Rapid and reliable diagnostic method to detect Zika virus by real-time fluorescence reverse transcription loop-mediated isothermal amplification
Guo et al. AMB Expr
Rapid and reliable diagnostic method to detect Zika virus by real-time fluorescence reverse transcription loop-mediated isothermal amplification
Xu‑Guang Guo 0 2
Yong‑Zhuo Zhou 1
Lei Zheng 0
Qian Wang 0
0 Laboratory Medicine Center, Nanfang Hospital, Southern Medical University , Guangzhou 510515, Guangdong Province , China
1 Department of Clinical Laboratory Medicine, The Third Affiliated Hospital of Southern Medical University , Guangzhou 510150, Guangdong Province , China
2 Department of Clinical Laboratory Medicine, The Third Affiliated Hospital of Guangzhou Medical University , Guangzhou 510150, Guangdong Province , China
To detect Zika virus more rapidly and accurately, we developed a novel method that utilized a real‑ time fluorescence reverse transcription loop‑ mediated isothermal amplification (LAMP) technique. The NS5 gene was amplified by a set of six specific primers that recognized six distinct sequences. The amplification process, including 60 min of thermostatic reaction with Bst DNA polymerase following real‑ time fluorescence reverse transcriptase using genomic Zika virus standard strain (MR766), was conducted through fluorescent signaling. Among the six pairs of primers that we designate here, NS5 was the most efficient with a high sensitivity of up to 3.3 ng/μl and reproducible specificity on eight pathogen samples that were used as negative controls. The real‑ time fluorescence reverse transcription LAMP detection process can be completed within 35 min. Our study demonstrated that real‑ time fluorescence reverse transcription LAMP could be highly beneficial and convenient clinical application to detect Zika virus due to its high specificity and stability.
Zika virus; NS5 gene; LAMP; Loop‑ mediated isothermal amplification; Real‑ time fluorescence reverse transcription
Zika virus is primarily transmitted by the bite of the
female Aedes aegypti mosquito in tropical regions, which
must feed on blood to lay eggs
(Gulland 2016; Walsh
. Other species of mosquito have been reported
such as Aedes polynesiensis and Aedes albopictus. The
virus has also been isolated from a number of arboreal
mosquito species in the Aedes genus.
Patients with active or recessive infection and
nonhuman primates infected with Zika virus are the source
of the disease. The symptoms of infection are similar to
dengue fever and include fever, rash, joint pain, muscle
pain, headache and conjunctivitis
Only 20% of infected people present clinical symptoms,
including fever, rash, joint pain and conjunctivitis. The
symptoms usually disappear in less than a week.
However, Zika virus could pose serious problems for
pregnant women and their fetus. Zika can be transmitted
to the fetus in the uterus or during the delivery process
(Chen and Tang 2016)
. Zika virus nucleic acid has been
detected in milk. In early 2015, a widespread epidemic
of Zika fever, caused by the Zika virus in Brazil, spread
to other parts of South and North America. The
incidence in China is relatively low but the number is
steadily increasing. Zika virus infection has become a serious
global public health threat to humans
The outbreak period is usually associated with the
mosquito breeding season. Thus, a fast and efficient method
to detect Zika virus is necessary.
Real-time loop mediated isothermal amplification
(LAMP) is a novel technology that has been developed in
(Xia et al. 2014)
. LAMP is a thermos-stable
nucleic acid amplification technique for gene
detection that requires 4–6 primers targeting 4–6 specific
(Mansour et al. 2015)
. It only takes 60 min for
the Bst DNA polymerase to amplify the target sequences.
LAMP technology has been widely used in detecting a
variety of viruses, bacteria and parasites, which has been
useful for pathogen identification in clinics
(Khan et al.
Compared with traditional serologic test, LAMP is fast
and efficient and may be a particularly useful method for
infectious disease diagnosis in low and middle-income
countries. LAMP does not require expensive equipment,
and the process can be performed with a water bath or
(Hattori et al. 2016)
. Thus far, detection
methods for Zika virus have primarily relied on traditional
real-time polymerase chain reaction (RT-PCR). In this
study, we demonstrated that Zika virus can be accurately
detected by LAMP and that it is less time-consuming and
has a higher sensitivity than traditional RT-PCR,
suggesting that this method could be beneficial tothe clinical
treatment and prevention of Zika infections.
Materials and methods
Zika virus and bacterial strains
The nucleic acid of Zika virus MR766 RNA was
purchased from Beijing Ruilimaidi Laboratory Co., Ltd.
Klebsiella pneumoniae ATCC700603, Acinetobacter
baumannii ATCC19606, Streptococcus pneumoniae
ATCC49619, Staphylococcus aureus ATCC25923,
Streptococcus mitis ATCC49456, Pseudomonas aeruginosa
ATCC27853, Haemophilus influenzae ATCC49766 and
Escherichia coli ATCC25922 were stored in the
microbiology room of the Third Affiliated Hospital of
Guangzhou Medical University.
Primer design and synthesis
The primers forward outer primer (F3), backward outer
primer (B3), forward internal primer (FIP), backward
internal primer (BIP) and loop backward primer (LB)
were designed by Guangzhou Deaou Biotechnology
Company Limited and synthesized by Tiangen
Biochemical Technology (Beijing) Company Limited. The
sequences of the primers used in this study are listed in
Tables 1, 2, 3 and 4.
LAMP reaction system preparation
Bacterial genomic DNA extraction kit was purchased
from Tiangen Biochemical Technology Company
Limited. The LAMP reaction system was prepared according
to the manual for the LAMP amplification kit.
Primer screening and dissociation curve analysis
Four sets of primers were designed: NS5-1, NS5-2,
NS53, and NS5-4. Each of the working fluids to Zika virus
RNA served as a template. The reaction procedure was
set to 63 °C 15 s, 63 °C 45 s as one cycle for 60 cycles,
95 °C 15 s to terminate the reaction, and 63 °C 45 s at the
collection of fluorescence signals. A reaction tube was
used for the dissolution curve detection and to compare
the amplification efficiency of four sets of primers while
screening out the highest amplification efficiency without
a primer dimer.
Sensitivity of LAMP for Zika virus
The RNA concentration of Zika virus was measured by
adjusting the RNA template concentration with 1 μl
of TE buffer to the Thermo Scientific Nanodrop 2000
microphotometer and 1 μl of RNA extract of Zika virus.
10 μl of RNA stock solution was diluted 10-fold with
ultra-pure water four times to obtain five RNA template
concentrations. This was added to the LAMP system
reaction, and the amplification curve was observed to
evaluate the sensitivity of the primers.
Specificity and repeatability of LAMP for Zika virus
The genomic DNA of Zika virus and other negative
pathogens were extracted and amplified according to the
reaction conditions of Real-LAMP. The specificity of the
primers was evaluated. One of the positive strains and
five negative strains were tested three times with LAMP.
The same primer used for the repeat test was used to
evaluate the reliability of LAMP.
Primer screening test of LAMP assay
Four sets of primers amplified without the loop primer
showed that the primers NS5-3 had a peak at about
39 min and that the NS5-1 primers had a peak at about
31 min. The efficiencies are both lower than those with
the loop primers. The NS5-2 and NS5-4 primers had no
peak (Figs. 1, 2).
At approximately 15 min after the initiation of the
reaction, the system of the primer NS5-3 peaked, and at
approximately 21 min, the reaction of the NS5-1 primers
peaked. The Bio-Rad CFX Manager detected the
amplification signal of the NS5 gene, NS5-2 and NS5-4 without
a peak (Fig. 3). Although the primer NS5-3 had a
relatively high amplification efficiency for the Zika virus NS5
gene, the fluorescence intensity of the negative control
curve was significantly decreased, possibly for the dimer
of primers (Fig. 4).
Sensitivity of LAMP for amplification of Zika virus
In this study, the clinical common pathogens were
selected as negative controls, but no Zika virus RNA was
amplified. The typical “S” curve showed that the
specificity of Zika virus RNA was better. The LAMP primer
NS5-1 has good specificity and does not cross-react with
non-target bacteria (Fig. 5).
Specificity of LAMP for amplification of Zika virus
Measured by micro-spectrophotometer, the experiment’s
Zika virus nucleic acid concentration is 33 ng/μl. Diluted
by 4 gradients, the resulting concentrations were 3.3,
0.33 ng/μl, 33 and 3.3 pg/μl. The limit of the detection
of this test can reach 3.3 ng/μl. Reliable test results are
shown in Fig. 6.
Repeatability of LAMP for amplification of Zika virus
Take Zika virus NS5 for three replicates, wherein two
peaks were at 23 min and another peak was at 20 min.
The difference in peak time is approximately 3 min. The
repeatability of the tests is shown in Fig. 7.
The Zika virus belongs to the Flaviviridae family and the
Flavivirus genus and is thus related to the dengue fever,
yellow fever, Japanese encephalitis, and West Nile viruses
(Zuanazzi et al. 2017)
. Most patients show no
symptoms or have mild symptoms
(Zhang et al. 2017)
20% of patients experience fever, rash, conjunctivitis and
joint pain, while in some cases, symptoms disappear
within a week
(Wilbe et al. 2017; Zamani and Zamani
. Therefore, to develop a rapid and robust nucleic
acid amplification assay to efficiently detect Zika virus
is critically important for clinical treatment and disease
In this study, the NS5 gene was selected as the target
gene and 8 clinical common pathogens were used as
negative controls. The yellow fever virus was theoretically
selected as the negative control.
NS5 gene sequences were successfully amplified and
displayed as a typical ‘S’ shape in Zika virus, except for
other negative pathogens, which were used as
negative controls, indicating that the NS5 gene sequence as
a highly specific candidate could be used in Zika
detection. The results showed that the ring primer could
significantly shorten the reaction time. The reaction of the
ring primer lasted approximately 22 min, and the peak
time of the ring primer was less than 10 min. The entire
reaction process can be completed within 35 min, which
is significantly shorter than that for PCR and is conducive
to the clinical diagnosis of Zika virus infection, the timely
detection of Zika virus infection, and timely treatment.
Traditional PCR methods are based on agarose gel
electrophoresis, which are cumbersome to operate
et al. 2017)
. Our current method, the LAMP
amplification technology, is superior due to its high sensitivity and
specificity and ease of operation. Generally, the
amplification could be visualized and quantified by
fluorescence dye intensity. Primers were specifically labeled with
fluorescent dye and embedded in the amplified
doublestranded DNA. False positive results could be obtained
by the dimer in the primer. In our study, we carefully
checked the dissolution curve for the NS5 primer. The
negative control curve was straight while the positive
control curve was nearly overlapped, indicating that the
NS5 primer contained no dimer.
LAMP detection technology has the following
advantages compared with other methods. The primers used
in the LAMP detection technique are based on six
primers designed for the target sequence
(Abdul-Ghani et al.
2012; Ahmad and Hashsham 2012; Deborggraeve and
Buscher 2012; Ebenezer et al. 2012; Njiru 2012)
addition, LAMP is superior to other methods due to its high
sensitivity, as high as 3.3 ng/μl. No positive signals were
observed in the negative control. These results were
reproducible, and we repeated them three times in our
study with no significant difference in peak time. An
important feature of the LAMP method is the very short
detection time and small amount of reagents needed.
LAMP is a consistent reaction system that avoids
temperature changes, which may influence accuracy.
However, there are limitations. For example,
inevitable aerosol pollution may lead to false positive results
(Asiello and Baeumner 2011; Fu et al. 2011)
. LAMP is
less common than PCR and is primarily used as a
diagnostic or detection technique, not for cloning (Beck
and Henrickson 2010). In addition, the LAMP reaction
system requires more primers, which may increase the
possibility to form dimers
(Gill and Ghaemi 2008; Mori
and Notomi 2009)
Our current study demonstrated that the application
of LAMP to detect Zika virus was superior to traditional
methods with a high sensitivity and specificity. As a rapid
and efficient novel method, LAMP may be a beneficial
tool for clinical application.
LAMP: loop‑mediated isothermal amplification.
XGG and QW designed the study. XGG, QL, WW, YZZ, JZW, LZ and QW col‑
lected and analyzed the data. YZZ advised on data analysis. XGG drafted
and wrote the manuscript. QL and QW revised the manuscript critically for
intellectual content. All authors gave intellectual input to the study. All authors
read and approved the final manuscript.
of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine,
Yale School of Medicine, Yale University, New Haven, CT 06520‑8057, USA.
5 Department of Laboratory Medicine, The First Affiliated Hospital of Shenzhen
University, Shenzhen 518035, Guangdong, China. 6 Department of Laboratory
Medicine, Shenzhen Second People’s Hospital, Shenzhen 518035, Guangdong,
China. 7 Center For Disease Control and Prevention of Chaozhou, Guang‑
zhou 521000, Guangdong Province, China.
This research was funded by the General Research Projects of Medical and
Health Sciences of Guangzhou City (Funding No: 20151A011083), Bureau
of Science, Technology and Information of Guangzhou (Funding Nos.
20151217081 and 2016080067).
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analyzed during this study are included within the
Consent for publication
All data published here are under consent for publication.
Ethics approval and consent to participate
The study was approved by the ethics committees of the participating hospi‑
tals and institutes. All procedures performed in the studies involving human
participants were in accordance with the ethical standards of the institutional
and national research committee and with the 1964 Helsinki declaration and
its later amendments or comparable ethical standards.
This study was supported by the General Research Projects of Medical and
Health Sciences of Guangzhou City (No. 20151A011083), the Bureau of
Science, Technology and Information of Liwan District of Guangzhou (Nos.
20151217081 and 2016080067), and the National Nature Science Foundation
Springer Nature remains neutral with regard to jurisdictional claims in pub‑
lished maps and institutional affiliations.
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