CoV-2 genome alignment also revealed that the
However, such studies must utilize representative datasets and mutations (Online Resource 2) were outside of the annealing
the correct analysis methods to produce results with the nec- sequences designed for primers and probes (Fig. 3), that is
essary robustness for monitoring lethal outbreaks, such as for except for the virus circulating in South Korea, which holds
the novel coronavirus [18–21]. a mutation in the region of a probe recommended by two
European organizations, the Charité - Universitätsmedizin
Berlin and the Pasteur Institute of France. Therefore, the tests
Mutations and molecular assays developed in-house developed by these institutions may be ineffective to accurate-
ly detect that virus version. Indeed, mutations lying within
The study of genome mutations of pathogens as epi- primer annealing sites have already been reported for SARS-
demics spread can help us better understand emerging CoV-2 [30].
outbreaks [26]. This type of data is essential, as it can
indicate the frequency and extent of the genetic variation Variations of PCR techniques and diagnosis methods
of novel viruses. The alignment of complete SARS-CoV-
2 genomes revealed that the new coronavirus had under- Since the genetic code of coronaviruses consists of RNA, a
gone several mutations (Online Resource 2). Korber et al. critical step of RT-qPCR protocols is RNA purification from
J Mol Med

Fig. 3 a Representation of the SARS-CoV-2 genome sequence depicting E was also tested in France. Germany developed two probes for the same
annotated genes. This scheme uses the nucleotide coordinates of a virus RdRP gene region indicated in the image, one being specific for SARS-
sequenced in China (NC_045512). b Positions of the primer/probe targets CoV-2 and another common for SARS-CoV-2, SARS-CoV, and bat
used for detection by institutions in six countries. Colors of primers and SARS-related coronavirus, but only one assay is indicated in the image.
probes correspond to countries: China, CDC (red); USA, CDC (blue); Japan also indicated assays for nested RT-PCR, but the image above only
Germany (yellow); China/Hong Kong (gray); France (pink); Japan represents RT-qPCR assays. An important aspect of RT-qPCR assay
(black); and Thailand (orange). The numbers above each primer and development for viral detection is confirming that primers and probes
probe indicate nucleotide position (coordinate) in the genome. are specific to the virus of interest and do not detect viruses from the same
Important details: France developed two assays for the RdRP gene (iden- family. This is particularly important for coronaviruses, since members of
tified in the figure as ORF1a and ORF1b, according to the reference this group already circulate in the human population
sequence [NC_045512] used). The assay developed by Germany for gene

the testing samples. Specific RNA isolation kits for SARS- amplicon) or via two-step RT-qPCR reaction (two successive
CoV-2 detection were officially recommended by institutions reactions with the cDNA synthesis via reverse transcription
in six countries (Table 3). All the protocols endorsed included preceding the PCR).
RNA extraction by affinity columns that allow automation to Thus, given the scarcity of detection kits officially recom-
optimize time and minimize errors. mended by each institution, analyzing the effectiveness of
All official institutions (Table 3) recommended using one- technique variations could help diagnosis, since the isolation
step RT-qPCR (the reverse transcriptase reaction automatical- of infected patients and epidemiological models depend on the
ly precedes the PCR phase in the same tube), which is availability of such data.
regarded as the gold standard for viral detection and diagnosis Much of the PCR work published on the development of
(Fig. 4). Most of the endorsed assays were designed to detect detecting other viruses can be adapted for the diagnosis of
two regions of the SARS-CoV-2 genome. However, the diag- SARS-CoV2. These studies include the use or comparison
nosis of SARS-CoV-2 can also be performed by conventional between PCR variations for the detection of viruses [31, 32],
PCR reaction after reverse transcription (RT-PCR), nested the use of PCR and the ELISA serological method [33, 34],
RT-PCR reaction (i.e., two successive PCRs with the second commercial kits for virus detection [35, 36], and the chemistry
reaction using primers that anneal internally to the first (e.g., the general double-strand DNA intercalating dye SYBR
 J Mol Med

Table 3 Kits for viral RNA extraction and detection used by institutions from six countries listed by WHO

Kits utilized (company) Country

Virus RNA extraction MagNA Pure 96 System (Roche) Germany

QIAamp Viral RNA Mini Kit (Qiagen) Hong Kong, Japan, USA
NucleoSpin RNA Virus - Macherey (Nagel) France, Thailand
QIAamp DSP Viral RNA Mini Kit (Qiagen) USA*
Virus detection: RT reaction SuperScript III One-Step RT-PCR System with Platinum Germany
and qPCR platform Taq DNA Polymerase (ThermoFisher)
TaqMan Fast Virus 1-Step Master Mix (ThermoFisher) Hong Kong
SuperScript III Platinum One-Step qRT-PCR Kit (ThermoFisher) France, Thailand
QuantiTect Probe RT-PCR Kit (Qiagen) Japan**
TaqPath 1-Step RT-qPCR Master Mix, CG (ThermoFisher) USA

*The US CDC indicates the possibility of using several RNA extraction kits. This table only lists the first kit indicated by each country
**The Japan NIID also indicates the reagents for nested RT-PCR

Green, or post-amplification exonuclease-based probes, such simple, fast, accurate, safe, and amenable to be used in local
as the TaqMan system) used for detecting and quantifying hospital and clinic settings bearing the burden of testing and
DNA amplification [37, 38]. Moreover, these studies aimed treating patients [47]. Recent studies evaluated different tests
to determine simple or multiplex reaction assays for virus available and explored the possibility of improving SARS-
detection and serotyping [39–42] and the development of tests CoV-2 diagnosis [47–49]. Tests based on biomarkers (e.g.,
for simultaneous detection of viruses [43–46]. serum porphobilinogen and aminolaevulinic acid) can be sen-
Currently, several integrated, random-access, point-of-care sitive, specific, and low cost, and could even be used to mon-
molecular devices are under development for the diagnosis of itor the response to treatments [50]. The international, non-
SARS-CoV-2 infections. These assays are expected to be profit organization Foundation for Innovative New
Diagnostics (FIND) has identified almost 800 testing pipe-
lines proposed to detect SARV-CoV-2 (for updates, cf.:
https://www.finddx.org/covid-19/pipeline). As of September
1, 2020, these included 403 immunoassays, 362 molecular
assays, 17 sample collections/inactivation, and 7 digital
solutions.

Discussion

Interpretation

Most of the internal diagnostic assays developed by several

groups around the globe were designed to detect two regions
of the SARS-CoV-2 genome. Here we present a workflow to
validate the internal tests indicated for the novel coronavirus
through a phylogenetic approach. It becomes evident that
Fig. 4 Variations of the polymerase chain reaction (PCR) used for virus monitoring the evolution of the virus is crucial for the valida-
diagnoses. In conventional PCR after reverse transcription (RT-PCR), the tion of internal tests and even for the development of new
target DNA is detected at the end of PCR amplification, requiring a post- protocols.
PCR process for visualization (e.g., DNA electrophoresis). The nested
RT-PCR significantly enhances the sensitivity of conventional PCR,
Furthermore, we suggest there is viability in using PCR
but it nearly doubles the amplification time. All qPCR protocols detect variations (RT-PCR, nested RT-PCR, and two-step RT-
and measure target DNA after each polymerization (extension) cycle qPCR) for the diagnosing of SARS-CoV-2 as a way to face
during the exponential amplification through fluorescence and do not one of the major bottlenecks that emerged with the new pan-
require post-PCR processing. While two-step RT-qPCR involves two
distinct stages of reverse transcription followed by qPCR, one-step RT-
demic, namely the availability of inputs to carry out one-step
involves a single reaction in which cDNA synthesis and amplification RT-qPCR, which is the gold standard of virus diagnosis.
occur successively Moreover, the investment to utilize the infrastructure already
J Mol Med

existent in many university campuses around the world, and are often not practical or even possible in some patients
especially in developing countries, of laboratories fully [56]. Suo et al. [57] pointed to the superiority of ddPCR
equipped to carry out RT-PCR or nested RT-PCR analyses for clinical diagnosis of SARS-CoV-2 to reduce false-
to diagnose SARS-CoV-2 is much lower and amenable than negative results [57]. However, we argue that the limited
creating at speed new labs with qPCR equipment while many availability of ddPCR instrumentation and expertise world-
academic labs are at a standstill situation during the adoption wide, as well as the higher analysis costs, still makes RT-
of quarantine restrictions. Express laboratory certifications PCR as the gold standard for diagnosing and monitoring
could be issued by regulatory agencies by using resources in viral pandemics, at least for the time being.
local offices, and personnel teams could be trained for virus Another technique based on nucleic acid detection that has
diagnosis to enable comprehensive testing of the population. been successfully used to detect SARS-CoV-2 [58–63] but
All in all, the validation of other PCR methods, mainly does not depend on sophisticated equipment is the reverse
regarding sensitivity and specificity for the diagnosis of transcription-loop-mediated isothermal amplification (RT-
SAR-CoV-2, could help countries handle higher volumes of LAMP) [64, 65]. Indeed, quantitative RT-LAMP has been
diagnosis by using alternative supplies and amplify the labo- utilized for diagnosing and the surveillance of viruses because
ratory network for the diagnosis, thus also reducing the test it is a highly sensitive and specific method allied to being
turnaround time. simple (one-step, single tube), fast, and low-cost [66].
Commercial RT-LAMP kits have already been developed
Limitations for SARS-CoV-2 during the course of the pandemic (cf.
https://www.finddx.org/covid-19/pipeline). We speculate
Studies on the use of RT-qPCR have sought to optimize the that RT-LAMP may soon become standard in comprehensive
assays, aiming mainly at increasing the efficiency of viral point-of-care testing and surveillance of ongoing pandemics.
detection [51]. However, due to the short time in which the
virus appeared and spread, few studies aimed to analyze the
assay variables, such as the PCR variations for viral load
detection, and the comparison between the molecular ge- Conclusion
netic methods and serological tests. Some studies aimed at
evaluating and comparing already established RT-qPCR An efficient strategy to enable and increase the efficiency of
tests [52] or molecular point-of-care tests (e.g., RT- molecular testing via PCR for viral detection is the examina-
LAMP—reverse transcriptase loop-mediated isothermal tion of the protocols endorsed by WHO collaborating institu-
amplification) with RT-qPCR [53]. Notwithstanding, the tions and the technique variations. Since many tests have been
broad spectrum of PCR inputs and methodologies poten- developed or are under development, the range of options will
tially available to efficiently detect SARS-CoV2 still war- eventually increase and can alleviate the scarcity of diagnostic
ranted further analysis. Consequently, studies developed kits, thus quickly relieving the demand for testing kits. The
for other viruses show some avenues that can be adapted present study underscores the importance of using phyloge-
to diagnosing SARS-CoV-2. netic approaches not only to understand the evolution of the
Therefore, it is clear that we can still improve the RT- virus but also to designing and reviewing primers and probes
qPCR technique, although it is currently considered the used for SARS-CoV-2 diagnosis via PCR. We also show the
gold standard for diagnosing patients with an ongoing viral versatility of RT-qPCR for the viral diagnosis during a pan-
infection. Another PCR variant called digital droplet PCR demic and identified testing alternatives that can be used in the
(ddPCR) has great potential in this area, especially to detect SARS-CoV-2 diagnosis.
low viral loads in samples, which is a major limitation of
the RT-qPCR [54]. Recently, Yu et al. [55] reported a study Acknowledgments The authors thank their respective institutions for
in which they analyzed viral loads of SARS-CoV-2 in in- time and scientific resources available to conduct this work.
fected patients and concluded that although the RT-qPCR is
sensitive and reliable, ddPCR was better to detect samples Authors’ contributions All authors contributed to the study conception
and design. Material preparation, data collection, and analysis were per-
with low viral loads [55]. Falzone et al. [56] also evaluated formed by Horllys Gomes Barreto, Matheus Martins Daúde, and Vagner
the ddPCR sensitivity and specificity for detecting SARS- Augusto Benedito. Flávio Augusto de Pádua Milagres and Gessi
CoV-2 comparatively to RT-qPCR and reported that Carvalho de Araújo Santos contributed with critical methodological con-
ddPCR was superior in both parameters. They noticed that cepts in the development of the study. The first draft of the manuscript
was written by Horllys Gomes Barreto and all authors contributed with
ddPCR could also be used to detect SARS-CoV-2 in blood editing. All authors read and approved the final manuscript.
and saliva samples, which have not been optimally
established for RT-qPCR. ddPCR could indeed improve Data availability All data used in this study is available to the scientific
the diagnostic procedures since rhino-pharyngeal swabs community upon request.
 J Mol Med

Compliance with ethical standards 12. Forster P, Forster L, Renfrew C, Forster M (2020) Phylogenetic
network analysis of SARS-CoV-2 genomes. Proc Natl Acad Sci
117:9241–9243
Conflict of interest The authors declare that they have no conflict of
13. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: im-
interest.
proving the sensitivity of progressive multiple sequence alignment
through sequence weighting, position-specific gap penalties and
Consent to participate Not applicable. weight matrix choice. Nucleic Acids Res 22:4673–4680
14. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolu-
Consent for publication Not applicable. tionary genetics analysis version 7.0 for bigger datasets. Mol Biol
Evol 33:1870–1874
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patient data. splitting by a molecular clock of mitochondrial DNA. J Mol Evol
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