A rapid, sensitive and carryover contamination-free loop-mediated

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A rapid, sensitive and carryover contamination-free loop-mediated isothermal amplification-coupled visual detection method for ‘Candidatus Liberibacter asiaticus’ Wenjuan Qian, Youqing Meng, Ying Lu, Cui Wu, Rui Wang, Liu Wang, Cheng Qian, Zunzhong Ye, Jian Wu, and Yibin Ying J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03490 • Publication Date (Web): 31 Aug 2017 Downloaded from http://pubs.acs.org on September 1, 2017

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Journal of Agricultural and Food Chemistry

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A rapid, sensitive and carryover contamination-free loop-mediated isothermal

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amplification-coupled visual detection method for ‘Candidatus Liberibacter

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asiaticus’

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Wenjuan Qian ab, Youqing Meng c, Ying Lu c, Cui Wu ab, Rui Wang ab, Liu Wang ab,

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Cheng Qian ab, Zunzhong Ye ab *, Jian Wu ab and Yibin Ying ad

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a

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Hangzhou 310058, China.

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b

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Ministry of Agriculture, Hangzhou 310058, China.

College of Biosystems Engineering and Food Science, Zhejiang University,

Key Laboratory of On Site Processing Equipment for Agricultural Products,

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c

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

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d

Zhejiang A&F University, Hangzhou, Zhejiang 311300, China.

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Correspondence

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(Z. Ye) E-mail: [email protected]. Tel: 0086-571-88982282

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Fax: 0086-571-88982282 College of Biosystems Engineering and Food Science,

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Zhejiang University, Hangzhou 310058, China.

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ACS Paragon Plus Environment


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ABSTRACT:

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Huanglongbing is a devastating citrus disease and ‘Candidatus Liberibacter

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asiaticus’ is the most prevalent Huanglongbing-associated bacterium. Its field

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detection remains challenging. In this work, a visual, rapid, sensitive and carryover

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contamination-free method was developed for field detection of Las. Leaf samples

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were treated with 500 µL 0.5 M sodium hydroxide solution for 3 min and 50-fold

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dilutions were directly amplified by loop-mediated isothermal amplification. Then, a

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novel SYTO 9 based visual detection method was used to evaluate amplification

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results without uncapping operation. Negative samples remained colorless while

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positive samples generated obvious green fluorescence which could be easily

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distinguished by naked eyes with a mini fluorescent-emission cartridge developed

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originally. The proposed detection method could be accomplished within 40 min and

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is about 100 times more sensitive than conventional TaqMan PCR. The reliability of

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this method was also verified by analyzing practical samples.

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Keywords: Huanglongbing, Candidatus Liberibacter asiaticus, field detection, crude

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DNA extraction, isothermal amplification, visual detection

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Introduction

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Citrus huanglongbing (HLB), also known as citrus greening disease, is one of the

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most destructive diseases of citrus worldwide.1 Three fastidious gram negative

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bacteria have been associated with citrus HLB: ‘Candidatus Liberibacter asiaticus’

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(Las), ‘Candidatus Liberibacter americanus’ (Lam) and ‘Candidatus Liberibacter

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africanus’ (Laf). Among them, Las is the most prevalent HLB-associated bacterium in

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Asia as well as in the western countries.2 Infected citrus can present a long latency

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period and will be destroyed or become unproductive in 5 to 8 years following

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infection.3, 4 However, there is no effective cure of the disease, and hence infected

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trees have to be removed immediately at early stage of the infection to prevent the

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spread of the disease.5 Thus, accurate and convenient detection methods play an

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important role in reducing the incidence of HLB. Some detection methods such as

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serologic assays, electron microscopy and biological assays have been developed, but

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the detection results are inconsistent due to the low concentration and uneven

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distribution of pathogens in infected citrus.6-11 Moreover, many of these methods have

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drawbacks of being time-consuming and requiring complex facilities which limit their

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application in field and places where complex facilities may not be available. Hence,

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it is urgent to develop a rapid, affordable, simple and reliable method for field

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detection of Las in a timely fashion.

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Currently, polymerase chain reaction (PCR) is widely used to detect Las in

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infected plants because of its high sensitivity and reproducibility.12-17 However, it is

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not applicable for field detection due to its reliance on non-portable thermal cycler 3



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and other basic apparatus for molecular biological experiments. The emergence of

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isothermal amplification like loop-mediated isothermal amplification (LAMP) has

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overcome constraints mentioned above since they can be conducted at a constant

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temperature.

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LAMP assay was first described by Notomi et al as a simple, rapid, sensitive and

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cost-effective technique for detection of specific DNA.18 It is conducted with 4~6

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primers recognizing 6~8 regions of the target DNA which confers extremely high

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specificity.

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between 60 to 65 °C in a simple and inexpensive device like a water bath or a heating

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block.

18,19

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has

already

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tufB-secE-nusG-rplKAJL-rpoB gene cluster.20,

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abandoned thermal cyclers, tedious gel electrophoresis analysis or optical instrument

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based fluorescent detection is unavoidable which greatly limits its field application.

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Visual detection of LAMP products is a promising approach for field detection since it

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is rapid, direct and simple. Aravind Ravindran et al have been reported to detect Las

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visually via direct observation of turbidity in LAMP reaction tubes22 but the turbidity

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produced by LAMP is difficult to identify by naked eyes when the amount of

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amplicons is insufficient23. They also enhanced visual detection of Las by adding

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manganese-quenched calcein to generate fluorescence upon amplification. However,

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the fluorescence difference between positive and sensitive samples was difficult to

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distinguish under normal light and an additional UV light was needed. Also,

18,19

In contrast to PCR, LAMP can be carried out at a single temperature

LAMP assay for the detection of Las in infected plants or vector insects been

reported

based

on

conserved 21

sequence

of

the

Although this method has

4


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manganese-quenched calcein detection is reported to suffer from inhibition by

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manganese to some extent.

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visual detection of LAMP products by employing intercalating dyes such as SYBR

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Green or SYTO dyes26-28 to observe a color change but the requirement of uncapping

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operation presents a high risk of carryover contamination.29, 30 Xuhui Wu et al have

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developed a SYBR Green I based LAMP visual detection method of Las without

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uncapping operation by adding SYBR Green I to the inside of the lid prior to

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amplification.31 However, an additional centrifugation operation is needed to mix the

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pre-added SYBR Green I to the reaction solution. What’s more, the color change

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resulting from intercalating dyes may be subtle for some users because of the high

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background signal and the adjacent colorimetric change.32-34 In addition, Rigano et al

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have developed a visual LAMP method combined with a lateral flow dipstick assay to

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detect a hypothetical protein region from Las.35 This method still presents a high risk

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of carryover contamination due to the uncapping operation.

24, 25

Some researchers have attempted to accomplish

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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 filed detection

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due to the cumbersome steps and long operation duration.36 Fortunately, LAMP shows

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high tolerance to interferences from biological contaminants37 and, hence, simplified

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sample preparation. So far, some DNA crude extraction methods have already been

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reported for LAMP detection of Las. 2, 35 A centrifuge is still necessary in these boiling

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methods.

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More convenient and simpler sample preparation methods are still needed.

In this content, we have developed a novel LAMP-coupled visual detection 5



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method which can overcome the weaknesses of methods mentioned above for rapid

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field detection of HLB associated with Las. The whole detection process (from leaf

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sampling up to result) of this SYTO 9 based method could be completed in 40

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minutes. This method is characterized by the novelties and advantages as following: (i)

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A very short (∼3 min) DNA extraction step was performed on leaf samples by simple

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treatment with sodium hydroxide (NaOH) solution without heating treatment or

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centrifugation; (ii) The novel visual detection method of LAMP products just needed

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low concentration fluorescent dye and SYTO 9 was added before amplification,

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preventing the risk of carryover contamination; (iii) Instead of former adjacent

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colorimetric change, positive samples emitted green fluorescent which could be easily

108

distinguished from colorless negative samples; (iv) No longer needing thermal cycler

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and other expensive or non-portable apparatuses, a portable heater and a mini

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fluorescent-emission cartridge (460 nm) invented by our Lab are enough for visual

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assay of LAMP.

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Materials and methods

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Plant material

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Asymptomatic and symptomatic disease leaf samples of infected citrus trees and

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352 blind citrus leaf samples obtained from 9 different regions in Zhejiang Province

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were collected by Zhejiang Plant protection and quarantine Bureau. Healthy citrus

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leaves as negative controls were bought from local supermarket.

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NaOH-based DNA crude extraction

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A piece of midrib tissues (around 0.6×2 cm, 50 mg) of a leaf sample was cut up 6


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and immerged in 500 µL 0.5 M NaOH in a mortar. After grinding for 1 min and

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standing for 9 min at room temperature, the supernatant of slurries was serially diluted

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(1:1, 1:5, 1:50, and 1:500) with Tris-EDTA (TE) buffer. 2.5 µL of the rude cell lysate

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was directly used as template for LAMP assay. The durations of lysis were studied by

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crushing the leaves in 0.5 M NaOH for 1 min and then letting the slurry stand for 0, 2,

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5 and 9 min, respectively. For a comparative test, standard DNA extraction was

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conducted using Plant Genomic DNA Kit (Tiangen Biotech Co., Ltd, Beijing, China).

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Primer designed for LAMP

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A DNA fragment of Las encompassing a bacteriophage-type DNA polymerase 2, 38

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region (GenBank NO. M94302) was selected as target sequence.

A set of six

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LAMP primers including a pair of inner primer (FIP and BIP), two out primers (F3

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and B3) and two loop primers (LF and LB) were designed using Primer Explorer ver.

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4 (http://primerexplorer.jp/ elamp4.0.0/index.html). The specificity of primers was

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insured by Blast analysis and sequences are displayed in Figure 1.

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LAMP Assay

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LAMP was performed in a total of 25 µL reaction mixture containing 1.6 µM of

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FIP and BIP, 0.2 µM of F3 and B3, 0.4 µM of LF and LB, 16 U of Bst DNA

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polymerase large fragment (New England Biolabs Inc., Ipswich, MA), 1.4 mM of

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dNTPs, 2.5 µL of 10 × Thermopol Reaction buffer (New England Biolabs Inc.,

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Ipswich, MA), 6.0 mM of MgSO4, 0.8 M of Betaine, 4µM of SYTO 9 (Thermo

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Fisher Scientific Inc., Waltham, MA USA) and 2.5 µL of target DNA. DNA extracted

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from healthy citrus leaves was used as negative controls. For LAMP-coupled visual 7



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detection, the mixture in each tube was incubated at 65 °C for 35 min on a

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conventional heating block (Sangon Biotech Co., Ltd., Shanghai, China) after

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layering with 10 µL of mineral oil to avoid evaporation. For real-time LAMP, the

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QuantStudioTM3 Real Time PCR System (Thermo Fisher Scientific Inc., Waltham,

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MA USA) was used to collect fluorescence signal every 60 s. Time threshold (Tt)

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value means the time that fluorescence reached the threshold.

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Real-time TaqMan PCR.

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Primer-probe sets, HLBaspr, HLBafpr and HLBampr, employed for real-time

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TaqMan PCR were reported in previous paper by Li W. et al10 and synthesized by

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Sangon Biotech (Shanghai, China). The 25 µL total reaction volumed 12.5 µL

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FastStart Essential Probes Master reaction mix (Roche Diagnostics Ltd., Switzerland),

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250 nM of each primer, 150 nM of target probe, 2.5 µL template and 8.7 µL Sterile

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water. The standard amplification protocol was 98 °C for 20 s followed by 45 cycles

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at 95 °C for 20 s and 58 °C for 40 s. Fluorescence was collected on the

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QuantStudioTM3 Real Time PCR System at the end of each cycle. Time consumed

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was defined as cycle number while Cycle threshold (Ct) value means the cycle

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number that fluorescence reached the threshold.

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Sensitivity of Real-time TaqMan PCR and LAMP

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To compare the sensitivity of real-time TaqMan PCR and LAMP, amplification

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was performed on a dilution series of DNA extracted from symptomatic leaves by

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Plant Genomic DNA Kit (Tiangen Biotech Co., Ltd, Beijing, China). DNA template

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was adjusted to the initial concentration of 100 ng/µL and diluted in 1:10 serials. 8


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Before DNA amplification, the diluents were mixed with host DNA extracted from

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Las-free citrus leaves to ensure that the total DNA concentration was 100 ng/µL to

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eliminate the effect of host DNA. For each reaction, 2.5 µL DNA templates were

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added for real-time TaqMan PCR and LAMP.

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Specificity of Las-LAMP

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In order to evaluate the specificity of Las-LAMP assay, several other main citrus

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diseases infected by bacterial or fungal plant pathogens, including citrus canker, citrus

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anthracnose, citrus scab, citrus black spot and citrus brown spot were tested by

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Las-LAMP primer. DNA from Las-free citrus samples was used as negative control.

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Visual detection of LAMP products

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4 µM of SYTO 9 was added into the reaction mixture before amplification. Then,

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with the help of mini fluorescent-emission cartridge (460 nm) developed by our lab,

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positive amplifications would emit green fluorescence (520 nm) while negative

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amplifications generated no fluorescence. To figure out the main reason for high

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background signal of negative samples, we detected the fluorescence of reaction

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mixtures at room temperature before LAMP amplification. Furthermore, to ensure the

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detection specificity and eliminate false-positive results, reaction tubes were

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fore-heated under 45 °C, 65 °C, 75 °C and 85 °C respectively for 2 min before visual

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detection.

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Detection of practical samples

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For LAMP assay, DNA was extracted from a piece of midrib tissues (around

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0.6×2 cm, 50 mg) of a citrus leaf with 500 µL 0.5 M NaOH for 3 min (including 1 9



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min of grinding and 2 min standing). Thereafter, the solution was 50-fold diluted in

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TE buffer and then amplified by LAMP at 65 °C on a heating block for 35 min. After

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the amplification, the mixture was immediately visual judged via the mini

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fluorescent-emission cartridge (Scheme 1).

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To evaluate the feasibility of visual detection of Las, a total of 60 samples with 30

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asymptomatic and 30 symptomatic plant materials from different infected trees were

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divided into 10 groups randomly and tested by LAMP-coupled visual detection

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method. Real-time TaqMan PCR and real-time LAMP were used as control.

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Finally, 352 blind field samples collected from 9 different regions in Zhejiang

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Province were tested to evaluate the HLB infection condition of Zhejiang Province.

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Results and discussions

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NaOH-based DNA crude extraction

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In order to simplify operation processes and reduce sampling time, DNA was

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extracted without purification. Different lysis time and dilution ratios were compared

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to evaluate the proposed NaOH-based DNA extraction method. As shown in Table 1,

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no positive signal was obtained when the crude cell lysates were directly used without

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dilution no matter how long the lysis time was. This phenomenon could be related to

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the presence of LAMP inhibitors which were introduced during the lysis process and

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compromised the analysis results such as carryover NaOH or the compound arising

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from the cytolysis. However, with appropriate dilution, the effect of inhibitor could be

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eliminated to some extent. According to the Tt values in Table 1, 5-fold dilution still

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showed negative impact on LAMP amplification while the result of 500-fold dilution 10


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was inferior to that of 50-fold dilution due to the low DNA concentration. Therefore,

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we chose 50-fold dilution in our further study.

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To shorten the whole detection time, different durations of lysis were also studied.

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Table 1 illustrated that there was no significant difference among 3 min, 6 min and 10

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min incubation. And the amplification ability of the crude extracted DNA had no

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significant difference with that of standard DNA extraction by commercial kit. For

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field detection, a method which requires less time is more efficient and convenient.

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Therefore, 3 min was applied for lysis.

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In conclusion, DNA crude extraction was performed with 500 µL 0.5 M NaOH

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for 3 min (including 1 min of grinding and 2 min standing) and 50-fold dilutions were

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applied for LAMP amplification and visual detection in the following assay.

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Sensitivity and Specificity of LAMP

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To compare the sensitivity of Las-LAMP and real-time TaqMan PCR, serial

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dilutions of purified DNA ranging from 1.0 ×102 ng/µL to 1.0×10-5 ng/µL extracted

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from a Las positive plant were used. As shown in Figure 2, the limit-of-detection

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(LOD) of real-time TaqMan PCR was 1.0 ×10-2 ng/µL while it was 1.0 ×10-4 ng/µL

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for real-time LAMP, suggesting that LAMP was potentially about 100 times more

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sensitive than real-time TaqMan PCR. LAMP products were also visually detected by

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mini fluorescent-emission cartridge and the results were consistent to real-time LAMP

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that products of the first 7 dilutions emitted an obvious green fluorescence while the

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others showed no fluorescence (Figure 2). The intensity of fluorescence emitted by

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products amplified from different concentration of initial target templates showed no 11



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significant difference and all could be easily distinguished from negative samples by

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naked eyes. This may be due to the substantial and almost the same amount of LAMP

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products according to the plateau of their amplification curves of real-time LAMP.

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The specificity of LAMP primers was tested on several other main citrus diseases

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infected by bacterial or fungal plant pathogens, including citrus canker, citrus

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anthracnose, citrus scab, citrus black spot and citrus brown spot. No positive result

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was observed except the Las-infected sample, indicating a high level of detection

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specificity of LAMP assay. (ESI, Table S1).

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Visual detection of LAMP products

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SYTO 9 fluorescent dye has already been reported to be used for visual detection

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of LAMP products.28 However, the detection results may be subtle for some users

241

because of the adjacent colorimetric change (from orange to green). Since high

242

concentration fluorescent dye has a negative impact on LAMP amplification, SYTO 9

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is added after amplification by uncapping operation which will cause carryover

244

contamination. In this paper, we developed a novel SYTO 9 based visual detection

245

method of LAMP products which just needed low concentration fluorescent dye. By

246

adding SYTO 9 fluorescent dye before amplification and giving an exciting light to

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amplification products, the color of positive samples changed from colorless to green,

248

which could easily distinguished by naked eyes with the mini fluorescent-emission

249

cartridge (460 nm) developed by our Lab.

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Before LAMP amplification, SYTO 9 with a concentration of 4 µM was added

251

into the reaction mixture, which would intercalate into gradually accumulated 12


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double-stranded DNA (ds DNA) as the reaction progress and generate fluorescence

253

simultaneously. Combined with the mini fluorescent-emission cartridge, we hoped

254

that the positive amplifications could be visually distinguished from the negative ones

255

but it failed at room temperature because both positive and negative samples

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generated green fluorescence easily observed by naked eyes (Figure 3c).

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Traditionally, the fluorescent signal of SYTO 9 for real-time DNA amplification

258

was collected at high temperature like 72 °C of extending in PCR and the background

259

signal was weak enough to be ignored. However, negative samples displayed high

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background signal at low temperature which was demonstrated by melt curve plot in

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Figure 3b. Since the fluorescent amplification curve of the negative samples in

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real-time LAMP remained flat during the whole amplification process (Figure 3a),

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we think that such high background signal had nothing to do with the non-specific

264

amplification. To figure out what is the main reason for the high background signal of

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negative samples, we detected the fluorescence of reaction mixtures before LAMP

266

amplification. As shown in Figure 3d, the positive sample, negative sample and even

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blank control which used water as template could emit obvious fluorescence without

268

amplification while no fluorescence was observed when there were no primers in

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reaction mixtures which indicated that the high background signal was highly related

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to the primers in reaction mixtures. Since SYTO 9 is a ds DNA-specific dye, its

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fluorescent signal should be low enough in the solution of primers. However, in

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LAMP system, the concentration of primers, especially the two inner primers which

273

are usually over 40 nt length, is relatively high. These long primers tend to be 13



274

presented as secondary structures at room temperature. Therefore, high fluorescence

275

intensity was produced by these primers. Once the temperature was raised up, the

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secondary structures of these primers were dissociated, and the fluorescent signal of

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the negative samples also decreased. Furthermore, the melting temperature of these

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primer secondary structures is far below that of the LAMP products (Figure 3b). So

279

an appropriate high temperature can subtract the background signal caused by these

280

secondary structures. Therefore，fluorescence observations of LAMP products must

281

be performed at elevated temperature for maximum discrimination between positive

282

and negative amplifications.

283

As seen in Figure 3e, the fluorescence from positive amplifications was obvious

284

and could be observed by naked eyes at all temperatures except 85 °C at which the

285

two strands of the desired products were dissociated so that the fluorescent signal was

286

also decreased. Both 65 °C and 75 °C were appropriate for visual detection because

287

background signal was totally eliminated while the fluorescent intensity of positive

288

products was still sufficiently strong. After comprehensive consideration, we chose

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65 °C for visual detection since it is also the temperature for LAMP amplification and

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will be more convenient to perform.

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This LAMP-coupled visual detection method can easily discriminate between

292

positive and negative reactions without requiring cumbersome manipulations and

293

elaborate instrumentation. The risk of carryover contamination is also eliminated.

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Detection of practical samples

295

For the citrus industry, the detection capability for asymptomatic samples of Las 14


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associated with the devastating disease HLB is vital. The feasibility for

297

LAMP-coupled visual detection method described here was validated by 60 samples

298

containing 30 asymptomatic and 30 symptomatic plant materials obtained from

299

different infected trees. The 60 samples were divided into 10 groups and the detection

300

result of one group is shown in Figure 4 (Detection results of other groups were

301

shown in ESI). All samples were tested positive by LAMP-coupled visual detection

302

method and real-time LAMP while 8 asymptomatic samples were missed by real-time

303

PCR for the lower sensitivity compared to LAMP-coupled visual detection method

304

and real-time LAMP. These results indicate that this LAMP-coupled visual detection

305

method can be deployable for the detection of Las in asymptomatic leaf samples and

306

it is feasible even using crude extracts, needing no expensive equipment and with less

307

time-consumption making this method very attractive to growers.

308

Subsequently, to investigate the distribution and severity of HLB in Zhejiang

309

Province, a systematic survey of HLB was conducted in the citrus-growing areas. A

310

total of 352 field samples from 9 different regions were collected and tested by

311

proposed LAMP-coupled visual detection method. Real-time TaqMan PCR and

312

real-time LAMP were used as control. As shown in Figure 5, among 352

313

field-collected samples, 36 samples were tested positive by LAMP-coupled visual

314

detection method and real-time LAMP while only 21 tested positive by real-time

315

TaqMan PCR. It is noted that all samples that were found to be positive by real-time

316

PCR were also positive by real-time LAMP and LAMP-coupled visual detection

317

method. The detection rate of 352 field samples of LAMP-coupled visual detection 15



318

method and real-time LAMP were both 10.23% (36/352) while only 5.97% (21/352)

319

of real-time TaqMan PCR. Therefore, the proposed LAMP-coupled visual detection

320

method showed great reliability for detection of Las compared to conventional

321

real-time TaqMan PCR and can be an alternative detection method used for field

322

detection of Las.

323

In summary, we have developed an extremely rapid, sensitive and carryover

324

contamination-free LAMP-coupled visual detection method for filed detection of Las

325

from citrus leaf samples. Cumbersome DNA purification steps in this method are

326

omitted and DNA extraction can be accomplished in only 3 min with NaOH solution,

327

making detection in field more efficient and convenient. Unlike existing SYTO 9

328

based visual detection methods, this innovative visual method can be performed

329

without exposing amplicons to the environment, reducing the risk of carryover

330

contamination. Instead of former adjacent colorimetric change, the color of positive

331

samples changes from colorless to green and can be easily observed by naked eyes.

332

No longer requiring the expensive thermal cycler, a portable heater and a mini

333

fluorescent-emission cartridge invented by our Lab are employed for visually assay of

334

LAMP. Compared to conventional real-time PCR, this LAMP-coupled visual

335

detection method of Las is more sensitive and rapid. The whole detection process can

336

be accomplished within 40 min from leaf sampling to result. The feasibly of this

337

method has been verified by analysis of practical field samples. Overall, the

338

affordable, sensitive, rapid and convenient method would contribute a lot to the citrus

339

planting industry by field detection of Las. 16


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341

Abbreviations Used HLB

Citrus huanglongbing

Las

Candidatus Liberibacter asiaticus

PCR

Polymerase chain reaction

LAMP

Loop-mediated isothermal amplification

NaOH

Sodium hydroxide

Tt

Time threshold

Ct

Cycle threshold

RSD

Relative standard deviation

SD

Standard deviation

TE

Tris-EDTA

LOD

Limit-of-detection

Acknowledgement

342

This work was supported by National Natural Science Foundation of China

343

(31571918) and Hong Kong, Macao and Taiwan Scientific and Technological

344

Cooperation Projects (2015DFT30150).

345

Supporting Information

346 347

Brief statement in nonsentence format listing the contents of the material supplied as Supporting Information.

348 349

References 17



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Candidatus Liberibacter asiaticus in the Asian citrus psyllid, Diaphorina citri Kuwayama. J. microbiol. Meth. 2014, 102, 15-22. (18) Notomi, T.; Okayama, H.; Masubuchi, H.; Yonekawa, T.; Watanabe, K.; Amino, N.; Hase, T., Loop-mediated isothermal amplification of DNA. Nucleic Acids res. 2000, 28, e63-e63. (19) Nagamine, K.; Hase, T.; Notomi, T., Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol. Cell. Probe. 2002, 16, 223-229. (20) 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. (21) 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. (22) Ravindran, A.; Levy, J.; Pierson, E.; Gross, D. C., Development of a loop-mediated isothermal amplification procedure as a sensitive and rapid method for detection of ‘Candidatus Liberibacter solanacearum’in potatoes and psyllids. Phytopathology 2012, 102, 899-907. (23) Mori, Y.; Nagamine, K.; Tomita, N.; Notomi, T., Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem. Bioph. Res. Co. 2001, 289, 150-154. (24) Goto, M.; Honda, E.; Ogura, A.; Nomoto, A.; Hanaki, K.-I., Short technical reports. Biotechniques 2009, 46, 167-172. (25) Tomita, N.; Mori, Y.; Kanda, H.; Notomi, T., Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat. Protoc. 2008, 3, 877-882. (26) Iwamoto, T.; Sonobe, T.; Hayashi, K., Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M. avium, and M. intracellulare in sputum samples. J. Clin. Microbiol. 2003, 41, 2616-2622. (27) Ball, C. S.; Light, Y. K.; Koh, C.-Y.; Wheeler, S. S.; Coffey, L. L.; Meagher, R. J., Quenching of unincorporated amplification signal reporters in Reverse-Transcription Loop-Mediated Isothermal Amplification enabling bright, single-step, closed-tube, and multiplexed detection of RNA viruses. Anal. Chem. 2016, 88, 3562-3568. (28) Njiru, Z. K.; Mikosza, A. S. J.; Armstrong, T.; Enyaru, J. C.; Ndung'u, J. M.; Thompson, A. R. C., Loop-mediated isothermal amplification (LAMP) method for rapid detection of Trypanosoma brucei rhodesiense. PLoS Negl. Trop Dis. 2008, 2, e147. (29) Kil, E.-J.; Kim, S.; Lee, Y.-J.; Kang, E.-H.; Lee, M.; Cho, S.-H.; Kim, M.-K.; Lee, K.-Y.; Heo, N.-Y.; Choi, H.-S., Advanced loop-mediated isothermal amplification method for sensitive and specific detection of Tomato chlorosis virus using a uracil DNA glycosylase to control carry-over contamination. J. Virol. Methods 2015, 213, 68-74. (30) 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, 3747-3749. (31) 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. (32) Zhang, X.; Lowe, S. B.; Gooding, J. J., Brief review of monitoring methods for loop-mediated isothermal amplification (LAMP). Biosens. Bioelectron. 2014, 61, 491-499. (33) Hill, J.; Beriwal, S.; Chandra, I.; Paul, V. K.; Kapil, A.; Singh, T.; Wadowsky, R. M.; Singh, V.; 19



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Goyal, A.; Jahnukainen, T., Loop-mediated isothermal amplification assay for rapid detection of

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Figure captions

459

Schemes 1 Schematic of LAMP-coupled visual detection method for field detection

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of Las from leave samples within only 40 min.

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Figure 1 Target sequence and primer locations for LAMP amplification. Nucleotide

462

positions are based on the sequence of Las encompassing a bacteriophage-type DNA

463

polymerase region (GenBank NO. M94302)

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Figure 2 Sensitivity comparisons of real-time TaqMan PCR (a), real-time LAMP (b)

465

and LAMP-coupled visual detection method (c). Serial dilutions of purified DNA

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extracted from symptomatic leaves ranging from 1.0 ×102 ng/µL to 1.0×10-5 ng/µL

467

(from left to right in each picture) were tested.

468

Figure 3 Visual detection of LAMP products. a) Fluorescent amplification curve of

469

LAMP amplification. b) Melting curve of LAMP amplification. c) Visual detection of

common strains of Escherichia coli. J. Clin. Microbiol. 2008, 46, 2800-2804. (34) Parida, M.; Horioke, K.; Ishida, H.; Dash, P. K.; Saxena, P.; Jana, A. M.; Islam, M. A.; Inoue, S.; Hosaka, N.; Morita, K., Rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay. J. Clin. Microbiol.2005, 43, 2895-2903. (35) 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. (36) 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. (37) Kaneko, H.; Kawana, T.; Fukushima, E.; Suzutani, T., Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances. J. Biochem. Bioph. Meth. 2007, 70, 499-501. (38) Tomimura, K.; Miyata, S.-i.; Furuya, N.; Kubota, K.; Okuda, M.; Subandiyah, S.; Hung, T.-H.; Su, H.-J.; Iwanami, T., Evaluation of genetic diversity among ‘Candidatus Liberibacter asiaticus’ isolates collected in Southeast Asia. Phytopathology 2009, 99, 1062-1069.

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470

positive and negative reactions at room temperature. d) Visual detection of LAMP

471

reaction mixture before amplification at room temperature. e) Visual detection of

472

positive and negative reactions at different temperatures.

473

Figure 4 Feasibility validation of LAMP-coupled visual detection method of Las. a)

474

Detection samples of one of the ten groups with 3 asymptomatic (A1, A2, A3) and 3

475

symptomatic leaves (S1, S2, S3). b) Visual detection for asymptomatic (A1, A2, A3) and

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symptomatic (S1, S2, S3) samples. c) Results of real-time LAMP.

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Figure 5 Detection result of 352 field samples in Zhejiang Province.

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Table 1 Datailed evaluation of NaOH-based DNA crude extraction from leave samples by real-time LAMP analyses. Tt Mean of each repeat

Mean of

Time for

Diluted

lysis (min)

multiples

Repeat 1

1

1

N

N

N

N

N

N

N

5

22.2

22.6

22.6

22.5

0.2

1.0

22.5

50

14.9

14.8

14.8

14.8 a

0.1

0.4

14.8

500

15.0

15.9

15.1

15.0

0.1

0.7

14.0

1

N

N

N

N

N

N

N

5

21.7

22.2

21.9

21.9

0.3

1.1

21.9

50

13.5

13.3

13.4

13.4 b

0.1

0.7

13.4

500

14.8

14.8

13.8

14.5

0.6

3.9

14.5

1

N

N

N

N

N

N

N

5

22.8

22.8

22.3

22.6

0.3

1.3

22.6

50

13.3

13.8

13.4

13.5 b

0.3

2.0

13.5

500

14.0

14.0

14.1

14.0

0.1

0.4

14.0

1

N

N

N

N

N

N

N

5

22.6

23.3

22.7

22.9

0.4

1.7

22.9

50

13.6

13.7

13.4

13.6 b

0.2

1.1

13.6

500

16.7

16.8

16.4

16.6

0.1

1.3

16.6

/

12.89

12.94

13.18

13.00 b

0.2

1.2

13.00

3

6

10

Commercial kit

Repeat 2

all

Repeat 3

SD

Tt values

RSD Time (%)

(min)

a, b The level of significant difference (p

A rapid, sensitive and carryover contamination-free loop-mediated

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