Field Detection of Citrus Huanglongbing ... - ACS Publications

May 21, 2018 - Zhejiang Plant Protection and Quarantine Bureau, Hangzhou 310020, ... effective approaches to control the disease once the plants have...
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Agricultural and Environmental Chemistry

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

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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*

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a

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Hangzhou 310058, China.

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b

College of Biosystems Engineering and Food Science, Zhejiang University,

Zhejiang Plant Protection and Quarantine Bureau, Hangzhou 310020, China.

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Correspondence

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(J. Wu) E-mail: [email protected]. Tel: 0086-571-88982180

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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

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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

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extraction by 50-fold diluting after 1 min of grinding in 0.5 M sodium hydroxide and

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visual detection via fluorescent DNA dye (positive samples display obvious green

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fluorescence while negative samples remain colorless), the whole detection process

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can be accomplished within 15 min. The sensitivity and specificity of this RPA-based

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method were evaluated, which were proven to be equal to real-time PCR. The

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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

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americanus(Lam), of which Las is the most prevalent species and has been spreading

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worldwide.2, 3 Infected citrus may exhibit long latency period and will be destroyed or

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become unproductive in 5−8 years following infection.4, 5 However, there are no

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effective approaches to control the disease once the plants have been infected.6 To

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prevent it from spreading, the current management strategy of HLB is to remove

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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

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management practices in a timely fashion.

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Nowadays, polymerase chain reaction (PCR) is widely used to detect Las because

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of its high sensitivity and reliability.7-12 Nevertheless, it is impractical for field

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detection due to the long-running operation, need for expertise and use of expensive

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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

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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

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polymerase amplification (RPA), has attracted extensive attention since it is a rapid,

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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

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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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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

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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

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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

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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

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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

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(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

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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

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Primer sets, OI1 (5’-GCGCGTATTTTATACGAGCGGCA-3’) and OI2c

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(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

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70°C for 1 min. For real-time PCR, fluorescence signal was collected at the end of

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each cycle on the QuantStudioTM3 Real Time PCR System. Time consumed was

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defined as cycle threshold (Ct) value. For conventional PCR, 2 µL of PCR products

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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

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100 ng/µL to eliminate the effect of host DNA. RPA amplification was performed at

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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

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other citrus samples infected by other bacterial or fungal plant pathogens, including

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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

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citrus leaf was used for DNA extraction with 500 µL of 0.5M NaOH for 1 min of

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grinding. A volume of 2 µL of 50-fold diluted rough cell lysate was amplified by RPA

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at 37 °C on a heating block for 10 min. After the amplification, the mixture was 2-fold

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diluted and mixed with 1 µL of SYBR Green I (200 µg/mL). The presence or absence

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of fluorescence was examined by visualization under UV light (354 nm) using a

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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

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symptomatic citrus trees and 150 filed leaf samples were detected to evaluate the

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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

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for the success of RPA. To ensure the successful RPA amplification, three pairs of

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RPA primers targeting the conserved sequence of tufB-secE-nusG-rplKAJL-rpoB

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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

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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

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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

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incubated at 37, 38, 39, 40, 41, and 42 °C since the working temperature of the

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recombinase and polymerase is between 37-42 °C. As shown in Figure 2a, visible

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target bands at expected size range were obtained by RPA at all of these six different

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temperatures. The reactions at 37 °C and 38 °C had better performance compared with

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other temperatures. As a result, 37 °C was applied for RPA amplification.

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Crude extraction of DNA for RPA

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Purification steps of DNA extraction were omitted in this paper. About 50 mg of

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leaf midrib tissue was ground in 500 µL of 0.5 M NaOH for 1 min. Then 2 µL of

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supernatant was collected after 0, 2, 5, and 9 min of standing and diluted in 100 µL of

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TE. 2 µL of the dilute extract was used for RPA reaction. In order to ensure the quality

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of crude-extracted DNA and make the extraction time as short as possible, different

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lysis time were studied. The results show that no incubation was necessary (Figure

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2b). In addition, the amplification appeared to have decreased with increased

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incubation time. This phenomenon could be explained that NaOH solution could

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cause DNA degradation. And more compound RPA inhibitors would rise from the

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cytolysis with the process of NaOH lysis. Since the amplification ability of 1 min of

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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.

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Visual detection of RPA amplicons

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Here, we employed florescent dye, SYBR Green I, for visual detection of Las by

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RPA for the first time. After RPA amplification, 1 µL of SYBR Green I (200 µg/ml)

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was added to reaction mixture. SYBR Green I would intercalate into double-stranded

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DNA and generate fluorescence. Then with the help of a mini-UV torch, the presence

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or absence of fluorescence was recorded. It was assumed that the amplicons of

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positive samples would emit green fluorescent which could be easily distinguished

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from colorless negative samples by naked eyes. However, it is reported that RPA has

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high background signal so that negative samples may emit fluorescence under some

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circumstances.19

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To eliminate background signal and optimize the reaction condition for visual

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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,

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with the amplification duration becoming longer and the concentration of primers

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getting higher, the background signal of negative samples was enhanced. Especially,

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both positive and negative samples could emit obvious fluorescence after 15 min

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amplification regardless of the primers concentration and all negative samples with

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0.78 µM of primers had high background even if the reaction time was only 8 min

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(Figure 3a). This phenomenon may be related to the relatively high concentration and

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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

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positive samples and low background signal for negative sample, the RPA reaction

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was determined to be performed for 10 min with 0.36 µM primers. To further

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eliminate the background signal and get the most distinct differences between positive

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and negative samples, we compared the difference among 2-fold, 5-fold and 10-fold

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dilution of the products. Figure 3b demonstrates that 2-fold diluted RPA amplicons

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were enough for visually distinguish positive and negative samples while the

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fluorescence intensity of positive samples got weak when 5 or 10-fold diluted.

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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

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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.

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However, as reported, the requirement of uncapping operation presents a high risk

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of amplicon contamination for DNA amplification.31 Thus, a convenient, portable and

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disposable cartridge developed by our group was introduced for visual detection of

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RPA products (Figure 4). (This portable cartridge also has another form for lateral

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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

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uncapping operation and prevent cross-contamination.

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Sensitivity and Specificity of RPA-based visual detection method

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To investigate the sensitivity of RPA-based visual detection method, a set of

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10-fold gradient dilutions of template DNA extracted from symptomatic leaves were

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amplified by RPA. PCR detection using primer sets, OI1 and OI2c, was used as

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control. As shown in Figure 5, the limit-of-detection (LOD) of gel electrophoresis

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images of RPA, visual detection of RPA, real-time PCR and gel electrophoresis

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images of PCR was around 1.0 ×10-1 ng/µL, suggesting that the sensitivity of this

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RPA-based visual detection method was equal to the conventional PCR method, while

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it took less time and easier to operate.

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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

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RPA-based visual detection method. (Figure S2).

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Detection of Las from field samples by RPA

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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

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consistent that all symptomatic and asymptomatic samples were tested positive,

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indicating that this proposed RPA-based visual detection method could be an

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alternative for real-time PCR (Table 1). Subsequently, a total of 150 blind

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field-collected samples from 9 different regions in Zhejiang Province, China were

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evaluated by the proposed RPA-based visual detection method in comparison with

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real-time PCR. As shown in Table 1, among 150 blind samples, the positive rates by

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these two methods were both 8.57 %. All samples that were found to be positive by

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RPA-based visual detection method were also positive by real-time PCR (Figure 6).

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The results illustrated that this RPA-based visual detection method also had high

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reliability for natural samples detection.

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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.

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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

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This work was supported by National Natural Science Foundation of China

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(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.

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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

407

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

436

RPA. b) Visual detection of RPA. c) Gel electrophoresis images of PCR. d) Real time

437

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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178

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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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