Field Detection of Citrus Huanglongbing Associated with 'Candidatus

Subscriber access provided by Kaohsiung Medical University

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29

Journal of Agricultural and Food Chemistry

1

Field Detection of Citrus Huanglongbing Associated with ‘Candidatus

2

Liberibacter Asiaticus’ by Recombinese Polymerase Amplification within 15 min

3 4

Wenjuan Qian a, Ying Lu b, Youqing Meng b, Zunzhong Ye a, Liu Wang a, Rui Wang a,

5

Qiqi Zheng a, Hui Wu a and Jian Wu a*

6

a

7

Hangzhou 310058, China.

8

b

College of Biosystems Engineering and Food Science, Zhejiang University,

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

9 10

Correspondence

11

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

12

Fax: 0086-571-88982180 College of Biosystems Engineering and Food Science,

13

Zhejiang University, Hangzhou 310058, China.

1

ACS Paragon Plus Environment


14

ABSTRACT:

15

Candidatus Liberibacter asiaticus（Las）is the most prevalent bacterium associated

16

with Huanglongbing which is one of the most destructive diseases of citrus. In this

17

paper, an extremely rapid and simple method for field detection of Las from leaf

18

samples based on recombinase polymerase amplification (RPA) is described. Three

19

RPA primer pairs were designed and evaluated. RPA amplification was optimized so

20

that it could be accomplished within 10 min. In combination with DNA crude

21

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

22

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

23

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

24

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

25

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

26

reliability of this method was also verified by analyzing field samples.

27 28

Keywords:

29

recombinase polymerase amplification, visual detection

Candidatus Liberibacter asiaticus, field detection, crude DNA extraction,

2


Page 2 of 29

Page 3 of 29


30

Introduction

31

Huanglongbing (HLB) is one of the most serious and destructive diseases of citrus

32

and causes huge economic losses in citrus-producing areas worldwide.1The infection

33

is associated with three yet-uncultured pathogen species: Candidatus Liberibacter

34

asiaticus (Las), Candidatus Liberibacter africanus (Laf), and Candidatus Liberibacter

35

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

36

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

37

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

38

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

39

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

40

infected citrus trees immediately. 6 Thus, rapid, sensitive, simple, low cost and easy to

41

operate method for field detection of Las would facilitate implementation of required

42

management practices in a timely fashion.

43

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

44

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

45

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

46

equipment like thermal cycler or fluorescent detection equipment. Apart from

47

PCR-based methods, isothermal nucleic acid amplification, such as loop-mediated

48

isothermal amplification (LAMP) has also been applied for HLB detection.13, 14 Our

49

group also reported a detection method for Las with visual detection based on

50

LAMP.15 Although LAMP has overcome the limitations of thermal cyclers and can be

3



51

used for field detection of Las, it still need at least half an hour to complete the

52

detection process and the design of the six primers is complex.15-18

53

Recently, a novel isothermal nucleic acid amplification technique, recombinase

54

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

55

sensitive and simple diagnostic approach.19 The reaction initiates with the

56

recombinase binding to the primer to form nucleoprotein filaments which scan the

57

template DNA for homologous sequences. Next, single-stranded DNA binding protein

58

helps template DNA melting thus primers and template DNA start pairing. Finally, the

59

strand-displacing polymerase adds nucleotide bases to the 3’ end of the primer for

60

extension. (Figure S1, Supporting Information).19-21 Exponential amplification can be

61

accomplished within 10 to 20 min by the circulation of this process.22, 23 RPA is a

62

good choice for field detection because it is isothermal, simple, rapid and sensitive.

63

Application of RPA for detection of plant pathogens such as Tomato mottle virus,

64

Rose rosette virus and Banana bunchy top virus have already been reported.24-27 Paul

65

F et al. have reported to implement the rapid on-site detection of Las by RPA.28

66

However, they used lateral flow dipstick (LFD) to detect RPA amplicons, which

67

usually requires costly nickase, complex probe design, and the uncapping operation.29,

68

30

69

on lateral flow dipstick. For visual detection in field, DNA florescent dye will be more

70

convenient and simple.31, 32 To the best of our knowledge, florescent dye based visual

71

detection method for field detection of Las by RPA has not been reported up in

72

literatures until now.

Furthermore, present field detection method for RPA amplificons is mostly based

4


Page 4 of 29

Page 5 of 29


73

Another obstacle for field detection of HLB is DNA extraction. Standard

74

extraction methods and commercial kits are not appropriate for rapid field detection

75

due to the redundant operation steps and long operation duration. Fortunately, RPA

76

amplification is very robust and has already been reported to work with DNA crude

77

extraction.33, 34

78

Here, we have developed a RPA-based visual detection method of Las from citrus

79

leaf samples. DNA was extracted from leaf samples by simple treatment with sodium

80

hydroxide (NaOH) solution and the 50-fold dilution was directly used as template for

81

RPA. After 10 min amplification, the reaction mixture, fluorescent dye, and sterile

82

water were mixed immediately. Then positive samples emit green fluorescence which

83

could be easily distinguished from colorless negative samples under a mini-UV torch

84

light. The whole process could be accomplished in 15 min and the amplicon

85

contamination could be avoided with a special closed cartridge developed by our

86

group.

87

Materials and methods

88

Plant material

89

40 leaf samples containing 20 asymptomatic and 20 symptomatic leaves of Las

90

symptomatic citrus trees and 150 blind leaf samples obtained from different regions in

91

Zhejiang Province, China were collected by Zhejiang Plant Protection and Quarantine

92

Bureau. Healthy citrus leaves bought from local supermarket were used as negative

93

controls.

94

NaOH-based DNA crude extraction 5



Page 6 of 29

95

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

96

up into pieces and ground in a mortar and immerged in 500 µL 0.5 M NaOH. After 1

97

min of grinding and several minutes of standing at room temperature, the supernatant

98

was diluted 50-fold with Tris-EDTA (TE) buffer (10 mM Tris, 1 mM EDTA, pH 8.0)

99

and was directly used as template for RPA assay. The durations of lysis were studied

100

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

101

2, 5 and 9 min, respectively. For a comparative test, standard DNA extraction was

102

conducted using a commercial DNA extraction kit (Tiangen Biotech Co., Ltd, Beijing,

103

China).

104

Primer design for RPA

105

Three

pairs

of

RPA

primers

targeting

the

conserved

sequence

of

106

tufB-secE-nusG-rplKAJL-rpoB gene cluster [GenBank: AY342001.1] in Las designed

107

for amplification of 144-178 bp products using Primer Premier 5.0.35 The specificity

108

of primers was ensured by Blast analysis and primer sequences are shown in Table 1.

109

RPA Assay

110

RPA reactions were carried out in a total of 25 µL reaction mixture according to

111

the manufacturer’s instructions (TwistAmp basic kit, TwistDx, U.K.). Each reaction

112

contained one pellet, 14.75 µL of rehydration buffer, 0.2-0.72 µM of each primer,

113

1.25µL of magnesium acetate, 2 µL of DNA template, and sterile water up to 25 µL.

114

The mixture was incubated at 37-42 °C for 8-15 min on a conventional heating block

115

(Sangon Biotech Co., Ltd., Shanghai, China). After amplification, 2 µL of RPA

116

products were electrophoresed at constant voltage (110 V) on a 3% (w/v) agarose gel 6


Page 7 of 29


117

for 35 min and then photographed with a ChemiDoc XRS +System (BioRad, Hercules,

118

CA, USA).

119

Visual detection of RPA amplificons

120

1 µL of SYBR Green I (1:50 dilution of 10 mg/mL stock solution, Sangon Biotech

121

Co., Ltd., Shanghai, China) was added to reaction mixture after amplification. The

122

amplicons were detected directly by fluorescence observation using a mini-UV torch

123

(Tantoo E-commerce Co., Ningbo, China). To optimize the condition of visual

124

detection, the RPA reactions were performed for 8, 10 and 15 min, respectively, with

125

varying primer concentrations (0.2, 0.36 and 0.78 µM each). Negative samples using

126

DNA extracted from healthy citrus leaves as template were used as control.

127

Furthermore, the amplicons were diluted in water at 1:2, 1:5, and 1:10 dilutions before

128

visualization under UV light in order to reduce the background fluorescence.

129

Visual detection of RPA products in a disposable cartridge

130

The disposable cartridge is made of plastic and 102L × 12W × 13H mm. The 25

131

µL reaction mixture, 1 µL SYBR Green I (200 µg/mL), and 25 µL sterile water was

132

pre-added into three commercial PCR tubes and the tubes were connected to the

133

cartridge. Once the reaction is over, turn the cartridge upside down and shake

134

intensively to mix all the agents thoroughly. Then the results were visually detected

135

under UV light.

136

PCR detection

137

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

138

(5’-GCCTCGCGACTTCGCAACCCAT-3’), employed for PCR were reported in 7



139

previous paper by Li W. et al14 and synthesized by Sangon Biotech (Shanghai, China).

140

The 25 µL total reaction volume 2.5 µL 10×PCR buffer, 2 µL dNTP (2.5 mM each),

141

0.125 µL TaKaRa Taq™ HS (TaKaRa Biotech Co., Ltd, Dalian, China), 250 nM of

142

each primer, 2 µM SYTO 9 (Thermo Fisher Scientific Inc. Waltham, MA USA), 2.5

143

µL template, and sterile water up to 25 µL. The thermal program consisted of a single

144

cycle at 98°C for 3 min followed by 40 cycles at 98°C for 10 s, 60 °C for 30 s, and

145

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

146

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

147

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

148

were electrophoresed after amplification.

149

Sensitivity of RPA-based visual detection method

150

DNA template extracted from symptomatic leaves by commercial kits was

151

adjusted to the initial concentration of 100 ng/µL and 1:10 serial dilutions were

152

prepared. Before RPA amplification, the dilutions were mixed with host DNA

153

extracted from healthy citrus leaves to ensure that the total DNA concentration was

154

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

155

37 °C for 10 min.

156

Specificity of RPA-based visual detection method

157

To evaluate the specificity of the RPA-based visual detection method, several

158

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

159

citrus canker, citrus anthracnose, citrus scab, citrus black spot, and citrus brown spot,

160

were tested by Las-RPA technology described above. DNA from healthy citrus 8


Page 8 of 29

Page 9 of 29


161

samples was used as a negative control.

162

Detection of Las by RPA from field samples

163

For RPA assay (Figure 1), a piece of midrib tissue (around 0.6 cm×2 cm, 50 mg) of

164

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

165

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

166

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

167

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

168

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

169

mini-UV torch. Since the UV light is very weak, protective equipment is needless.

170

Finally, 40 leaf samples (20 asymptomatic and 20 symptomatic leaves) of Las

171

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

172

feasibility of RPA-based visual method for the detection of Las. Real-time PCR was

173

used as control.

174

Results and discussions

175

Optimization of reaction condition for RPA

176

RPA primers are relatively long (~30 bp) compared with PCR primers and vital

177

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

178

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

179

gene cluster [GenBank: AY342001.1] in Las were designed (Table 1).

180

In order to select the optimal primer pair for RPA, RPA reactions were conducted

181

using each of the three primer pairs described above and analyzed by electrophoresis.

182

A visible target band was displayed for positive samples in the gel electrophoresis 9



183

image no matter which primer pair was used while negative samples displayed no

184

bands. And target band of the amplicons amplified from primer pair I was the

185

brightest, indicating better performance compared with other two primer pairs (data

186

not shown). Thus, primer F I and R I were selected for the following assay.

187

Subsequently, to find out the optimal temperature for RPA assay, the mixture was

188

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

189

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

190

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

191

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

192

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

193

Crude extraction of DNA for RPA

194

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

195

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

196

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

197

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

198

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

199

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

200

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

201

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

202

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

203

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

204

grinding without incubation had no significant difference with that of standard DNA 10


Page 10 of 29

Page 11 of 29


205

extraction (Figure 2b), this NaOH-based DNA crude extraction method (1 min of

206

grinding followed by 50-fold dilution) was applied in the following assay.

207

Visual detection of RPA amplicons

208

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

209

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

210

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

211

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

212

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

213

positive samples would emit green fluorescent which could be easily distinguished

214

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

215

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

216

circumstances.19

217

To eliminate background signal and optimize the reaction condition for visual

218

detection, RPA reactions were performed for 8, 10 and 15 min, respectively, with

219

varying the primer concentration (0.2, 0.36 and 0.78 µM). As shown in Figure 3a,

220

with the amplification duration becoming longer and the concentration of primers

221

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

222

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

223

amplification regardless of the primers concentration and all negative samples with

224

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

225

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

226

long primers in RPA mixture which tend to form secondary structures and cause 11



227

non-specificity amplification. To obtain relatively high fluorescence intensity for

228

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

229

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

230

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

231

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

232

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

233

were enough for visually distinguish positive and negative samples while the

234

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

235

Therefore, the RPA-based visual detection method of Las was determined as

236

following: a piece of midrib tissues (around 0.6 cm×2 cm) of citrus leaf was used for

237

DNA extraction with 0.5M NaOH for 1 min of grinding. 2 µL of 50-fold diluted rough

238

cell lysate was amplified by RPA at 37 °C with 0.36 µM primers on a heating block

239

for 10 min. After the amplification, the mixture was 2-fold diluted and mixed with 1

240

µL of SYBR Green I (200 µg/ml). The detection result was visually judged under a

241

mini-UV torch.

242

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

243

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

244

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

245

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

246

flow dipstick detection which has been reported previously.36) The 25 µL reaction

247

mixture, 1 µL fluorescent dye, and 25 µL sterile water are pre-added into three

248

commercial PCR tubes. Then PCR tubes are connected to the cartridge tightly so that 12


Page 12 of 29

Page 13 of 29


249

the reaction space is sealed. The cartridge can be directly incubated at 37 °C for RPA.

250

After amplification, turn the cartridge upside down and shake intensively to mix all

251

the agents thoroughly. Finally, the visually detection can be performed without

252

uncapping operation and prevent cross-contamination.

253

Sensitivity and Specificity of RPA-based visual detection method

254

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

255

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

256

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

257

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

258

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

259

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

260

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

261

it took less time and easier to operate.

262

The specificity of RPA primers was tested on several other citrus samples infected

263

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

264

citrus scab, citrus black spot and citrus brown spot. No positive result was observed

265

except with the Las-infected sample, indicating a high level of detection specificity of

266

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

267

Detection of Las from field samples by RPA

268

Finally, to evaluate the feasibility of proposed RPA-based visual detection

269

method, 40 samples containing 20 asymptomatic and 20 symptomatic plant materials

270

obtained from different infected trees were tested by RPA-based visual detection 13



271

method with real-time PCR as control. The detection results of the two methods were

272

consistent that all symptomatic and asymptomatic samples were tested positive,

273

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

274

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

275

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

276

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

277

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

278

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

279

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

280

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

281

reliability for natural samples detection.

282

In summary, we have developed an extremely rapid, simple and visual method

283

for field detection of Las without carry-over contamination based on RPA

284

amplification in this paper. DNA extracted from leaf samples by simple treatment

285

with NaOH solution can be directly used as template for RPA. RPA amplification was

286

optimized and can be accomplished within 10 min. Then SYBR Green I was

287

employed for end-point RPA detection so that the result of the amplification can be

288

accurately judged by naked eyes under UV light using a mini-UV torch. The whole

289

detection process from leaf sampling to result can be completed in 15 minutes.

290

Without any sophisticated complex instruments, a heater and a mini-UV torch are

291

enough to perform the detection process, making this method be an alternative

292

method for field detection. And the amplicon contamination can be avoided with an 14


Page 14 of 29

Page 15 of 29


293

enclosed cartridge. The feasibly of this method has also been verified by analysis of

294

field samples.

295

Abbreviations Used

296

HLB

Citrus huanglongbing

Las

Candidatus Liberibacter asiaticus

PCR

Polymerase chain reaction

LAMP

Loop-mediated isothermal amplification

RPA

Recombinase polymerase amplification

NaOH

Sodium hydroxide

LFD

Lateral flow dipstick

TE

Tris-EDTA

LOD

Limit-of-detection

Funding

297

This work was supported by National Natural Science Foundation of China

298

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

299

Cooperation Projects (2015DFT30150).

300

Supporting Information

301

Brief statement in nonsentence format listing the contents of the material supplied

302

as Supporting Information.

303

References

304

(1)

Freitas, D. d. S.; Carlos, E. F.; Gil, M. r. C. S. d. S.; Vieira, L. G. E.; Alcantara, G. B., 15



305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322

NMR-Based Metabolomic analysis of Huanglongbing-asymptomatic and-symptomatic citrus trees. J.

323

(9) Wang, Z.; Yin, Y.; Hu, H.; Yuan, Q.; Peng, G.; Xia, Y., Development and application of molecular‐

324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347

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


Page 16 of 29

Page 17 of 29


348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391

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



392 393 394 395 396 397 398 399 400 401 402 403

(33) Lorraine, L.; Dara, L.; Singhal, M. C.; Jason, C.; Jered, S.; Paul, L.; Anthony, T.; Olaf, P.; Mathew,

404

Figure captions

405

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

408

diluted rough cell lysate was amplified by RPA at 37 °C for 10 min. After the

409

amplification, turn the cartridge upside down and shake intensively to mix the mixture,

410

fluorescent dye and sterilize water thoroughly. The presence or absence of

411

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

412

mini-UV torch.

413

Figure 2 Visualization of RPA products by agarose gel electrophoresis. a)

414

Temperature optimization for RPA amplification. Lanes 1-6, positive samples

415

amplified by RPA at 37, 38, 39, 40, 41 and 42 °C, respectively. Lanes 7-12, negative

416

samples amplified by RPA at 37, 38, 39, 40, 41 and 42 °C, respectively. b) Evaluation

417

of different lysis time of NaOH-based DNA extraction for RPA amplification. Lane

418

1-2 were standard DNA extraction using a commercial kit. Lane 3-4, 5-6, 7-8, 9-10

419

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.

18


Page 18 of 29

Page 19 of 29


420

standing, respectively. The odd numbered wells contain amplicons from Las-free leaf

421

samples, and even numbered lanes contain amplicons from Las positive leaf samples.

422

Figure 3 Effect of reaction time, primer concentration, and amplicon dilution on

423

visual detection by RPA. a) Visual detection of RPA amplicons amplified from RPA

424

reactions performed for 8, 10 and 15 min, respectively, with varying the primer

425

concentration (0.2, 0.36 and 0.78 µM). b) Visual detection of RPA amplicons

426

amplified from PRA reaction mixture with 0.36 µM of primers for 10 min after 2-fold,

427

5-fold and 10-fold dilution.

428

Figure 4 Enclosed disposable cartridge integrated with RPA amplification and visual

429

detection. The numbers in the figure display the size of the cartridge (mm). RPA

430

reaction mixture, fluorescent dye, and sterile water are pre-added into the three PCR

431

tubes, respectively. Then PCR tubes are connected to the cartridge tightly and the

432

cartridge is directly incubated at 37 °C for RPA. After amplification, turn the cartridge

433

upside down and shake intensively to mix all the agents thoroughly for visual

434

detection.

435

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

439

added as negative control.

440

Figure 6 12 positive leaf samples in 150 blind field samples and their detection

441

results by real-time PCR and RPA-based visual detection method. Real time 19



442

amplification graph, the RPA reaction tube, and a representative leaf from

443

corresponding sample are shown in each panel.

20


Page 20 of 29

Page 21 of 29


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

21


Amplicon sizes

172

178

144


Page 22 of 29

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%

22


Page 23 of 29


Figure 1

23



Figure 2

24


Page 24 of 29

Page 25 of 29


Figure 3

25



Figure 4

26


Page 26 of 29

Page 27 of 29


Figure 5

27



Figure 6

28


Page 28 of 29

Page 29 of 29


For Table of Contents Only

29


Field Detection of Citrus Huanglongbing Associated with 'Candidatus

Recommend Documents