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Author's Personal Copy Journal of Virological Methods 240 (2017) 49–53
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One-step reverse transcription loop-mediated isothermal amplification for the detection of Maize chlorotic mottle virus in maize Ling Chen a , Zhiyuan Jiao a , Dongmei Liu a , Xingliang Liu b , Zihao Xia a , Congliang Deng b , Tao Zhou a , Zaifeng Fan a,∗ a State Key Laboratory of Agro-biotechnology and Ministry of Agriculture Key Laboratory for Plant Pathology, China Agricultural University, Beijing 100193, China b Beijing Entry-exit Inspection and Quarantine Bureau, Beijing 100016, China
a b s t r a c t Article history: Received 8 November 2015 Received in revised form 2 October 2016 Accepted 26 November 2016 Available online 27 November 2016 Keywords: Maize chlorotic mottle virus (MCMV) Loop-mediated isothermal amplification (LAMP) Maize Detection
Maize chlorotic mottle virus (MCMV) is spreading in many regions worldwide, causing maize lethal necrosis when co-infected with a potyvirid. In this study, one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect MCMV in maize. A set of four specific primers was designed based on the conserved coat protein gene sequences of MCMV. The RT-LAMP could be completed within 60 min under isothermal condition at 63 ◦ C. The sensitivity test showed that the RTLAMP was about 10-fold more sensitive than RT-PCR and no cross-reactivity was detected with other viral pathogens infecting maize in China. Moreover, the results of RT-LAMP could be visually inspected by SYBR Green I staining in a closed-tube, facilitating high-throughput application of MCMV detection. This method was further verified by testing field-collected samples. These results suggested that the developed MCMV RT-LAMP technique is a rapid, efficient and sensitive method which could be used as a routine screen for MCMV infection. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Maize (Zea mays L.) is an important cereal crop worldwide. Recently, Maize chlorotic mottle virus (MCMV) has been found in southwest China as a quarantine virus (Wu et al., 2013; Xie et al., 2011). MCMV in the genus Machlomovirus of the family Tombusviridae can infect maize and lead to typical symptoms like mild mosaic, severe stunting, and leaf necrosis (Niblett and Claflin, 1978; Uyemoto et al., 1981). Moreover, MCMV causes corn lethal necrosis (CLN) disease by synergistic infection with a maize-infecting potyvirid such as Maize dwarf mosaic virus (Goldberg and Brakke, 1987), Wheat streak mosaic virus (Scheets, 1998; Stenger et al., 2007) or Sugarcane mosaic virus (Adams et al., 2013; Wangai et al., 2012; Xia et al., 2016), resulting in severe yield losses. CLN was first described in maize from Peru in 1974 (Castillo and Hebert, 1974), thereafter, this disease was reported on maize plants in the United States (Niblett and Claflin, 1978). MCMV mainly distributed in America (Jensen et al., 1991; Jiang et al., 1992), but it has been found in several maize planting regions in Asia and Africa (Deng et al., 2014; Lukanda et al., 2014; Mahuku et al., 2015), suggesting
∗ Corresponding author. E-mail address:
[email protected] (Z. Fan). http://dx.doi.org/10.1016/j.jviromet.2016.11.012 0166-0934/© 2016 Elsevier B.V. All rights reserved.
that MCMV is spreading in major maize-planting regions worldwide. Meanwhile, there is a high risk of MCMV rapidly spread throughout China because of a large scale planting of its natural hosts (such as maize and sorghum) and its diverse transmission by thrips/beetles, seed and mechanical inoculation (Jiang et al., 1992; Jensen et al., 1991; Nault et al., 1979; Zhang et al., 2011). Consequently, there is a great potential threat of MCMV to maize production in many regions where this virus is emerging. Several traditional methods for detecting MCMV have been reported including enzyme-linked immunosorbent assay (Uyemoto, 1980), immunofluorescence (Nault et al., 1979), RTPCR, Real-time TaqMan RT-PCR (Zhang et al., 2011), and the next generation sequencing (Adams et al., 2013). However, these existing methods have limitations to detect MCMV. The results of immunological assay depend on the quality and availability of antibodies and false positives often make the results unreliable. RT-PCR is time consuming and the sensitivity is limited. Real-time RT-PCR is a relatively sensitive technique to detect MCMV, but it also requires expensive equipment in a laboratory setting. Next generation sequencing was mainly applied in the identification of plants infected with unknown virus because of its high costs and complex data analysis. Therefore, it is necessary to establish a rapid and effective technique to detect MCMV for controlling the spread of this virus.
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One-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay is suitable for detecting virus and measuring field samples because of its characteristics of rapidity, efficiency, simplification, high sensitivity and specificity (Nagamine et al., 2002; Notomi et al., 2000). This study was to develop a one-step RT-LAMP assay for rapid and sensitive detection of MCMV in field-grown maize.
2. Materials and methods 2.1. Virus sources, virus inoculations and RNA isolation MCMV was multiplied from the full-length cDNA clone (pMCM41) provided by Dr Kay Scheets. Sugarcane mosaic virus (SCMV), Pennisetum mosaic virus (PenMV) and Rice black streaked dwarf virus (RBSDV) were from previously published sources (Fan et al., 2003, 2004; Jia et al., 2012). Field maize samples were collected from Yuxi region of Yunnan Province. Maize inbred line B73 and cv. Va35 plants were grown in a growth chamber (28 ◦ C day and 22 ◦ C night, 16 h light and 8 h night cycles) for virus inoculation. Crude extracts were prepared by homogenizing the MCMV-, SCMV- or PenMV-infected maize leaf tissues in 0.01 M phosphate buffer (0.01 M KH2 PO4 : 0.01 M Na2 HPO4 = 49: 51 (V/V), pH 7.0) at a ratio of 1:10 (w/v). The crude extracts were rub-inoculated to the first true leaves of one-weekold B73 maize seedlings and the systemically infected leaves were harvested at 10 days post inoculation (dpi). For RBSDV inoculation, one-week-old Va35 seedlings were exposed to small brown planthoppers carrying RBSDV (five insects on each seedling) for 3 days in specific inoculation chambers and the systemically infected leaves were harvested at 30 dpi. Total RNA was extracted from infected and healthy maize leaves using Trizol reagent (Invitrogen, Carlsbad, CA, USA).
2.2. Primer design Available sequences of MCMV isolates were aligned to obtain the conserved coat protein (CP) gene sequences (accession nos. NC 003627, JQ982468, KF010583, JQ982470, JQ982469, GU13867, KJ782300, KP772217, EU358605, KF744394, KP851970) and then the RT-LAMP primers were designed according to the conserved CP gene sequences using the program Explorer V4 (http://primerexplorer.jp/elamp4.0.0/index.html). A forward inner primer (FIP, 5 -GCGCACACTGGAATCTCGAGAATTCTCCATGTCCGAAATTCTGC-3 , nt 3598-3575/nt 3527-3546) consisted of F1c (the complementary sequence of F1) and F2, and a reverse inner primer (BIP, 5 -CAAATGGCTGGCAGCACAAnt 3647-3665/nt GAATTC-GATACGCACAGAGTTGAACA-3 , 3707-3688, GAATCC represents the EcoR I sites) consisted of B1c (the complementary sequence of B1) and B2. The outer primers F3 (5 -GAGCTATTCGAGCCAACC-3 , nt 3451-3468) and B3 (5 -TAGTGGTGTCTGCTGTGA-3 , nt 3738-3721) were used for the initiation of the RT-LAMP reaction. The conventional RT-PCR primers were also designed according to the same and expanded conserved CP gene sequences of MCMV compared with that of RT-LAMP (Gong et al., 2010). The forward primer MCMV F (5 -ATGGCGGCAAGTAGCCGGTCTACCCGAGGTAGAA-3 , nt 3384-3417) and reverse primer MCMV R (5 TCAATGATTTGCCAGCCCTGGCCTGGAACCAGG-3 , nt 4094-4061) were used for RT-PCR reaction. Genome position listed according to the complete genome sequence of MCMV (accession no. JQ982468).
2.3. Conventional RT-PCR detection of MCMV The first-strand cDNA of MCMV-infected maize leaves was synthesized using M-MLV reverse transcriptase according to the instructions of the manufacturer (Promega, Madison, WI, USA). An RT reaction mixture (20 L final volume) included 5 L of total RNA (2.5 g), 4 L of dNTP mixture (2.5 mM), 1 L (200U) of MMLV reverse transcriptase, 4 L of M-MLV 5× reaction buffer, 1 L of random primers (10 M), 1 L (40U) of recombinant RNasin ribonuclease inhibitor, and 4 L of sterile, RNase-free water. The mixture was incubated at 37 ◦ C for 60 min. After the RT reaction, 1 L cDNA product was added to 24 L of the PCR mixture which consisted of 12.5 L of Premix (Takara Bio Inc., Dalian, China), 0.5 L of forward primer (10 M), 0.5 L of reverse primer (10 M), and 10.5 L of sterile distilled water. Thermal cycling conditions used for MCMV detection consisted of 94 ◦ C for 5 min, 30 cycles at 94 ◦ C for 30 s, 58 ◦ C for 30 s, and 72 ◦ C for 45 s. A final elongation step was performed at 72 ◦ C for 10 min. The PCR products (5 L) were analyzed by electrophoresis on 1% agarose gels, followed by ethidium bromide staining. 2.4. Optimized RT-LAMP conditions RNA Amplification Kit (Eiken, Shanghai, China) was used for the RT-LAMP reactions. The concentration of total RNA was 500 ng/L. The RT-LAMP reaction mixture contained 12.5 L of 2 × Reaction Mix, 5 L of total RNA template (2.5 g), 1 L of Enzyme Mix, 1 L of Fluorescent Detection Reagent, 1 L each of the FIP and BIP primers (10 M), 1.25 L each of the F3 and B3 primers (1 M), 1 L of distilled water. Six different temperatures (from 60 to 65 ◦ C) and five different durations of reactions (15 min, 30 min, 45 min, 60 min, and 75 min) were performed to optimize the reaction conditions. The mixtures were incubated at 95 ◦ C for 2 min to terminate the reactions. The optimized RT-LAMP was carried out at 63 ◦ C for 60 min and terminated at 95 ◦ C for 2 min. 2.5. Analysis of RT-LAMP products The RT-LAMP products (0.5 L) were analyzed by electrophoresis on a 2% agarose gel and subsequently stained with ethidium bromide. Lanes containing a laddered amplification pattern were considered positive, while lanes containing no visible bands were regarded as negative. Meanwhile, the reaction tubes were visualized under UV light (3UV Transilluminator, wavelength: 365 nm), and positive samples showed green fluorescence, whereas negative samples remained original orange. 2.6. Specificity of RT-LAMP To confirm the specificity of the RT-LAMP, the reaction products were purified, digested with EcoR I, repurified, and cloned into the vector pUC-18, which was pre-digested with EcoR I, purified, dephosphorylated with Thermosensitive Alkaline Phosphatase (Promega, Madison, WI, USA) and repurified. The recombinant plasmids were subsequently sequenced. The specificity of the assay was also tested by RT-LAMP reactions that used maize-infecting viruses (SCMV, PenMV, RBSDV), healthy maize plants and distilled water. The presences of MCMV, SCMV, PenMV and RBSDV in the samples was determined by RT-PCR using total RNA extracted from the systemically infected leaves of MCMV-, SCMV-, PenMV- and RBSDV-inoculated maize plants, respectively, and the primers used are provided in Supplementary Table 1. All of the RT-LAMP assays were performed as the optimized reaction system and conditions described above.
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Fig. 1. Test of the specificity for RT-LAMP. (A) Agarose gel electrophoresis of the RT-LAMP products. (B) Visual fluorescence detection of the RT-LAMP products. Lane M, Trans 2 K Plus DNA marker; lane 1, MCMV-infected maize leaves; lane 2, RBSDVinfected maize leaves; lane 3, SCMV-infected maize leaves; lane 4, PenMV-infected maize leaves; lane 5, healthy maize leaves; lane 6, distilled water.
2.7. Sensitivity of RT-LAMP and RT-PCR To compare the relative sensitivity of the RT-LAMP and RTPCR, 10-fold serial dilutions of the virus-positive total RNA extracts (2.5–2.5 × 10−9 g) were used. Both the RT-PCR and RT-LAMP reactions were carried out as described above. 3. Results 3.1. Specificity of RT-LAMP In the specificity test, the presence of MCMV, SCMV, PenMV and RBSDV was determined by RT-PCR, respectively (Fig. S1). However, only RT-LAMP products from MCMV-infected maize leaves showed a typical ladder-like pattern, while no amplicons were detected for other maize-infecting viral pathogens or the control (Fig. 1A). After adding SYBR Green I, only the reaction products of MCMVinfected samples turned green in accordance with the results of gel-based analysis (Fig. 1B). The small fragments from the RT-LAMP products were cloned into the vector pUC-18 and subsequently sequenced. The sequencing results of four positive clones showed that the length of the fragments had over 98% nucleotide sequence identity to the known MCMV sequences (Fig. S2). These results demonstrated that the established RT-LAMP procedure for MCMV detection was highly specific and no cross-reactivity with the other three maize-infecting viruses was detected (Fig. 1). 3.2. Sensitivity of RT-LAMP The sensitivity of the RT-LAMP assay and RT-PCR was compared using a dilution series of total RNA extracted from MCMV-infected maize leaves. The results of agarose gel electrophoresis and SYBR Green I staining showed that the RNA at dilutions of up to 2.5 × 10−6 g was detectable by RT-LAMP, while RT-PCR only gave positive results up to dilutions of 2.5 × 10−5 g (Fig. 2). These results showed that this RT-LAMP assay was about 10-fold more sensitive than RT-PCR.
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Fig. 2. Comparison of the relative sensitivity of RT-LAMP and RT-PCR for MCMV detection. (A) Sensitivity of RT-LAMP analyzed by agarose gel electrophoresis. (B) Sensitivity of RT-PCR analyzed by agarose gel electrophoresis. (C) Visual fluorescence detection of the RT-LAMP products corresponding to those in agarose gel electrophoresis analysis. Lane M, Trans 2 K Plus DNA marker; lanes 1–10, 10-fold serial dilutions of RNA isolated from MCMV-infected maize leaves.
3.3. Field sample detection with RT-LAMP To evaluate the feasibility of the established one-step RT-LAMP assay for the detection of MCMV in the field, 23 maize samples showing MCMV or virus-like symptoms were tested by RT-LAMP and RT-PCR, respectively. The results showed that 16 samples were detected MCMV positive by RT-LAMP assay, and only 14 samples were positive by RT-PCR. However, other two tested samples of maize plants developed typical symptoms soon, thus can be an indicator that they were infected when the samples were collected. In addition, the results of selected samples indicated that the RTLAMP products showed clear ladder patterns in the agarose gel electrophoresis and green fluorescence in the SYBR Green I staining (Fig. 3A and C). Therefore, the developed RT-LAMP was more sensitive than RT-PCR for detecting MCMV in the field. 4. Discussion MCMV, an important maize-infecting virus, has been spreading in major maize-planting regions worldwide. It can be a devastating disaster for maize production, because the severe CLN disease causing by synergistic interaction between MCMV and a potyvirid could lead to about 90% yield loss (Castillo and Hebert, 1974; Goldberg and Brakke, 1987; Niblett and Claflin, 1978). Moreover, MCMV is widely spread as a result of the increased international trade, largescale planting of MCMV natural hosts and the diverse transmission. In recent years, MCMV has been reported in many countries especially in Asia and Africa (Deng et al., 2014; Lukanda et al., 2014; Mahuku et al., 2015), highlighting the importance of MCMV surveillance. Therefore, it is necessary to develop a simple, rapid, sensitive, and efficient diagnostic technique to detect this virus. In this study, one-step RT-LAMP was established to successfully detect MCMV. The optimized RT-LAMP was performed rapidly and simply compared with other methods such as enzyme-linked
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simultaneously (Hasiów-Jaroszewska et al., 2015). Therefore, it is necessary to determine whether this RT-LAMP can detect MCMV from other hosts or simultaneously detect a potyvirid which acts synergistically with MCMV. The sensitivity of the established RTLAMP assay was also demonstrated using field-grown samples (Fig. 3), and one positive sample detected by RT-LAMP gave false negative results in the RT-PCR assay (Fig. 2, lane 7; Fig. 3, lane 9). Another advantage of one-step RT-LAMP is its rapidity, and much more time could be saved once the rapid RNA isolation method is developed. In conclusion, a one-step RT-LAMP assay was developed for rapid and sensitive detection of MCMV in field-grown maize to prevent the possible spread of this virus. The visual LAMP method could be used for high-throughput screening for field samples. Acknowledgements We thank Dr Kay Scheets (Oklahoma State University) for providing the infectious cDNA clone of MCMV (pMCM41). This work was supported by grants from the Ministry of Agriculture (2014ZX08003-001) and the Ministry of Education (the 111 Project B13006), and a Special Fund for Detecting Plant Viruses (201310068, 2012BAK11B02) from the General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China. Fig. 3. Detection of natural viral infection in selected field-grown maize samples with RT-LAMP and RT-PCR. (A) Using RT-LAMP method. (B) Using RT-PCR method. (C) Visual fluorescence detection of the RT-LAMP products corresponding to those in agarose gel electrophoresis analysis. Lane M, D2000 DNA marker; lanes 1–9, random field samples collected from Yuxi region; lane 10, healthy maize leaves; lane 11, distilled water.
immunosorbent assay (Uyemoto, 1980), immunofluorescence (Nault et al., 1979), RT-PCR, Real-time TaqMan RT-PCR (Zhang et al., 2011), and next generation sequencing (Adams et al., 2013). The reaction products of RT-LAMP could be visualized using agarose gel electrophoresis and a fluorescent dye, allowing the results to be observed visually. The use of agarose gel electrophoresis as a visualization method is relatively time-consuming; however, each positive reaction can be observed. The visualization method using the addition of SYBR Green I can show a positive reaction directly without the use of any instruments, but subtle differences between positive and negative samples cannot be distinguished easily (Fig. 3, lane 9) as previously reported (Zhao et al., 2015). Thus, addition of a fluorescent dye facilitates high-throughput screening of field-grown maize samples, and in some cases, agarose gel electrophoresis can be used to confirm the results such as quarantine detection of MCMV. The specificity of the primers used is a key issue of the successful LAMP assay. The four primers were designed based on the sequences of highly conserved regions of the MCMV CP gene, which were obtained from available sequences of different MCMV isolates. In addition, MCMV from the full-length cDNA clone and field maize samples are different isolates, suggesting that this RT-LAMP procedure could be used to detect different MCMV isolates from maize samples. Meanwhile, this RT-LAMP procedure had no crossreactivity with other maize-infecting viral pathogens especially SCMV, which acts synergistically with MCMV leading to CLN disease. In addition, PenMV also belongs to the genus Potyvirus, family Potyviridae, which may have a potential co-infection with MCMV to cause CLN disease. Thus, this RT-LAMP procedure could be used effectively to detect MCMV from field maize samples. Recently, RT-LAMP procedures have been established to detect Tomato black ring virus isolates collected from different hosts (Keizerweerd et al., 2015), and detect Sugarcane mosaic virus and Sorghum mosaic virus
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