Colorimetric detection of Maize chlorotic mottle virus by reverse transcription loop-mediated isothermal amplification (RT-LAMP) with hydroxynapthol blue dye

Abstract

Maize chlorotic mottle virus causes corn lethal necrosis disease, and can be transmitted via infected maize seeds. It remains a challenge to detect this virus to prevent its introduction, infection and wide transmission in fields. For this purpose, a colorimetric assay for the detection of Maize chlorotic mottle virus was developed, which utilises RT-LAMP and hydroxynapthol blue dye (HNB). The reaction was performed for amplification in one step in a single tube at the optimum conditions (64 °C for 60 min, 150 μM HNB and 2 mM MgSO₄). Samples infected with MCMV developed a characteristic sky blue color after the reaction but those uninfected with MCMV or infected with other plant pathogenic viruses did not. The results of the HNB staining method were reconfirmed through gel electrophoresis of the RT-LAMP products. The sensitivity of this assay was 4.8 pg μl⁻¹ of RNA of Maize chlorotic mottle virus per reaction, which was approximately 10-fold higher sensitivity over a conventional RT-PCR test. The results indicate that this assay is highly species-specific, simple, low-cost, and visual for easy detection of Maize chlorotic mottle virus in plant tissues. Therefore, colorimetric detection of Maize chlorotic mottle virus is a potentially useful tool for middle or small-scale corporations and entry-exit inspection and quarantine bureaus to detect maize seeds or plant tissues infected with Maize chlorotic mottle virus.

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Introduction

Maize chlorotic mottle virus (MCMV) is the only species in the genus Machlomo virus (family Tombusviridae), and it is a single strand RNA virus. As an important plant pathogenic virus, MCMV was first reported to infect Zea mays in Peru¹ where it caused losses of 10–15% in floury and sweet corn cultivars. The combination of MCMV with the Maize dwarf mosaic virus, Sugarcane mosaic virus, or Wheat streak mosaic virus may also cause a severe symptom known as maize lethal necrosis.² In addition, this virus can be introduced readily into other countries by seeds or vectors.^2,3 Because of the potential threat to the production of maize crops, it was listed as a quarantine pest by the Chinese government in 2007, and was identified in maize seeds imported from the United States, Germany, and Mexico, indicating a high risk of MCMV introduction with the increasing international exchange of maize seeds. In order to prevent the introduction of MCMV through the international exchange of maize seeds and its wide transmission in the fields, many kinds of assays have been used for the detection of MCMV, including biological indexing,⁴ ELISA,⁵ electron microscopy,⁶ real-time RT-PCR,⁷ and surface plasmon resonance.⁵ However, biological indexing is time-consuming, and labor-intensive; the results of ELISA are dependent on the quality and availability of expensive antibodies; electron microscopy, real-time RT-PCR and surface plasmon resonance require very expensive equipment; gold nanoparticles are not extremely stable; RT-PCR entails the requirement of running gels, which increases the risk of contamination during post-PCR manipulations.

More and more assays have been developed for the detection of biomolecules, such as DNA/RNA,^8–11 proteins,¹² dopamine,¹³ interleukin-6 ¹⁴ and cysteine.¹⁵ Furthermore, loop-mediated isothermal amplification (LAMP), a novel constant temperature nucleic acid amplification technique, has been demonstrated to be a rapid, low-cost, easy to operate, highly sensitive and specific detection method, applicable in several fields.^16,17 Under isothermal conditions, the target sequences are amplified with a high efficiency, rapidity and specificity according to 5 primers (two internal and two external primers, and one loop primer) designed for several regions of the target gene and Bst DNA polymerase with strain displacement activities. In addition, accelerated amplification via loop primers reduces the building of artefacts to a minimum.¹⁸ Furthermore, LAMP also offers the possibility of analysis without gel electrophoresis, for instance by measuring the turbidity of magnesium pyrophosphate that is formed as a side product in positive reactions using dyes like SYBR-green¹⁹ or hydroxynapthol blue (HNB).²⁰ The simplicity of the LAMP method, which does not require special equipment including a thermal cycler, makes it suitable for field testing. To date, this method has been successfully used for detections of Mycobacterium tuberculosis, and Listeria monocytogenes.^19,21 In this paper, we developed a rapid LAMP method for diagnosis of MCMV and explored the optimal reaction conditions to establish a detection system for MCMV and verified its reliability through detection of contaminated samples. Moreover HNB is used in our assays, which enables an easier, cheaper and more sensitive discrimination of positive (blue) and negative (violet) reactions with the naked eye compared to other methods for LAMP product detection.

Materials and methods

Materials and reagents

MCMV-Agdia2219 was purchased from Agdia, USA; MCMV-ZJ was provided by Beijing Entry-Exit Inspection and Quarantine Bureau, Beijing, China; MCMV-BJ was obtained from China Agricultural University; MCMV-field was obtained from the field; MCMV-1087, MCMV-2094, and MCMV-1907 isolates were supplied by Shanghai Entry-Exit Inspection and Quarantine Bureau, Shanghai, China. Carnation ringspot virus (CRSV), Odontoglossum ringspot virus (ORSV), Cucumber green mottle mosaic virus (CGMMV), Lily symptomless virus (LSV), Cymbidium mosaic virus (CymMV), and Southern bean mosaic virus (SBMV) isolates were kept in Shanghai Entry-Exit Inspection and Quarantine Bureau, Shanghai, China. MCMV was inoculated in Zea mays, and the infected tissues were harvested. Arabis mosaic virus (ArMV), Carnation ringspot virus (CRSV), and Tobacco rattle virus (TRV) were inoculated in Chenopodium quino, CGMMV was inoculated in Cucumissativus, and the infected tissues were harvested after 14 days. The leaf of Phalaenopsis aphrodite with ORSV, and lily bulbs with LSV were intercepted in entry plants at a port. DNA Marker I was purchased from TIANGEN Biotech (Beijing) Co., Ltd (Beijing. China), Bst DNA polymerase was purchased from NEB, and betaine and HNB were purchased from Sigma Co., Ltd. AMV reverse transcriptase was purchased from Takara Biotech Co., Ltd (Dalian, China).

Viral RNA extraction

Viral genomic RNA was extracted from 100 mg of leaves infected with the virus, using the TIANamp Virus RNA Kit (Beijing. China) according to the manufacturer’s instructions. The RNA was eluted in 70 μl of RNase-free water and stored at −80 °C.

Primer design

A sequence alignment of the 400-bp sequences located at nucleotide position 4001–4400 of nine MCMV genomes (GenBank accession no. KP851970.1, NC_003627.1, KJ782300.1, KF010583.1, JQ982470.1, JQ982469.1, GU138674.1, EU358605.1 and X14736.2) was undertaken using Vector NTI Advance version 11 (Invitrogen, Auckland, New Zealand). A reference template was generated according to the conserved region of the alignment. Primer explorer v3 software (http://primerexplorer.jp/e/index.html) was used to design the MCMV specific primers. The primers consisted of two outer primers (F3 and B3), two inner primers (FIP and BIP), and one loop primer (LF). The specificity of the primer and probe sequences was analyzed using the Basic Local Alignment Search Tool (BLAST). The sequences of the oligonucleotide primers were synthesized by Sangon Biotech (Shanghai, China) and are shown in Table 1, and Fig. 1 describes primers for the RT-LAMP method.

Table 1 The RT-LAMP primers used in this study

Primers	Sequences
F3	5′-AGACCGGAATAACCAGTCCT-3′
B3	5′-TGCCCCAGGGTTAAGTGTA-3′
LF	5′-CGATTTAGGCTCCCAAACA C-3′
FIP	5′-CTCCAGTCATGGTCATCACGCATTTTGGCAGAGTCCTGCCAATC-3′
BIP	5′-CAACCGCAGACTGGGCGTATTTTTGCACCGTTCGTAAGTACGT-3′

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Fig. 1 Position and orientation of MCMV RT-LAMP primers within the reverse nucleotide sequence of MCMV (GenBank accession no. EU358605.1). Nucleotide positions from 40 001 to 4400 are shown.

Optimization of RT-LAMP reaction conditions

Optimization of the concentration of the MgSO₄ in the reaction system was performed as followed. The RT-LAMP reaction was carried out in 25 μl of a reaction mixture containing 20 mM Tris–HCl (pH 8.8), 10 mM KCl, 10 mM (NH₄)₂SO₄, 0.1% Triton X-100, 0.8 M betaine, 1.2 mM dNTPs, 1.6 μM FIP primer, 1.6 μM BIP primer, 0.2 μM F3 primer, 0.2 μM B3 primer, 0.8 μM LF primer, 1 μl template RNA, 10 U Bst DNA polymerase, 5 U AMV reverse transcriptase, dNTPs (2.5 mM), and 0, 2 mM, 3 mM, 4 mM, 6 mM, or 8 mM MgSO₄, with the addition of ultrapure water to 25 μl. The RT-LAMP reaction was performed at 64 °C for 60 min and then heated at 80 °C for 5 min to terminate the reaction. The amplified products were analyzed using 2% agarose gel electrophoresis and the results were documented using a gel imaging system.

Optimization of the reaction temperature of the reaction system was carried out according to the procedure below, the RT-LAMP reaction was carried out in 25 μl of a reaction mixture containing 20 mM Tris–HCl (pH 8.8), 10 mM KCl, 10 mM (NH₄)₂SO₄, 0.1% TritonX-100, 0.8 M betaine, 1.2 mM dNTPs, 1.6 μM FIP primer, 1.6 μM BIP primer, 0.2 μM F3 primer, 0.2 μM B3 primer, 0.8 μM of LF primer, 1 μl template RNA, 10 U Bst DNA polymerase, 5 U AMV reverse transcriptase, dNTPs (2.5 mM), 2 mM MgSO₄ and ultrapure water was added to 25 μl. The RT-LAMP reaction was performed at 60, 62, 64, and 66 °C for 60 min and then heated for 5 min at 80 °C to terminate the reaction. The amplified products were analyzed using 2% agarose gel electrophoresis and the results were documented using a gel imaging system.

Optimization of the reaction time of the reaction system was followed using the protocol below, the RT-LAMP reaction was carried out in 25 μl of a reaction mixture containing 20 mM Tris–HCl (pH 8.8), 10 mM KCl, 10 mM (NH₄)₂SO₄, 0.1% Triton X-100, 0.8 M betaine, ​1.2 mM dNTPs, 1.6 μM FIP primer, 1.6 μM BIP primer, 0.2 μM F3 primer, 0.2 μM B3 primer, 0.8 μM of LF primer, 1 μl template RNA, 10 U Bst DNA polymerase, 5 U AMV reverse transcriptase, dNTPs (2.5 mM), 2 mM MgSO₄ and ultrapure water was added to 25 μl. The LAMP reaction was performed at 64 °C for 30, 45, 60, 75 or 90 min, and then heated at 80 °C for 5 min to terminate the reaction. The amplified products were analyzed using 2% agarose gel electrophoresis and the results were documented using a gel imaging system.

Optimization of the concentration of the HNB in the reaction system was performed as follows, the RT-LAMP reaction was carried out in 25 μl of a reaction mixture containing 20 mM Tris–HCl (pH 8.8), 10 mM KCl, 10 mM (NH₄)₂SO₄, 0.1% TritonX-100, 0.8 M betaine, 1.2 mM dNTPs, 1.6 μM FIP primer, 1.6 μM BIP primer, 0.2 μM F3 primer, 0.2 μM B3 primer, 0.8 μM LF primer, 1 μl template RNA, 10 U Bst DNA polymerase, 5 U AMV reverse transcriptase, dNTPs (2.5 mM), 2 mM MgSO₄, and 100, 120, 150, or 200 μM HNB, and ultrapure water was added to 25 μl. The RT-LAMP reaction was performed at 64 °C for 60 min and then heated at 80 °C for 5 min to terminate the reaction. The amplified products were analyzed using 2% agarose gel electrophoresis and the results were documented using a gel imaging system. In addition, the amplification product could also be visually inspected according to a color change from violet to sky blue, while the negative control remained violet. The tubes were observed by the naked eye and photographed under natural light.

Specificity of colorimetric detection

The specificity of the RT-LAMP was verified by performing the assay with the RNA of MCMV-Agdia2219 and other viral samples, including CRSV, ORSV, CGMMV, LSV, CymMV and SBMV. The RT-LAMP assay was done as described earlier. As before, the assays were assessed using gel electrophoresis and the HNB-visualized color change.

Sensitivity of colorimetric detection

The sensitivity of the RT-LAMP was estimated through detecting seven serial dilutions containing serial 10-fold dilutions of RNA (48 ng μl⁻¹ to 4.8 fg μl⁻¹). The RT-LAMP assay was done as described earlier. As before, the assays were assessed using gel electrophoresis and the HNB-visualized color change.

Colorimetric detection of MCMV from artificially contaminated maize leaves

In order to evaluate the capability of the colorimetric detection of MCMV, six maize leaves contaminated with viruses analyzed using conventional RT-PCR were tested as described above.

Results

Determination of the optimal reaction conditions of RT-LAMP for MCMV

The RT-LAMP was performed using MCMV genomic RNA as the template to determine the optimal temperature, reaction time and concentration of MgSO₄ in the study. The assay was also used to optimize the concentration of HNB for an optimal hydroxynaphthol blue color change during the reaction. A successful RT-LAMP with specific primers at 60–66 °C for 60 min produced many bands of different sizes upon agarose electrophoresis because the RT-LAMP products consisted of several inverted-repeat structures (Fig. 2). It was evident that the distinct bands at 64 °C were brighter than those of the other reaction temperatures (Fig. 2), so the optimal temperature for this RT-LAMP is 64 °C. The optimum concentration of MgSO₄ required for amplification by the RT-LAMP was investigated in the study, using the range from 0 to 8 mM, and the results suggested that efficient amplification of template RNA could be obtained at 2 mM MgSO₄ (Fig. 3). However, positive reactions were detected using different reaction times between 30 and 90 min (Fig. 4). No difference was observed when the RT-LAMP assays were performed in 60, 75 and 90 min. No amplification was observed for 30 min and the amplifications achieved at 45 min were less than others. Hence, a reaction time of 60 min was selected as the optimum reaction time for the RT-LAMP assay. Optimization of the RT-LAMP reaction conditions (temperature, time and concentration of MgSO₄) revealed that the ideal settings for the primer set were 64 °C for 60 min and a concentration of MgSO₄ of 2 mM. Therefore, the RT-LAMP assays were subsequently performed under these conditions. According to the optimal reaction conditions of the RT-LAMP for MCMV, the concentration of HNB was tested and optimized within the range of 100 to 200 μM and was optimum at 150 μM. The RT-LAMP products were detected both by agarose gel electrophoresis and by visual inspection (Fig. 5A and B).

Fig. 2 Temperature optimization results of RT-LAMP amplified MCMV. Lane M: DNA marker 600; lane 1–4: 60, 62, 64, and 66 °C, respectively.

Fig. 3 Mg²⁺ optimization results of RT-LAMP amplified MCMV. Lane M: DNA marker 600; lane 1–6: 0, 2, 3, 4, 6, and 8 mM, respectively.

Fig. 4 Time optimization results of RT-LAMP amplified MCMV. Lane M: DNA marker 600; lane 1–5: 30, 45, 60, 75, and 90 min, respectively.

Fig. 5 HNB optimization results of RT-LAMP detection for MCMV. (a) RT-LAMP detection of MCMV using electrophoresis; (b) color reaction with HNB. Lane M: DNA marker 600; lines 1, 3, 5, 7: positive reaction with HNB; lines 2, 4, 6, 8: negative reaction with HNB; line 1, 2: 100 μM; line 3, 4: 120 μM; line 5, 6: 150 μM; line 7, 8: 200 μM.

Specificity test results

The specificity of the RT-LAMP was verified by performing the assay with the RNA of MCMV-Agdia2219 and other viral samples, including CRSV, ORSV, CGMMV, LSV, CymMV and SBMV. The RT-LAMP products were detected both by agarose gel electrophoresis and by visual inspection (Fig. 6), and the results show that only maize leaves infected with MCMV could cause amplification of the bright, specific band in lane 1, as shown in Fig. 6A, and a blue color change can be seen in the corresponding sample in Fig. 6B. The other viruses and ddH₂O could not amplify the products as shown in Fig. 6A and remained violet, as shown in Fig. 6B. The RT-LAMP amplified only the MCMV genome with no cross-reactivity with the other viruses tested in the colorimetric assay. The colorimetric assay can be used to visually observe a change of color and no electrophoresis instrument is needed. The results suggested the colorimetric detection of MCMV was highly specific to MCMV.

Fig. 6 Specificity analyses of colorimetric detection of MCMV. (A) RT-LAMP detection of MCMV using electrophoresis; (B) color reaction with HNB. Lane M: 600 bp DNA marker; lane 1: MCMV-Agdia2219; lane 2: CRSV; lane 3: ORSV; lane 4: CGMMV; lane 5: LSV; lane 6: CymMV; lane 7: SBMV; lane 8: ddH₂O.

Sensitivity of colorimetric detection of MCMV

In order to determine the sensitivity of the colorimetric detection of MCMV, RT-LAMP was performed using 10-fold serial dilutions of MCMV-Agdia2219 genomic RNA. The RT-LAMP products were detected both by agarose gel electrophoresis and by visual inspection (Fig. 7). The RT-LAMP showed successful amplification when the concentrations of template were no less than 4.8 pg μl⁻¹ (Fig. 7A). Simultaneously, the reaction products were blue (Fig. 7B). Therefore, the sensitivity of the colorimetric detection was 4.8 pg μl⁻¹ MCMV genomic RNA.

Fig. 7 Sensitivity analysis of colorimetric detection of MCMV. (A) RT-LAMP detection of MCMV using electrophoresis; (B) color reaction with HNB. Lane M: DNA marker 600; lane 1–8: diluted samples of MCMV RNA crude solution, 48 ng μl⁻¹, 4.8 ng μl⁻¹, 480 pg μl⁻¹, 48 pg μl⁻¹, 4.8 pg μl⁻¹, 480 fg μl⁻¹, 48 fg μl⁻¹, 4.8 fg μl⁻¹, respectively. Lane 9: negative reaction.

Detection MCMV in crude extractions from artificially contaminated maize leaves

The utility of the colorimetric detection assay for MCMV was examined using five artificially contaminated maize leaves, one field leaf infected with MCMV and six other viruses (CRSV, ORSV, CGMMV, LSV, CymMV and SBMV). The results indicated that in this test, six samples were MCMV positive as tested by the RT-LAMP and colorimetric assay (Fig. 8A and B). These samples had also been analyzed using conventional RT-PCR (data unpublished). The detection accuracy of the colorimetric assay was 100% when compared with the RT-PCR. The results showed that it is reliable to use a colorimetric assay to detect MCMV in samples.

Fig. 8 Results of colorimetric assay for MCMV detection. (a) RT-LAMP detection of MCMV using electrophoresis; (b) color reaction with HNB. Lane M: DNA marker 600; lane 1: MCMV-ZJ; lane 2: MCMV-1087; lane 3: MCMV-C2094; lane 4: MCMV-C1907; lane 5: MCMV-BJ; lane 6: MCMV-field; lane 7: CRSV; lane 8: ORSV; lane 9: CGMMV; lane 10: LSV; lane 11: CYmMV; lane 12: SBMV; lane 13: ddH₂O.

Discussion

Recently, the moving protein gene has been used as a marker for the Cucumber green mottle mosaic virus,^22,23 the signal transduction gene, vicK has been identified as a molecular marker for the detection of Staphylococcus aureus and the lmo0460 sequence has been identified as a molecular marker for the detection of Listeria monocytogenes in our previous research based on comparative genome and sequence alignment analysis,^21,24,25 which implies that some sequences in the genome can be used as molecular markers for viruses except for the coat protein gene.^21,24,26 Based on a similar strategy, a 400-bp sequence (X14736.2) has been identified as a novel molecular marker for the detection of MCMV by means of bioinformatics. Simultaneously, RT-LAMP also confirmed the result in our study.

Huang et al. reported that the sensitivity of a quartz crystal microbalance based sensor for the detection of MCMV is 250 pg μl⁻¹, which is similar to that of the existing ELISA method.²⁷ However, in this study, a colorimetric assay was developed to detect MCMV with the sensitivity to detect as low as 4.8 pg of total RNA in the reaction system, which was more sensitive than a quartz crystal microbalance based sensor for MCMV diagnosis (Table 2). Compared with conventional RT-PCR (30 pg of total RNA), the colorimetric detection of MCMV had significantly higher sensitivity levels. This result was concordant with previous reports of the LAMP method for detecting Listeria monocytogenes.²⁸

Table 2 Several assays for detection of MCMV^a

Methods	Materials	Linear range	Detection limit	References
a ***** show no data.
Biological indexing	Maize leaf	*****	*****	4
ELISA	Maize leaf	1–1000 ppb	100 ppb	5
Electron microscopy	Maize leaf	*****	*****	6
Real-time RT-PCR	Maize seeds	≥4 fg	4 fg	7
SPR	Maize leaf	1–1000 ppb	100 ppb	5
RT-LAMP	Maize leaf	≥4.8 pg	4.8 pg	In this study

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The reliability of the performance was evaluated using eleven maize leaves artificially contaminated with viruses, and one field sample, all of which had been analysed using conventional RT-PCR. Of all the analysed samples, the rate of coincidence in terms of consistent results between the RT-PCR and the colorimetric assay was 12 out of 12 samples (100%), and the results indicated that the colorimetric assay is valid. China has strengthened the prevention and control of the introduction of maize seed through the international exchange of maize seeds so there is seldom a report on MCMV transmission, and it is very difficult to gain field isolates. Therefore, MCMV transmission in the corn field was simulated in our lab, where artificially infected samples were prepared to evaluate the novel assay for MCMV detection. The results showed that the positive results of the infected samples were the same, which was due to the specificity and reliability of the RT-LAMP. Since we did not have access to more field isolates, the current method has only been tested on the limited number of known MCMV isolates. Therefore, we hope to be able to conduct more trials with field isolated viruses to establish the fundamentals for assay applications.

In recent years, severe chlorotic mottle symptoms found in sweet corn or sugarcane have been observed at the base of leaves infected with MCMV in many countries and regions.^26,29,30 Sugarcane and corn in fields were also found to be infected with MCMV in the Yunnan province, China in 2013. This colorimetric assay only required a simple sample preparation and the results were obtained in less than two hours, which make it a rapid, sensitive, and simple detection of MCMV. This colorimetric assay for MCMV detection is very suitable for detection in field leaf samples compared to other assays and requires only sample preparation and RT-LAMP.

Conclusion

In this work, we took full advantage of RT-LAMP combined with a HNB-based color change and provided a low-cost, easy to operate, and sensitive assay for the visual detection of MCMV. The assay described is easily read with the naked eye. In comparison with other methods for the detection of MCMV, the method is more attractive because of its high sensitivity, low cost, ready availability and simple manipulation. This is the first application of RT-LAMP with HNB-based color change for the detection of MCMV by the naked eye without the need for expensive detection instruments.

Acknowledgements

This research was financed by grants from the Science Project of General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China (2014IK014), and Natural Science Foundation of Shanghai (15ZR1415600).

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