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Field Detection of Citrus Huanglongbing Associated with ‘Candidatus Liberibacter Asiaticus’ by Recombinese Polymerase Amplification within 15 min Wenjuan Qian, Ying Lu, Youqing Meng, Zunzhong Ye, Liu Wang, Rui Wang, Qiqi Zheng, and Jian Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01015 • Publication Date (Web): 21 May 2018 Downloaded from http://pubs.acs.org on May 21, 2018
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Field Detection of Citrus Huanglongbing Associated with ‘Candidatus
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Liberibacter Asiaticus’ by Recombinese Polymerase Amplification within 15 min
3 4
Wenjuan Qian a, Ying Lu b, Youqing Meng b, Zunzhong Ye a, Liu Wang a, Rui Wang a,
5
Qiqi Zheng a, Hui Wu a and Jian Wu a*
6
a
7
Hangzhou 310058, China.
8
b
College of Biosystems Engineering and Food Science, Zhejiang University,
Zhejiang Plant Protection and Quarantine Bureau, Hangzhou 310020, China.
9 10
Correspondence
11
(J. Wu) E-mail:
[email protected]. Tel: 0086-571-88982180
12
Fax: 0086-571-88982180 College of Biosystems Engineering and Food Science,
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Zhejiang University, Hangzhou 310058, China.
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ABSTRACT:
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Candidatus Liberibacter asiaticus(Las)is the most prevalent bacterium associated
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with Huanglongbing which is one of the most destructive diseases of citrus. In this
17
paper, an extremely rapid and simple method for field detection of Las from leaf
18
samples based on recombinase polymerase amplification (RPA) is described. Three
19
RPA primer pairs were designed and evaluated. RPA amplification was optimized so
20
that it could be accomplished within 10 min. In combination with DNA crude
21
extraction by 50-fold diluting after 1 min of grinding in 0.5 M sodium hydroxide and
22
visual detection via fluorescent DNA dye (positive samples display obvious green
23
fluorescence while negative samples remain colorless), the whole detection process
24
can be accomplished within 15 min. The sensitivity and specificity of this RPA-based
25
method were evaluated, which were proven to be equal to real-time PCR. The
26
reliability of this method was also verified by analyzing field samples.
27 28
Keywords:
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recombinase polymerase amplification, visual detection
Candidatus Liberibacter asiaticus, field detection, crude DNA extraction,
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Introduction
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Huanglongbing (HLB) is one of the most serious and destructive diseases of citrus
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and causes huge economic losses in citrus-producing areas worldwide.1The infection
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is associated with three yet-uncultured pathogen species: Candidatus Liberibacter
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asiaticus (Las), Candidatus Liberibacter africanus (Laf), and Candidatus Liberibacter
35
americanus(Lam), of which Las is the most prevalent species and has been spreading
36
worldwide.2, 3 Infected citrus may exhibit long latency period and will be destroyed or
37
become unproductive in 5−8 years following infection.4, 5 However, there are no
38
effective approaches to control the disease once the plants have been infected.6 To
39
prevent it from spreading, the current management strategy of HLB is to remove
40
infected citrus trees immediately. 6 Thus, rapid, sensitive, simple, low cost and easy to
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operate method for field detection of Las would facilitate implementation of required
42
management practices in a timely fashion.
43
Nowadays, polymerase chain reaction (PCR) is widely used to detect Las because
44
of its high sensitivity and reliability.7-12 Nevertheless, it is impractical for field
45
detection due to the long-running operation, need for expertise and use of expensive
46
equipment like thermal cycler or fluorescent detection equipment. Apart from
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PCR-based methods, isothermal nucleic acid amplification, such as loop-mediated
48
isothermal amplification (LAMP) has also been applied for HLB detection.13, 14 Our
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group also reported a detection method for Las with visual detection based on
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LAMP.15 Although LAMP has overcome the limitations of thermal cyclers and can be
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used for field detection of Las, it still need at least half an hour to complete the
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detection process and the design of the six primers is complex.15-18
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Recently, a novel isothermal nucleic acid amplification technique, recombinase
54
polymerase amplification (RPA), has attracted extensive attention since it is a rapid,
55
sensitive and simple diagnostic approach.19 The reaction initiates with the
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recombinase binding to the primer to form nucleoprotein filaments which scan the
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template DNA for homologous sequences. Next, single-stranded DNA binding protein
58
helps template DNA melting thus primers and template DNA start pairing. Finally, the
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strand-displacing polymerase adds nucleotide bases to the 3’ end of the primer for
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extension. (Figure S1, Supporting Information).19-21 Exponential amplification can be
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accomplished within 10 to 20 min by the circulation of this process.22, 23 RPA is a
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good choice for field detection because it is isothermal, simple, rapid and sensitive.
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Application of RPA for detection of plant pathogens such as Tomato mottle virus,
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Rose rosette virus and Banana bunchy top virus have already been reported.24-27 Paul
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F et al. have reported to implement the rapid on-site detection of Las by RPA.28
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However, they used lateral flow dipstick (LFD) to detect RPA amplicons, which
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usually requires costly nickase, complex probe design, and the uncapping operation.29,
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30
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on lateral flow dipstick. For visual detection in field, DNA florescent dye will be more
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convenient and simple.31, 32 To the best of our knowledge, florescent dye based visual
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detection method for field detection of Las by RPA has not been reported up in
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literatures until now.
Furthermore, present field detection method for RPA amplificons is mostly based
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Another obstacle for field detection of HLB is DNA extraction. Standard
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extraction methods and commercial kits are not appropriate for rapid field detection
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due to the redundant operation steps and long operation duration. Fortunately, RPA
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amplification is very robust and has already been reported to work with DNA crude
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extraction.33, 34
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Here, we have developed a RPA-based visual detection method of Las from citrus
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leaf samples. DNA was extracted from leaf samples by simple treatment with sodium
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hydroxide (NaOH) solution and the 50-fold dilution was directly used as template for
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RPA. After 10 min amplification, the reaction mixture, fluorescent dye, and sterile
82
water were mixed immediately. Then positive samples emit green fluorescence which
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could be easily distinguished from colorless negative samples under a mini-UV torch
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light. The whole process could be accomplished in 15 min and the amplicon
85
contamination could be avoided with a special closed cartridge developed by our
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group.
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Materials and methods
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Plant material
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40 leaf samples containing 20 asymptomatic and 20 symptomatic leaves of Las
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symptomatic citrus trees and 150 blind leaf samples obtained from different regions in
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Zhejiang Province, China were collected by Zhejiang Plant Protection and Quarantine
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Bureau. Healthy citrus leaves bought from local supermarket were used as negative
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controls.
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NaOH-based DNA crude extraction 5
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A piece of midrib tissues (around 0.6 cm×2 cm, 50 mg) of a leaf sample was cut
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up into pieces and ground in a mortar and immerged in 500 µL 0.5 M NaOH. After 1
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min of grinding and several minutes of standing at room temperature, the supernatant
98
was diluted 50-fold with Tris-EDTA (TE) buffer (10 mM Tris, 1 mM EDTA, pH 8.0)
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and was directly used as template for RPA assay. The durations of lysis were studied
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by crushing the leaves in 0.5 M NaOH for 1 min and then letting the slurry stand for 0,
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2, 5 and 9 min, respectively. For a comparative test, standard DNA extraction was
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conducted using a commercial DNA extraction kit (Tiangen Biotech Co., Ltd, Beijing,
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China).
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Primer design for RPA
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Three
pairs
of
RPA
primers
targeting
the
conserved
sequence
of
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tufB-secE-nusG-rplKAJL-rpoB gene cluster [GenBank: AY342001.1] in Las designed
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for amplification of 144-178 bp products using Primer Premier 5.0.35 The specificity
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of primers was ensured by Blast analysis and primer sequences are shown in Table 1.
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RPA Assay
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RPA reactions were carried out in a total of 25 µL reaction mixture according to
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the manufacturer’s instructions (TwistAmp basic kit, TwistDx, U.K.). Each reaction
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contained one pellet, 14.75 µL of rehydration buffer, 0.2-0.72 µM of each primer,
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1.25µL of magnesium acetate, 2 µL of DNA template, and sterile water up to 25 µL.
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The mixture was incubated at 37-42 °C for 8-15 min on a conventional heating block
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(Sangon Biotech Co., Ltd., Shanghai, China). After amplification, 2 µL of RPA
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products were electrophoresed at constant voltage (110 V) on a 3% (w/v) agarose gel 6
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for 35 min and then photographed with a ChemiDoc XRS +System (BioRad, Hercules,
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CA, USA).
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Visual detection of RPA amplificons
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1 µL of SYBR Green I (1:50 dilution of 10 mg/mL stock solution, Sangon Biotech
121
Co., Ltd., Shanghai, China) was added to reaction mixture after amplification. The
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amplicons were detected directly by fluorescence observation using a mini-UV torch
123
(Tantoo E-commerce Co., Ningbo, China). To optimize the condition of visual
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detection, the RPA reactions were performed for 8, 10 and 15 min, respectively, with
125
varying primer concentrations (0.2, 0.36 and 0.78 µM each). Negative samples using
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DNA extracted from healthy citrus leaves as template were used as control.
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Furthermore, the amplicons were diluted in water at 1:2, 1:5, and 1:10 dilutions before
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visualization under UV light in order to reduce the background fluorescence.
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Visual detection of RPA products in a disposable cartridge
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The disposable cartridge is made of plastic and 102L × 12W × 13H mm. The 25
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µL reaction mixture, 1 µL SYBR Green I (200 µg/mL), and 25 µL sterile water was
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pre-added into three commercial PCR tubes and the tubes were connected to the
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cartridge. Once the reaction is over, turn the cartridge upside down and shake
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intensively to mix all the agents thoroughly. Then the results were visually detected
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under UV light.
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PCR detection
137
Primer sets, OI1 (5’-GCGCGTATTTTATACGAGCGGCA-3’) and OI2c
138
(5’-GCCTCGCGACTTCGCAACCCAT-3’), employed for PCR were reported in 7
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previous paper by Li W. et al14 and synthesized by Sangon Biotech (Shanghai, China).
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The 25 µL total reaction volume 2.5 µL 10×PCR buffer, 2 µL dNTP (2.5 mM each),
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0.125 µL TaKaRa Taq™ HS (TaKaRa Biotech Co., Ltd, Dalian, China), 250 nM of
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each primer, 2 µM SYTO 9 (Thermo Fisher Scientific Inc. Waltham, MA USA), 2.5
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µL template, and sterile water up to 25 µL. The thermal program consisted of a single
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cycle at 98°C for 3 min followed by 40 cycles at 98°C for 10 s, 60 °C for 30 s, and
145
70°C for 1 min. For real-time PCR, fluorescence signal was collected at the end of
146
each cycle on the QuantStudioTM3 Real Time PCR System. Time consumed was
147
defined as cycle threshold (Ct) value. For conventional PCR, 2 µL of PCR products
148
were electrophoresed after amplification.
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Sensitivity of RPA-based visual detection method
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DNA template extracted from symptomatic leaves by commercial kits was
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adjusted to the initial concentration of 100 ng/µL and 1:10 serial dilutions were
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prepared. Before RPA amplification, the dilutions were mixed with host DNA
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extracted from healthy citrus leaves to ensure that the total DNA concentration was
154
100 ng/µL to eliminate the effect of host DNA. RPA amplification was performed at
155
37 °C for 10 min.
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Specificity of RPA-based visual detection method
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To evaluate the specificity of the RPA-based visual detection method, several
158
other citrus samples infected by other bacterial or fungal plant pathogens, including
159
citrus canker, citrus anthracnose, citrus scab, citrus black spot, and citrus brown spot,
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were tested by Las-RPA technology described above. DNA from healthy citrus 8
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samples was used as a negative control.
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Detection of Las by RPA from field samples
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For RPA assay (Figure 1), a piece of midrib tissue (around 0.6 cm×2 cm, 50 mg) of
164
citrus leaf was used for DNA extraction with 500 µL of 0.5M NaOH for 1 min of
165
grinding. A volume of 2 µL of 50-fold diluted rough cell lysate was amplified by RPA
166
at 37 °C on a heating block for 10 min. After the amplification, the mixture was 2-fold
167
diluted and mixed with 1 µL of SYBR Green I (200 µg/mL). The presence or absence
168
of fluorescence was examined by visualization under UV light (354 nm) using a
169
mini-UV torch. Since the UV light is very weak, protective equipment is needless.
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Finally, 40 leaf samples (20 asymptomatic and 20 symptomatic leaves) of Las
171
symptomatic citrus trees and 150 filed leaf samples were detected to evaluate the
172
feasibility of RPA-based visual method for the detection of Las. Real-time PCR was
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used as control.
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Results and discussions
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Optimization of reaction condition for RPA
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RPA primers are relatively long (~30 bp) compared with PCR primers and vital
177
for the success of RPA. To ensure the successful RPA amplification, three pairs of
178
RPA primers targeting the conserved sequence of tufB-secE-nusG-rplKAJL-rpoB
179
gene cluster [GenBank: AY342001.1] in Las were designed (Table 1).
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In order to select the optimal primer pair for RPA, RPA reactions were conducted
181
using each of the three primer pairs described above and analyzed by electrophoresis.
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A visible target band was displayed for positive samples in the gel electrophoresis 9
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image no matter which primer pair was used while negative samples displayed no
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bands. And target band of the amplicons amplified from primer pair I was the
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brightest, indicating better performance compared with other two primer pairs (data
186
not shown). Thus, primer F I and R I were selected for the following assay.
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Subsequently, to find out the optimal temperature for RPA assay, the mixture was
188
incubated at 37, 38, 39, 40, 41, and 42 °C since the working temperature of the
189
recombinase and polymerase is between 37-42 °C. As shown in Figure 2a, visible
190
target bands at expected size range were obtained by RPA at all of these six different
191
temperatures. The reactions at 37 °C and 38 °C had better performance compared with
192
other temperatures. As a result, 37 °C was applied for RPA amplification.
193
Crude extraction of DNA for RPA
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Purification steps of DNA extraction were omitted in this paper. About 50 mg of
195
leaf midrib tissue was ground in 500 µL of 0.5 M NaOH for 1 min. Then 2 µL of
196
supernatant was collected after 0, 2, 5, and 9 min of standing and diluted in 100 µL of
197
TE. 2 µL of the dilute extract was used for RPA reaction. In order to ensure the quality
198
of crude-extracted DNA and make the extraction time as short as possible, different
199
lysis time were studied. The results show that no incubation was necessary (Figure
200
2b). In addition, the amplification appeared to have decreased with increased
201
incubation time. This phenomenon could be explained that NaOH solution could
202
cause DNA degradation. And more compound RPA inhibitors would rise from the
203
cytolysis with the process of NaOH lysis. Since the amplification ability of 1 min of
204
grinding without incubation had no significant difference with that of standard DNA 10
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extraction (Figure 2b), this NaOH-based DNA crude extraction method (1 min of
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grinding followed by 50-fold dilution) was applied in the following assay.
207
Visual detection of RPA amplicons
208
Here, we employed florescent dye, SYBR Green I, for visual detection of Las by
209
RPA for the first time. After RPA amplification, 1 µL of SYBR Green I (200 µg/ml)
210
was added to reaction mixture. SYBR Green I would intercalate into double-stranded
211
DNA and generate fluorescence. Then with the help of a mini-UV torch, the presence
212
or absence of fluorescence was recorded. It was assumed that the amplicons of
213
positive samples would emit green fluorescent which could be easily distinguished
214
from colorless negative samples by naked eyes. However, it is reported that RPA has
215
high background signal so that negative samples may emit fluorescence under some
216
circumstances.19
217
To eliminate background signal and optimize the reaction condition for visual
218
detection, RPA reactions were performed for 8, 10 and 15 min, respectively, with
219
varying the primer concentration (0.2, 0.36 and 0.78 µM). As shown in Figure 3a,
220
with the amplification duration becoming longer and the concentration of primers
221
getting higher, the background signal of negative samples was enhanced. Especially,
222
both positive and negative samples could emit obvious fluorescence after 15 min
223
amplification regardless of the primers concentration and all negative samples with
224
0.78 µM of primers had high background even if the reaction time was only 8 min
225
(Figure 3a). This phenomenon may be related to the relatively high concentration and
226
long primers in RPA mixture which tend to form secondary structures and cause 11
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non-specificity amplification. To obtain relatively high fluorescence intensity for
228
positive samples and low background signal for negative sample, the RPA reaction
229
was determined to be performed for 10 min with 0.36 µM primers. To further
230
eliminate the background signal and get the most distinct differences between positive
231
and negative samples, we compared the difference among 2-fold, 5-fold and 10-fold
232
dilution of the products. Figure 3b demonstrates that 2-fold diluted RPA amplicons
233
were enough for visually distinguish positive and negative samples while the
234
fluorescence intensity of positive samples got weak when 5 or 10-fold diluted.
235
Therefore, the RPA-based visual detection method of Las was determined as
236
following: a piece of midrib tissues (around 0.6 cm×2 cm) of citrus leaf was used for
237
DNA extraction with 0.5M NaOH for 1 min of grinding. 2 µL of 50-fold diluted rough
238
cell lysate was amplified by RPA at 37 °C with 0.36 µM primers on a heating block
239
for 10 min. After the amplification, the mixture was 2-fold diluted and mixed with 1
240
µL of SYBR Green I (200 µg/ml). The detection result was visually judged under a
241
mini-UV torch.
242
However, as reported, the requirement of uncapping operation presents a high risk
243
of amplicon contamination for DNA amplification.31 Thus, a convenient, portable and
244
disposable cartridge developed by our group was introduced for visual detection of
245
RPA products (Figure 4). (This portable cartridge also has another form for lateral
246
flow dipstick detection which has been reported previously.36) The 25 µL reaction
247
mixture, 1 µL fluorescent dye, and 25 µL sterile water are pre-added into three
248
commercial PCR tubes. Then PCR tubes are connected to the cartridge tightly so that 12
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the reaction space is sealed. The cartridge can be directly incubated at 37 °C for RPA.
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After amplification, turn the cartridge upside down and shake intensively to mix all
251
the agents thoroughly. Finally, the visually detection can be performed without
252
uncapping operation and prevent cross-contamination.
253
Sensitivity and Specificity of RPA-based visual detection method
254
To investigate the sensitivity of RPA-based visual detection method, a set of
255
10-fold gradient dilutions of template DNA extracted from symptomatic leaves were
256
amplified by RPA. PCR detection using primer sets, OI1 and OI2c, was used as
257
control. As shown in Figure 5, the limit-of-detection (LOD) of gel electrophoresis
258
images of RPA, visual detection of RPA, real-time PCR and gel electrophoresis
259
images of PCR was around 1.0 ×10-1 ng/µL, suggesting that the sensitivity of this
260
RPA-based visual detection method was equal to the conventional PCR method, while
261
it took less time and easier to operate.
262
The specificity of RPA primers was tested on several other citrus samples infected
263
by bacterial or fungal plant pathogens, including citrus canker, citrus anthracnose,
264
citrus scab, citrus black spot and citrus brown spot. No positive result was observed
265
except with the Las-infected sample, indicating a high level of detection specificity of
266
RPA-based visual detection method. (Figure S2).
267
Detection of Las from field samples by RPA
268
Finally, to evaluate the feasibility of proposed RPA-based visual detection
269
method, 40 samples containing 20 asymptomatic and 20 symptomatic plant materials
270
obtained from different infected trees were tested by RPA-based visual detection 13
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method with real-time PCR as control. The detection results of the two methods were
272
consistent that all symptomatic and asymptomatic samples were tested positive,
273
indicating that this proposed RPA-based visual detection method could be an
274
alternative for real-time PCR (Table 1). Subsequently, a total of 150 blind
275
field-collected samples from 9 different regions in Zhejiang Province, China were
276
evaluated by the proposed RPA-based visual detection method in comparison with
277
real-time PCR. As shown in Table 1, among 150 blind samples, the positive rates by
278
these two methods were both 8.57 %. All samples that were found to be positive by
279
RPA-based visual detection method were also positive by real-time PCR (Figure 6).
280
The results illustrated that this RPA-based visual detection method also had high
281
reliability for natural samples detection.
282
In summary, we have developed an extremely rapid, simple and visual method
283
for field detection of Las without carry-over contamination based on RPA
284
amplification in this paper. DNA extracted from leaf samples by simple treatment
285
with NaOH solution can be directly used as template for RPA. RPA amplification was
286
optimized and can be accomplished within 10 min. Then SYBR Green I was
287
employed for end-point RPA detection so that the result of the amplification can be
288
accurately judged by naked eyes under UV light using a mini-UV torch. The whole
289
detection process from leaf sampling to result can be completed in 15 minutes.
290
Without any sophisticated complex instruments, a heater and a mini-UV torch are
291
enough to perform the detection process, making this method be an alternative
292
method for field detection. And the amplicon contamination can be avoided with an 14
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enclosed cartridge. The feasibly of this method has also been verified by analysis of
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field samples.
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Abbreviations Used
296
HLB
Citrus huanglongbing
Las
Candidatus Liberibacter asiaticus
PCR
Polymerase chain reaction
LAMP
Loop-mediated isothermal amplification
RPA
Recombinase polymerase amplification
NaOH
Sodium hydroxide
LFD
Lateral flow dipstick
TE
Tris-EDTA
LOD
Limit-of-detection
Funding
297
This work was supported by National Natural Science Foundation of China
298
(31571918) and Hong Kong, Macao and Taiwan Scientific and Technological
299
Cooperation Projects (2015DFT30150).
300
Supporting Information
301
Brief statement in nonsentence format listing the contents of the material supplied
302
as Supporting Information.
303
References
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(1)
Freitas, D. d. S.; Carlos, E. F.; Gil, M. r. C. S. d. S.; Vieira, L. G. E.; Alcantara, G. B., 15
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NMR-Based Metabolomic analysis of Huanglongbing-asymptomatic and-symptomatic citrus trees. J.
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(9) Wang, Z.; Yin, Y.; Hu, H.; Yuan, Q.; Peng, G.; Xia, Y., Development and application of molecular‐
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based diagnosis for ‘Candidatus Liberibacter asiaticus’, the causal pathogen of citrus huanglongbing.
Agric. Food Chem. 2015, 63, 7582-7588. (2) Bové, J. M., Huanglongbing: A destructive, newly-emerging, century-old disease of citrus. J. Plant Pathol. 2006, 88, 7-37. (3) Graca, J. V., Citrus Greening Disease. Annu. Rev. Phytopathology. 1991, 29, 109-136. (4) Chiyaka, C.; Singer, B. H.; Halbert, S. E.; Morris, J. G.; van Bruggen, A. H., Modeling huanglongbing transmission within a citrus tree. P. Natl. Acad. Sci.USA.2012, 109, 12213-12218. (5) Gottwald, T.; Aubert, B.; Zhao, X.-Y., Preliminary analysis of Citrus greening(huanglungbin) epidemics in the People's Republic of China and French Reunion Island. Phytopathology 1989, 79, 687-693. (6) Kim, J. S.; Wang, N., Characterization of copy numbers of 16S rDNA and 16S rRNA of Candidatus Liberibacter asiaticus and the implication in detection in planta using quantitative PCR. Bmc Res. Notes 2009, 2, 37. (7) Fujikawa, T.; Miyata, S.-I.; Iwanami, T., Convenient detection of the citrus greening (huanglongbing) bacterium ‘Candidatus Liberibacter asiaticus’ by direct PCR from the midrib extract. PloS one 2013, 8, e57011. (8) Li, W.; Levy, L.; Hartung, J. S., Quantitative distribution of ‘Candidatus Liberibacter asiaticus’ in citrus plants with citrus huanglongbing. Phytopathology 2009, 99, 139-144.
Plant Pathol. 2006, 55, 630-638. (10) Ananthakrishnan, G.; Choudhary, N.; Roy, A.; Sengoda, V.; Postnikova, E.; Hartung, J.; Stone, A.; Damsteegt, V.; Schneider, W.; Munyaneza, J., Development of primers and probes for genus and species specific detection of ‘Candidatus Liberibacter species’ by real-time PCR. Plant Dis. 2013, 97, 1235-1243. (11) Jagoueix, S.; Bové, J. M.; Garnier, M., PCR detection of the two «Candidatus» liberobacter species associated with greening disease of citrus. Mol.Cell.Probe. 1996, 10, 43-50. (12) Coy, M.; Hoffmann, M.; Gibbard, H. K.; Kuhns, E.; Pelz-Stelinski, K.; Stelinski, L., Nested-quantitative PCR approach with improved sensitivity for the detection of low titer levels of Candidatus Liberibacter asiaticus in the Asian citrus psyllid, Diaphorina citri Kuwayama. J. microbiol. Meth. 2014, 102, 15-22. (13) Okuda, M.; Matsumoto, M.; Tanaka, Y.; Subandiyah, S.; Iwanami, T., Characterization of the tufB-secE-nusG-rplKAJL-rpoB gene cluster of the citrus greening organism and detection by loop-mediated isothermal amplification. Plant Dis. 2007, 89, 705-711. (14) Li, W.; Hartung, J. S.; Levy, L., Evaluation of DNA amplification methods for improved detection of “Candidatus Liberibacter species” associated with citrus huanglongbing. Plant Dis. 2007, 91, 51-58. (15) Qian, W.; Meng, Y.; Lu, Y.; Wu, C.; Wang, R.; Wang, L.; Qian, C.; Ye, Z.; Wu, J.; Ying, Y., A rapid, sensitive and carryover contamination-free loop-mediated isothermal amplification-coupled visual detection method for 'Candidatus Liberibacter asiaticus'. J. Agric. Food Chem. 2017, 65, 8302-8310. (16) Wu, X.; Meng, C.; Wang, G.; Liu, Y.; Zhang, X.; Yi, K.; Peng, J., Rapid and quantitative detection of citrus huanglongbing bacterium ‘Candidatus Liberibacter asiaticus’ by real-time fluorescent loop-mediated isothermal amplification assay in China. Physiol. Mol. Plant P. 2016, 94, 1-7. (17) Keremane, M. L.; Ramadugu, C.; Rod.riguez, E.; Kubota, R.; Shibata, S.; Hall, D. G.; Roose, M. 16
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L.; Jenkins, D.; Lee, R. F., A rapid field detection system for citrus huanglongbing associated ‘Candidatus Liberibacter asiaticus’ from the psyllid vector, Diaphorina citri Kuwayama and its implications in disease management. Crop Prot. 2015, 68, 41-48. (18) Rigano, L. A.; Malamud, F.; Orce, I. G.; Filippone, M. P.; Marano, M. R.; Do Amaral, A. M.; Castagnaro, A. P.; Vojnov, A. A., Rapid and sensitive detection of Candidatus Liberibacter asiaticus by loop mediated isothermal amplification combined with a lateral flow dipstick. BMC Microbiol. 2014, 14, 86. (19) Piepenburg, O.; Williams, C. H.; Stemple, D. L.; Armes, N. A., DNA Detection Using Recombination Proteins. Plos Bio. 2006, 4, 1115-1121. (20) Wang, J.; Wang, J.; Liu, L.; Li, R.; Yuan, W., Rapid detection of Porcine circovirus 2 by recombinase polymerase amplification. Arch. Virol. 2016, 161, 1015-1018. (21) Chandu, D.; Paul, S.; Parker, M.; Dudin, Y.; Kingsitzes, J.; Perez, T.; Mittanck, D. W.; Shah, M.; Glenn, K. C.; Piepenburg, O., Development of a Rapid Point-of-Use DNA Test for the Screening of Genuity® Roundup Ready 2 Yield® Soybean in Seed Samples. Biomed Res. Int. 2016, 2016, 3145921. (22) Xu, C., Event-specific Real-time RPA Detection of Transgenic Rice Kefeng 6. Mol. Plant Bre. 2014, 05, 0001 (23) Boyle, D. S.; Mcnerney, R.; Teng, L. H.; Leader, B. T.; Pérezosorio, A. C.; Meyer, J. C.; O'Sullivan, D. M.; Brooks, D. G.; Piepenburg, O.; Forrest, M. S., Rapid Detection of Mycobacterium tuberculosis by Recombinase Polymerase Amplification. PLoS ONE,9,8(2014-8-13) 2014, 9, e103091. (24) Londoño, M. A.; Harmon, C. L.; Polston, J. E., Evaluation of recombinase polymerase amplification for detection of begomoviruses by plant diagnostic clinics. Virol. J. 2016, 13(1), 1-9. (25) Babu, B.; Washburn, B. K.; Miller, S. H.; et al., A rapid assay for detection of Rose rosette virus, using reverse transcription-recombinase polymerase amplification using multiple gene targets. J. Virol. Methods, 2016, 240:78. (26) Babu, B.; Washburn, B. K.; Ertek T. S.; et al., A field based detection method for Rose rosette virus, using isothermal probe-based Reverse transcription-recombinase polymerase amplification assay. J. Virol. Methods, 2017, 247:81-90. (27) Kapoor, R.; Srivastava, N.; Kumar S.; Saritha, R. K.; Sharma, S. K.; Jain R. K.; et al., Development of a recombinase polymerase amplification assay for the diagnosis of banana bunchy top virus in different banana cultivars. Arch. Virol, 2017, 162(9), 2791-2796. (28) Russell, P. F.; Mcowen, N.; Bohannon S.; Amato M. A.; Bohannon R., Rapid on-site detection of the huanglongbing/citrus greening causal agent 'liberibacter asiaticus' by amplifyrp, a novel rapid isothermal nucleic acid amplification platform. BBA-Mol. Cell Res. 2008, 1783(11), 2100-2107. (29) Crannell, Z. A.; Cabada, M. M.; Castellanos-Gonzalez, A.; Irani, A.; White, A. C.; Richards-Kortum, R., Recombinase Polymerase Amplification-Based Assay to Diagnose Giardia in Stool Samples. Am J. Trop. Med. Hyg. 2015, 92, 583-587. (30) ZA, C.; A, C.-G.; A, I.; B, R.; AC, W.; R, R.-K., Nucleic acid test to diagnose cryptosporidiosis: lab assessment in animal and patient specimens. Anal. Chem. 2014, 86, 2565-71. (31) Hsieh, K.; Mage, P. L.; Csordas, A. T.; Eisenstein, M.; Soh, H. T., Simultaneous elimination of carryover contamination and detection of DNA with uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification (UDG-LAMP). Chem.Commun. 2014, 50, 3747. (32) Wang, R.; Xiao, X.; Chen, Y.; Wu, J.; Qian, W.; Wang, L.; Liu, Y.; Ji, F.; Wu, J., A loop-mediated, isothermal amplification-based method for visual detection of Vibrio parahaemolyticus within only 1 h, from shrimp sampling to results. Anal. Methods 2017, 9, 1695-1701. 17
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(33) Lorraine, L.; Dara, L.; Singhal, M. C.; Jason, C.; Jered, S.; Paul, L.; Anthony, T.; Olaf, P.; Mathew,
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Figure captions
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Figure 1 Schematic of RPA-based visual detection method for field detection of Las
406
from leave samples within 15 min. a piece of midrib tissue (around 0.6 cm×2 cm, 50
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mg) of citrus leaf was ground in 500 µL of 0.5M NaOH for 1 min. 2 µL of 50-fold
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diluted rough cell lysate was amplified by RPA at 37 °C for 10 min. After the
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amplification, turn the cartridge upside down and shake intensively to mix the mixture,
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fluorescent dye and sterilize water thoroughly. The presence or absence of
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fluorescence was examined by visualization under UV light (354 nm) using a
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mini-UV torch.
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Figure 2 Visualization of RPA products by agarose gel electrophoresis. a)
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Temperature optimization for RPA amplification. Lanes 1-6, positive samples
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amplified by RPA at 37, 38, 39, 40, 41 and 42 °C, respectively. Lanes 7-12, negative
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samples amplified by RPA at 37, 38, 39, 40, 41 and 42 °C, respectively. b) Evaluation
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of different lysis time of NaOH-based DNA extraction for RPA amplification. Lane
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1-2 were standard DNA extraction using a commercial kit. Lane 3-4, 5-6, 7-8, 9-10
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were samples treated with 0.5 M NaOH for 1 min of grinding and 0, 2, 5 and 9 min of
P.; Robert, W., Non-Instrumented Incubation of a Recombinase Polymerase Amplification Assay for the Rapid and Sensitive Detection of Proviral HIV-1 DNA. Plos One 2014, 9, e108189. (34) Wang, R.; Zhang F.; Wang L.; Qian W.; Qian C.; Wu J Qian W.; Ying Y., Instant, Visual, and Instrument-Free Method for On-Site Screening of GTS 40-3-2 Soybean Based on Body-Heat Triggered Recombinase Polymerase Amplification. Anal. chem. 2017, 89, 4413-4418. (35) Okuda, M.; Matsumoto, M.; Tanaka, Y.; Subandiyah, S.; Iwanami, T., Characterization of the tuf B-sec E-nus G-rpl KAJL-rpo B gene cluster of the citrus greening organism and detection by loop-mediated isothermal amplification. Plant Dis. 2005, 89, 705-711. (36) Wang L.; Qian C.; Qian W.; Wang R.; Wu J.; Ying Y., A Highly Specific Strategy for In Suit Detection of DNA with Nicking Enzyme Assisted Amplification and Lateral Flow. Sensor. Actuat. B-Chem. 2017, 253, 258-265.
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standing, respectively. The odd numbered wells contain amplicons from Las-free leaf
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samples, and even numbered lanes contain amplicons from Las positive leaf samples.
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Figure 3 Effect of reaction time, primer concentration, and amplicon dilution on
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visual detection by RPA. a) Visual detection of RPA amplicons amplified from RPA
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reactions performed for 8, 10 and 15 min, respectively, with varying the primer
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concentration (0.2, 0.36 and 0.78 µM). b) Visual detection of RPA amplicons
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amplified from PRA reaction mixture with 0.36 µM of primers for 10 min after 2-fold,
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5-fold and 10-fold dilution.
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Figure 4 Enclosed disposable cartridge integrated with RPA amplification and visual
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detection. The numbers in the figure display the size of the cartridge (mm). RPA
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reaction mixture, fluorescent dye, and sterile water are pre-added into the three PCR
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tubes, respectively. Then PCR tubes are connected to the cartridge tightly and the
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cartridge is directly incubated at 37 °C for RPA. After amplification, turn the cartridge
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upside down and shake intensively to mix all the agents thoroughly for visual
434
detection.
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Figure 5 Comparison of sensitivity of RPA and PCR. a) Gel electrophoresis images of
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RPA. b) Visual detection of RPA. c) Gel electrophoresis images of PCR. d) Real time
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amplification graph of PCR. In each figure, from left to right, the initial templates
438
were 10-fold serially diluted from 1.0 ×102 ng/µL to 1.0×10-1 ng/µL, with no template
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added as negative control.
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Figure 6 12 positive leaf samples in 150 blind field samples and their detection
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results by real-time PCR and RPA-based visual detection method. Real time 19
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amplification graph, the RPA reaction tube, and a representative leaf from
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corresponding sample are shown in each panel.
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Table 1 Sequences of RPA primers used in this study. The primer length, position of the primers in the reference genome of Las (NC_012985.3), and amplicon sizes are shown. Primer
Sequence (5’-3’)
Position
FI
CCTAGATTCATTGCTATCTCAACTGTTTCA
30
RI
GGGTATAAGTGTGGTGGATTAATATGGTGT
30
F II
AATGCCTAGATTCATTGCTATCTCAACTGTTT
32
R II
ATGGGTATAAGTGTGGTGGATTAATATGGTGT
32
F III
CTAGCCGTAGCACGCTCTTTAAGCATAA
28
R III
TGTTCAATGGGTATAAGTGTGGTGGATT
28
Name
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Table 2 Detection results of 20 asymptomatic and 20 symptomatic samples from known infected trees and 150 field samples of unknown status. Samples
Real-time PCR
RPA-based visual detection
Positive
Negative
Positive rate
Positive
Negative
Positive rate
20 symptomatic samples
20
0
100%
20
0
100%
20 asymptomatic samples
20
0
100%
20
0
100%
150 blind field samples
12
138
8.57%
12
138
8.57%
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