- Email: [email protected]
Rapid detection of Cucumber green mottle mosaic virus by reverse transcription recombinase polymerase amplification
Accepted Manuscript Rapid detection of Cucumber green mottle mosaic virus by reverse transcription recombinase polymerase amplification Rong Zeng, Jinyan Luo, Shigang Gao, Lihui Xu, Zhiwei Song, Fuming Dai PII:
S0890-8508(18)30309-8
DOI:
https://doi.org/10.1016/j.mcp.2018.12.005
Reference:
YMCPR 1379
To appear in:
Molecular and Cellular Probes
Received Date: 21 November 2018 Revised Date:
17 December 2018
Accepted Date: 21 December 2018
Please cite this article as: Zeng R, Luo J, Gao S, Xu L, Song Z, Dai F, Rapid detection of Cucumber green mottle mosaic virus by reverse transcription recombinase polymerase amplification, Molecular and Cellular Probes (2019), doi: https://doi.org/10.1016/j.mcp.2018.12.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Rapid detection of Cucumber green mottle mosaic virus by reverse transcription recombinase polymerase amplification
3
Rong Zenga,1, Jinyan Luob, Shigang Gaoa,1, Lihui Xua, Zhiwei Songa, Fuming Daia,*
4 5 6
a, Institute of Eco-Environment and Plant Protection, Shanghai Key Laboratory of Protection Horticultural Technology, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China
7
b, Shanghai Extension and Service Center of Agriculture Technical, Shanghai 201103, China.
8 9 10
E-mail addresses: [email protected] (R. Zeng), [email protected] (J.Y. Luo), [email protected] (S.G. Gao), [email protected] (L.H Xu), [email protected] (Z.W. Song), [email protected] (F.M. Dai).
11
1 These authors contributed equally to this work.
12
∗ Corresponding author.
SC
M AN U
13
RI PT
1 2
Abstract Reverse transcription recombinase polymerase amplification (RT-RPA) for detection of Cucumber green mottle mosaic virus (CGMMV) was developed in this study. It was found to be specific, with a limit of detection of 0.5 pg of total CGMMV RNA. CGMMV on inoculated leaves was tested using RT-RPA assay, suggesting that this method is suitable for CGMMV detection from leaves.
20
Keywords: CGMMV; isothermal amplification; specificity
21
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14 15 16 17 18 19
Cucumber green mottle mosaic virus (CGMMV) infects cucurbit crops all over the world and is a typical tobamovirus that can be transmitted by mechanical contact and by seeds [1]. CGMMV causes severe mosaic symptoms on infected cucumber leaves and fruits. CGMMV induces green mottle on watermelon leaves at an early time and fruit decay in the harvest period. Due to threats on cucurbit production, CGMMV is regarded as a virus with significance for plant quarantine in China [2]. Some available methods for CGMMV detection, such as ELISA and real-time RT-PCR, are time-consuming and require expensive equipment. Hence, it is necessary to develop a rapid, sensitive and specific method for CGMMV identification.
31 32 33 34 35 36 37 38
Recombinase polymerase amplification (RPA) is an isothermal amplification system developed by TwistDx (Cambridge, United Kingdom) [3]. RPA, as a highly sensitive and rapid detection method, has been used in a wide range of applications, including pathogen detection, transgene detection, food safety testing [4-6]. It can amplify nucleic acids at 37-42 ˚C within 3-10 min. RPA detection have applied for some plant virus like Tomato yellow leaf curl virus (TYLCV) [7], Little cherry virus 2 (LChV2) [8], Rose rosette virus (RRV) [9], Rice black-streaked dwarf virus (RBSDV) [10] etc, but not for CGMMV. In this study, an RT-RPA isothermal amplification method for CGMMV detection was evaluated.
AC C
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22 23 24 25 26 27 28 29 30
1
ACCEPTED MANUSCRIPT RNA of virus-infected leaves was extracted using TRIzol Reagent (Life, USA) according to the manufacturer’s instructions. Total RNA quality and concentrations were tested by Tecan Infinite M200 PRO (Tecan, Switzerland). cDNA was synthesized by reverse transcription using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, Japan). PCR was conducted using 2×TransStart FastPfu Fly PCR SuperMix (Transgen, China). The RPA was performed in a total volume of 50 µL containing 30.0 µL of rehydration buffer, 12.5 µL of ddH2O, 0.5 µmol·L-1 each forward and reverse primer, 1.0 µL of cDNA, and 0.28 µmol·L-1 magnesium acetate. The mixture was incubated in a conventional laboratory water bath at 37 °C for 15 min. Then, 50 µL of chloroform was added and the solution was mixed and centrifuged at 12,000 rpm for 1 min. The supernatants were analysed by electrophoresis on a 1.5% agarose gel. To negate nonspecific amplification and avoid any possible carry-over contamination, all steps were followed strict safety operations and laboratory practices.
51 52 53 54 55
A set of primers were designed to anneal onto a conserved region in the CGMMV movement protein (MP). The sequences of the two primers are as follows: CGMMV-5144 (5’-GAATCCCTCGTGCCTGTCAAGTTGTTGCGTGGT-3’), CGMMV-5346 (5’-CGGACGGTGGCGGGAGCTGAAAATTTGCATATA-3’). Other primer sequences and product length details for the RT-PCR assay were described previously [11].
56 57 58 59 60 61 62
The specificity of the RPA assay was evaluated by detecting CGMMV and 3 other reference viruses with indistinguishable symptoms, including Zucchini yellow mosaic virus (ZYMV), Cucumber mosaic virus (CMV), and Melon yellow spot virus (MYSV). RNA from healthy leaves was used as the negative control. Positive results were observed for CGMMV samples (approximately 200 bp products), but not for the reference samples or the negative control (Fig. 1A). The specificity of the assay was also verified by RT-PCR (Fig. 1B). These results suggested that RT-RPA is a specific method for the detection of CGMMV.
63 64 65 66 67 68
The RPA and PCR limits of detection for the CGMMV assay were determined by using 10-fold serial dilutions of total RNA (50 - 5×10-7 ng) extracted from CGMMV-infected cucumber leaves. The RPA assay was found to be specific, with a sensitivity limit of detection of 0.5 pg of total RNA (Fig. 2A), and PCR was found to detect CGMMV from 50 pg total RNA (Fig. 2B). Therefore, RT-RPA was 100-fold more sensitive than the normal RT-PCR assay.
69 70 71 72 73
Based on the established CGMMV RPA assay method, the CGMMV-inoculated leaves of watermelon and Nicotiana benthamiana were tested. The results showed that CGMMV could be detected in the CGMMV-inoculated samples by the RPA assay (Fig. 3). This result demonstrated that the developed CGMMV RPA method could be used as a promising diagnostic tool in less well-equipped laboratories or in the field.
74 75 76 77 78 79
Molecular detection using PCR/RT-PCR requires high quality and quantity of nucleic acids [12]. Numerous reports showed that RPA could rapidly detect low copy samples and exhibit a higher discriminative power than PCR/RT-PCR [3-5]. RPA is also similar to PCR/RT-PCR, where multiplex detection can be performed in the same tube [9,13]. Moreover, the products of RPA could be used for sequencing, enzyme digestion, cloning, etc. in subsequent assays.
AC C
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ACCEPTED MANUSCRIPT In summary, the CGMMV RT-RPA assay developed here could be applied for the detection of CGMMV, which causes similar symptoms to MYSV, ZYMV, and CMV in the field. As a new method, it has the advantages of being time-saving and having a high sensitivity and low requirements for equipment.
84
Competing interests
85
The authors declare that they have no competing interests.
86
Acknowledgments
87 88 89 90
This work was supported by the Shanghai Agriculture Applied Technology Development Program, China (Grant No.2014070301 ), Modern science and technology innovation project of Shanghai science and Technology Commission (16391905202), SAAS Program for Excellent Research Team (Grant No. 2018(B-01)).
91
References
SC
[1] A. Dombrovsky, L.T.T. Tran-Nguyen, R.A.C. Jones, Cucumber green mottle mosaic virus: Rapidly
M AN U
Increasing Global Distribution, Etiology, Epidemiology, and Management, Annu Rev Phytopathol 55 (2017) 231-256.
[2] H.Y. Chen, W.J. Zhao, Y. Cheng, M.F. Li, S.F. Zhu, Molecular identifcation of the virus causing watermelon mosaic disease in Mid-Liaoning, Acta Phytopath Sin 36 (2016) 306-309. [3] O. Piepenburg, C.H. Williams, D.L. Stemple, N.A. Armes, DNA detection using recombination proteins. , PLoS biology 4(7) (2006) e204.
[4] G. Nair, M. Rebolledo, A.C. White, Jr., Z. Crannell, R.R. Richards-Kortum, A.E. Pinilla, J.D.
TE D
Ramirez, M.C. Lopez, A. Castellanos-Gonzalez, Detection of Entamoeba histolytica by Recombinase Polymerase Amplification, Am J Trop Med Hyg 93(3) (2015) 591-5. [5] Y. Yang, X. Qin, G. Wang, Y. Zhang, Y. Shang, Z. Zhang, Development of a fluorescent probe-based recombinase polymerase amplification assay for rapid detection of Orf virus, Virol J 12 (2015) 206.
EP
[6] A. James, J. Macdonald, Recombinase polymerase amplification: Emergence as a critical molecular technology for rapid, low-resource diagnostics, Expert Rev Mol Diagn 15(11) (2015) 1475-89. [7] J. Wang, J. Wang, R. Li, R. Shi, L. Liu, W. Yuan, Evaluation of an incubation instrument-free reverse transcription recombinase polymerase amplification assay for rapid and point-of-need
AC C
92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109
RI PT
80 81 82 83
detection of canine distemper virus, J Virol Methods 260 (2018) 56-61.
110 111 112
[8] T.A. Mekuria, S. Zhang, K.C. Eastwell, Rapid and sensitive detection of Little cherry virus 2 using
113 114 115
[9] B. Babu, B.K. Washburn, S.H. Miller, K. Poduch, T. Sarigul, G.W. Knox, F.M. Ochoa-Corona, M.L.
116 117 118
[10] C. Zhao, F. Sun, X. Li, Y. Lan, L. Du, T. Zhou, Y. Zhou, Reverse transcription-recombinase
isothermal reverse transcription-recombinase polymerase amplification, J Virol Methods 205 (2014) 24-30.
Paret, A rapid assay for detection of Rose rosette virus using reverse transcription-recombinase polymerase amplification using multiple gene targets, J Virol Methods 240 (2017) 78-84.
polymerase amplification combined with lateral flow strip for detection of rice black-streaked dwarf virus in plants, J Virol Methods 263 (2019) 96-100. 3
ACCEPTED MANUSCRIPT [11] R. Zeng, L. H. Xu, S.-g. Gao, X.H. Ni, C.L. Chen, J.C. Chen, F.M. Dai, One-step reverse transcription loop-mediated isothermal amplification assay for rapid detection of melon yellow spot virus, Eur J Plant Pathol 145 (2016) 119-124. [12] R. Li, R. Mock, Q. Huang, J. Abad, J. Hartung, G. Kinard, A reliable and inexpensive method of nucleic acid extraction for the PCR-based detection of diverse plant pathogens, Journal of virological methods 154(1-2) (2008) 48-55. [13] S. Santiago-Felipe, L.A. Tortajada-Genaro, S. Morais, R. Puchades, A. Maquieira, Isothermal
RI PT
DNA amplification strategies for duplex microorganism detection, Food chemistry 174 (2015)
EP
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509-515.
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119 120 121 122 123 124 125 126 127 128 129
4
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ACCEPTED MANUSCRIPT
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Fig. 1 Specificity analysis of the CGMMV RT-RPA assay. A: Specificity analysis of CGMMV
RT-RPA assay. B: Virus-positive controls amplified by the specific RT-PCR assay. M: DNA Ladder.
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Lane 1–4: CGMMV, MYSV, ZYMV, and CMV. Lane 6: negative control with RNA from healthy cucumber leaves.
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131 132 133 134
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Fig. 2 Sensitivity comparison of RT-RPA (A) and RT-PCR (B) for the detection of CGMMV by agarose gel electrophoresis. M: DNA Ladder. Lane 1-9: ten-fold dilution of total RNA (50 ng - 5 × 10-7 ng) from CGMMV-infected cucumber leaves. Lane 10: negative control with RNA from healthy cucumber leaves.
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136 137 138 139
140 141 142 143 144
Fig. 3 Sample test of CGMMV by RT-RPA. M: DNA Ladder. Lane 1-4: positive control with RNA from CGMMV-inoculated cucumber leaves, CGMMV-inoculated watermelon leaves, CGMMV-inoculated Nicotiana benthamiana leaves, and negative control with RNA from healthy watermelon leaves. 5
ACCEPTED MANUSCRIPT Highlights
1.
Reverse transcription recombinase polymerase amplification (RT-RPA), as a new method, it has the advantages of being time-saving and having a high sensitivity.
EP
TE D
M AN U
SC
RI PT
This developed CGMMV RPA method could be used as a promising diagnostic tool in less well-equipped laboratories or in the field.
AC C
2.
S0890-8508(18)30309-8
DOI:
https://doi.org/10.1016/j.mcp.2018.12.005
Reference:
YMCPR 1379
To appear in:
Molecular and Cellular Probes
Received Date: 21 November 2018 Revised Date:
17 December 2018
Accepted Date: 21 December 2018
Please cite this article as: Zeng R, Luo J, Gao S, Xu L, Song Z, Dai F, Rapid detection of Cucumber green mottle mosaic virus by reverse transcription recombinase polymerase amplification, Molecular and Cellular Probes (2019), doi: https://doi.org/10.1016/j.mcp.2018.12.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Rapid detection of Cucumber green mottle mosaic virus by reverse transcription recombinase polymerase amplification
3
Rong Zenga,1, Jinyan Luob, Shigang Gaoa,1, Lihui Xua, Zhiwei Songa, Fuming Daia,*
4 5 6
a, Institute of Eco-Environment and Plant Protection, Shanghai Key Laboratory of Protection Horticultural Technology, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China
7
b, Shanghai Extension and Service Center of Agriculture Technical, Shanghai 201103, China.
8 9 10
E-mail addresses: [email protected] (R. Zeng), [email protected] (J.Y. Luo), [email protected] (S.G. Gao), [email protected] (L.H Xu), [email protected] (Z.W. Song), [email protected] (F.M. Dai).
11
1 These authors contributed equally to this work.
12
∗ Corresponding author.
SC
M AN U
13
RI PT
1 2
Abstract Reverse transcription recombinase polymerase amplification (RT-RPA) for detection of Cucumber green mottle mosaic virus (CGMMV) was developed in this study. It was found to be specific, with a limit of detection of 0.5 pg of total CGMMV RNA. CGMMV on inoculated leaves was tested using RT-RPA assay, suggesting that this method is suitable for CGMMV detection from leaves.
20
Keywords: CGMMV; isothermal amplification; specificity
21
TE D
14 15 16 17 18 19
Cucumber green mottle mosaic virus (CGMMV) infects cucurbit crops all over the world and is a typical tobamovirus that can be transmitted by mechanical contact and by seeds [1]. CGMMV causes severe mosaic symptoms on infected cucumber leaves and fruits. CGMMV induces green mottle on watermelon leaves at an early time and fruit decay in the harvest period. Due to threats on cucurbit production, CGMMV is regarded as a virus with significance for plant quarantine in China [2]. Some available methods for CGMMV detection, such as ELISA and real-time RT-PCR, are time-consuming and require expensive equipment. Hence, it is necessary to develop a rapid, sensitive and specific method for CGMMV identification.
31 32 33 34 35 36 37 38
Recombinase polymerase amplification (RPA) is an isothermal amplification system developed by TwistDx (Cambridge, United Kingdom) [3]. RPA, as a highly sensitive and rapid detection method, has been used in a wide range of applications, including pathogen detection, transgene detection, food safety testing [4-6]. It can amplify nucleic acids at 37-42 ˚C within 3-10 min. RPA detection have applied for some plant virus like Tomato yellow leaf curl virus (TYLCV) [7], Little cherry virus 2 (LChV2) [8], Rose rosette virus (RRV) [9], Rice black-streaked dwarf virus (RBSDV) [10] etc, but not for CGMMV. In this study, an RT-RPA isothermal amplification method for CGMMV detection was evaluated.
AC C
EP
22 23 24 25 26 27 28 29 30
1
ACCEPTED MANUSCRIPT RNA of virus-infected leaves was extracted using TRIzol Reagent (Life, USA) according to the manufacturer’s instructions. Total RNA quality and concentrations were tested by Tecan Infinite M200 PRO (Tecan, Switzerland). cDNA was synthesized by reverse transcription using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, Japan). PCR was conducted using 2×TransStart FastPfu Fly PCR SuperMix (Transgen, China). The RPA was performed in a total volume of 50 µL containing 30.0 µL of rehydration buffer, 12.5 µL of ddH2O, 0.5 µmol·L-1 each forward and reverse primer, 1.0 µL of cDNA, and 0.28 µmol·L-1 magnesium acetate. The mixture was incubated in a conventional laboratory water bath at 37 °C for 15 min. Then, 50 µL of chloroform was added and the solution was mixed and centrifuged at 12,000 rpm for 1 min. The supernatants were analysed by electrophoresis on a 1.5% agarose gel. To negate nonspecific amplification and avoid any possible carry-over contamination, all steps were followed strict safety operations and laboratory practices.
51 52 53 54 55
A set of primers were designed to anneal onto a conserved region in the CGMMV movement protein (MP). The sequences of the two primers are as follows: CGMMV-5144 (5’-GAATCCCTCGTGCCTGTCAAGTTGTTGCGTGGT-3’), CGMMV-5346 (5’-CGGACGGTGGCGGGAGCTGAAAATTTGCATATA-3’). Other primer sequences and product length details for the RT-PCR assay were described previously [11].
56 57 58 59 60 61 62
The specificity of the RPA assay was evaluated by detecting CGMMV and 3 other reference viruses with indistinguishable symptoms, including Zucchini yellow mosaic virus (ZYMV), Cucumber mosaic virus (CMV), and Melon yellow spot virus (MYSV). RNA from healthy leaves was used as the negative control. Positive results were observed for CGMMV samples (approximately 200 bp products), but not for the reference samples or the negative control (Fig. 1A). The specificity of the assay was also verified by RT-PCR (Fig. 1B). These results suggested that RT-RPA is a specific method for the detection of CGMMV.
63 64 65 66 67 68
The RPA and PCR limits of detection for the CGMMV assay were determined by using 10-fold serial dilutions of total RNA (50 - 5×10-7 ng) extracted from CGMMV-infected cucumber leaves. The RPA assay was found to be specific, with a sensitivity limit of detection of 0.5 pg of total RNA (Fig. 2A), and PCR was found to detect CGMMV from 50 pg total RNA (Fig. 2B). Therefore, RT-RPA was 100-fold more sensitive than the normal RT-PCR assay.
69 70 71 72 73
Based on the established CGMMV RPA assay method, the CGMMV-inoculated leaves of watermelon and Nicotiana benthamiana were tested. The results showed that CGMMV could be detected in the CGMMV-inoculated samples by the RPA assay (Fig. 3). This result demonstrated that the developed CGMMV RPA method could be used as a promising diagnostic tool in less well-equipped laboratories or in the field.
74 75 76 77 78 79
Molecular detection using PCR/RT-PCR requires high quality and quantity of nucleic acids [12]. Numerous reports showed that RPA could rapidly detect low copy samples and exhibit a higher discriminative power than PCR/RT-PCR [3-5]. RPA is also similar to PCR/RT-PCR, where multiplex detection can be performed in the same tube [9,13]. Moreover, the products of RPA could be used for sequencing, enzyme digestion, cloning, etc. in subsequent assays.
AC C
EP
TE D
M AN U
SC
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39 40 41 42 43 44 45 46 47 48 49 50
2
ACCEPTED MANUSCRIPT In summary, the CGMMV RT-RPA assay developed here could be applied for the detection of CGMMV, which causes similar symptoms to MYSV, ZYMV, and CMV in the field. As a new method, it has the advantages of being time-saving and having a high sensitivity and low requirements for equipment.
84
Competing interests
85
The authors declare that they have no competing interests.
86
Acknowledgments
87 88 89 90
This work was supported by the Shanghai Agriculture Applied Technology Development Program, China (Grant No.2014070301 ), Modern science and technology innovation project of Shanghai science and Technology Commission (16391905202), SAAS Program for Excellent Research Team (Grant No. 2018(B-01)).
91
References
SC
[1] A. Dombrovsky, L.T.T. Tran-Nguyen, R.A.C. Jones, Cucumber green mottle mosaic virus: Rapidly
M AN U
Increasing Global Distribution, Etiology, Epidemiology, and Management, Annu Rev Phytopathol 55 (2017) 231-256.
[2] H.Y. Chen, W.J. Zhao, Y. Cheng, M.F. Li, S.F. Zhu, Molecular identifcation of the virus causing watermelon mosaic disease in Mid-Liaoning, Acta Phytopath Sin 36 (2016) 306-309. [3] O. Piepenburg, C.H. Williams, D.L. Stemple, N.A. Armes, DNA detection using recombination proteins. , PLoS biology 4(7) (2006) e204.
[4] G. Nair, M. Rebolledo, A.C. White, Jr., Z. Crannell, R.R. Richards-Kortum, A.E. Pinilla, J.D.
TE D
Ramirez, M.C. Lopez, A. Castellanos-Gonzalez, Detection of Entamoeba histolytica by Recombinase Polymerase Amplification, Am J Trop Med Hyg 93(3) (2015) 591-5. [5] Y. Yang, X. Qin, G. Wang, Y. Zhang, Y. Shang, Z. Zhang, Development of a fluorescent probe-based recombinase polymerase amplification assay for rapid detection of Orf virus, Virol J 12 (2015) 206.
EP
[6] A. James, J. Macdonald, Recombinase polymerase amplification: Emergence as a critical molecular technology for rapid, low-resource diagnostics, Expert Rev Mol Diagn 15(11) (2015) 1475-89. [7] J. Wang, J. Wang, R. Li, R. Shi, L. Liu, W. Yuan, Evaluation of an incubation instrument-free reverse transcription recombinase polymerase amplification assay for rapid and point-of-need
AC C
92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109
RI PT
80 81 82 83
detection of canine distemper virus, J Virol Methods 260 (2018) 56-61.
110 111 112
[8] T.A. Mekuria, S. Zhang, K.C. Eastwell, Rapid and sensitive detection of Little cherry virus 2 using
113 114 115
[9] B. Babu, B.K. Washburn, S.H. Miller, K. Poduch, T. Sarigul, G.W. Knox, F.M. Ochoa-Corona, M.L.
116 117 118
[10] C. Zhao, F. Sun, X. Li, Y. Lan, L. Du, T. Zhou, Y. Zhou, Reverse transcription-recombinase
isothermal reverse transcription-recombinase polymerase amplification, J Virol Methods 205 (2014) 24-30.
Paret, A rapid assay for detection of Rose rosette virus using reverse transcription-recombinase polymerase amplification using multiple gene targets, J Virol Methods 240 (2017) 78-84.
polymerase amplification combined with lateral flow strip for detection of rice black-streaked dwarf virus in plants, J Virol Methods 263 (2019) 96-100. 3
ACCEPTED MANUSCRIPT [11] R. Zeng, L. H. Xu, S.-g. Gao, X.H. Ni, C.L. Chen, J.C. Chen, F.M. Dai, One-step reverse transcription loop-mediated isothermal amplification assay for rapid detection of melon yellow spot virus, Eur J Plant Pathol 145 (2016) 119-124. [12] R. Li, R. Mock, Q. Huang, J. Abad, J. Hartung, G. Kinard, A reliable and inexpensive method of nucleic acid extraction for the PCR-based detection of diverse plant pathogens, Journal of virological methods 154(1-2) (2008) 48-55. [13] S. Santiago-Felipe, L.A. Tortajada-Genaro, S. Morais, R. Puchades, A. Maquieira, Isothermal
RI PT
DNA amplification strategies for duplex microorganism detection, Food chemistry 174 (2015)
EP
TE D
M AN U
SC
509-515.
AC C
119 120 121 122 123 124 125 126 127 128 129
4
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ACCEPTED MANUSCRIPT
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Fig. 1 Specificity analysis of the CGMMV RT-RPA assay. A: Specificity analysis of CGMMV
RT-RPA assay. B: Virus-positive controls amplified by the specific RT-PCR assay. M: DNA Ladder.
SC
Lane 1–4: CGMMV, MYSV, ZYMV, and CMV. Lane 6: negative control with RNA from healthy cucumber leaves.
TE D
M AN U
131 132 133 134
135
Fig. 2 Sensitivity comparison of RT-RPA (A) and RT-PCR (B) for the detection of CGMMV by agarose gel electrophoresis. M: DNA Ladder. Lane 1-9: ten-fold dilution of total RNA (50 ng - 5 × 10-7 ng) from CGMMV-infected cucumber leaves. Lane 10: negative control with RNA from healthy cucumber leaves.
AC C
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136 137 138 139
140 141 142 143 144
Fig. 3 Sample test of CGMMV by RT-RPA. M: DNA Ladder. Lane 1-4: positive control with RNA from CGMMV-inoculated cucumber leaves, CGMMV-inoculated watermelon leaves, CGMMV-inoculated Nicotiana benthamiana leaves, and negative control with RNA from healthy watermelon leaves. 5
ACCEPTED MANUSCRIPT Highlights
1.
Reverse transcription recombinase polymerase amplification (RT-RPA), as a new method, it has the advantages of being time-saving and having a high sensitivity.
EP
TE D
M AN U
SC
RI PT
This developed CGMMV RPA method could be used as a promising diagnostic tool in less well-equipped laboratories or in the field.
AC C
2.