Rapid diagnosis of Tobacco mosaic virus in tobacco using time-resolved fluorescence immunoassay

ABSTRACT

Tobacco mosaic virus (TMV) causes a significant yield loss in solanaceous crops. Accurate and rapid diagnosis of TMV is an essential pre-requisite for proactive and tailored disease management strategies, filling the gap between more rigorous laws regarding pesticide use and maintaining crop production. Herein, we developed a time-resolved fluorescence immunoassay (TRFIA) to quantitatively detect TMV in tobacco. TMV virions were used as immunogens to develop monoclonal antibodies, which were then conjugated to Eu³⁺ nanospheres. The detection limit of the TRFIA platform was 0.24 ng/mL, with recovery rates in the range of 90.71–108.11% in spiked tobacco. The specificity was confirmed using ten species of tobacco-infecting phytopathogens. In-field applicability was implemented by screening for TMV in 10,000 tobacco leaf samples collected from five fields in Kunming, China. Overall, the proposed TRFIA platform is rapid, accurate, and end-user friendly, demonstrating its application in TMV onsite testing in tobacco.

KEYWORDS:

• Tobacco mosaic virus
• time-resolved fluorescence immunoassay
• rapid diagnostic test
• lateral flow immunoassay
• field detection

Introduction

Tobacco mosaic virus (TMV) is a positive-sense RNA virus that causes substantial losses in yield and quality of tobacco (Nicotiana tabacum L.) and other Solanaceae members (Johnson & Main, 1983). The virus is highly stable and hardy as it remains viable on work surfaces and clothing, even for decades in tobacco products (Balique et al., 2012; Lee et al., 2021). The symptoms of a tobacco plant infected with TMV vary greatly from leaf mottling or curling to stunted growth – these phenotypes are depending upon factors such as viral strains, the plant cultivar / growth stage and various environmental stimuli. TMV-mediated phenotypes can be difficult to distinguish from those caused by other viruses and thus far, there are very few effective treatments post infection.

Rapid and accurate diagnosis of TMV infections are important pre-requisites in the prevention and control of large-scale outbreaks, which poses a significant challenge since during the early stages of infection plant tissues have low viral titres and lack the quantifiable phenotypes that are apparent in the later stages of infection. Currently, common detection methods for TMV include electron microscopy techniques (Fromm et al., 2015), genetic screening using PCR (Kumar et al., 2011; Li et al., 2018; Liu et al., 2019; Yang et al., 2012), and ELISA-based immunoassays (Kumar & Prakash, 2012; Lamptey et al., 2013). Reverse transcription PCR (RT–PCR) is considered the gold standard for the detection of RNA viruses at the species or genus level, which is achieved by amplification of viral cDNA (Li et al., 2018). Techniques based on nucleic acid amplification such as real-time PCR (Ellis et al., 2020), multiplex RT–PCR (Liu et al., 2019), immunocapture PCR (Yang et al., 2012), reverse transcription loop-mediated isothermal amplification (RT-LAMP), and reverse transcription recombinase polymerase amplification-CRISPR/Cas12a (Aman et al., 2020) have been applied for the detection of TMV with additional advantages. For example, coupled with post amplification detection approaches, RT-LAMP not only offers better sensitivity and efficiency than quantitative RT–PCR in real samples, but also eliminates the need of thermal cyclers, thus allowing fast in-field detection (Foo et al., 2020; Liu et al., 2010; Zhao et al., 2012). Nevertheless, these molecular methods still need RNA extraction, reverse transcription, and amplification procedures which take hours to complete (Malik et al., 2019).

Serological methods such as ELISA and lateral flow immunoassay (LFA) have been widely used in onsite plant virus diagnosis (Guo et al., 2022; Mehetre et al., 2021). Compared to ELISA, LFA strips provide benefits such as simple test procedures, rapid diagnosis, low cost, and long shelf life, meeting the need of end-users under in-field or resource-poor environments (Byzova et al., 2009; Posthuma-Trumpie et al., 2009). Although traditional LFA test strips are less sensitive than molecular methods, the advancement of nanoprobes has remarkably improved the detection limits and fulfilled quantitative requirements (Parolo et al., 2020; Porras et al., 2021; Xie et al., 2014). Notably, lanthanide chelates-based time-resolved fluorescence immunoassay (TRFIA) offers unique characteristics including long fluorescence lifetime, a large Stokes shift, broad excitation spectrum, and narrow emission spectrum. Coupled with a time-resolved fluorometer, TRFIA can be applied for rapid diagnosis test with high sensitivity, accuracy, a wide detection range, and low background interference (Bian et al., 2021; Chen et al., 2020).

Currently, no quantitative lateral flow test strip for TMV detection is available. Constant monitoring can provide insights of viral load, which is essential to deduce the risk of disease from quantitative observations. In this study, a TRFIA system for TMV detection was fabricated, and demonstrated its high specificity, sensitivity, great convenience, and quick turnaround by successful application in onsite diagnosis.

Materials and methods

Virus purification

TMV (GenBank HE818413.1) was inoculated in tobacco cultivar Samsun, followed by propagation in tobacco cultivar K326 in a phytotron with a 16 h light / 8 h dark cycle (Guo et al., 2022). TMV virions were purified using a method with minor modification (Zhou et al., 1994).

Antibody development

The procedure of anti-TMV monoclonal antibody (mAb) preparation was performed in accordance with the principle of the Helsinki Accords and approved by the Animal Experimentation Ethics Committee of Guizhou Academy of Tobacco Science, China. Eight-week-old female BALB/c mice (Beijing Vital River Laboratory Animal Technology Co., Ltd) were immunised with purified virions as described previously (Song et al., 2017). Hybridomas producing the desired mAbs were screened using indirect competitive ELISA (Xiao et al., 2018). mAbs were tested pairwise using colloidal gold lateral flow strips to identify pairs that exhibited strong binding to TMV.

Preparation of Eu³⁺ nanosphere-labelled detection mAb conjugates

Eu³⁺ nanosphere-labelled detection mAb conjugates were prepared as described previously (Ji et al., 2022). Briefly, Eu³⁺ time-resolved fluorescent nanospheres (100 μL, Microdetection Bio-Tech, Nanjing, China) were added to 400 μL borate buffer (50 mM, pH 8.0) and centrifuged at 17,000 g for 15 min at 10°C. The pellet was mixed with 500 μL borate buffer and sonicated for 1 min. Five microliters of 1-(3-Dimethylaminopropyl)−3-ethylcarbodiimide hydrochloride solution (1 mg/mL, J&K Scientific, Beijing, China) were added to the nanosphere solution and vortexed for 15 min. At the end of activation, nanospheres were rinsed once with 500 μL borate buffer, and mixed with the primary mAb (80 μg), followed by 2 h vortexing. Fifty-five microliters of borate buffer containing 10% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) were added to the mixture, which was further vortexed for 2 h. After the blocking step, the resulting solution was rinsed twice with borate buffer and finally resuspended in 500 μL borate buffer.

Fabrication of TRFIA test strips

The conjugate pad was drenched in Tris-HCl (50 mM, pH 8.0) and dried at 37°C. Eu³⁺ nanospheres-labelled detection mAb was diluted 10 times with 10 mM Tris buffer (pH 8.0, supplemented with 2% bovine serum albumin) sprayed onto the conjugate pad at a dispense rate of 2.5 μL/cm. Capture mAb against TMV and goat anti-mouse secondary antibody (GAM, Solarbio Life Sciences, Beijing, China) were diluted to 150 and 40 μg/mL with PBS (10 mM, pH 7.4), respectively. Diluted capture mAb and GAM were coated onto the nitrocellulose film (Sartorius, Goettingen, Germany) in parallel at 1 μL/cm to form a test line (T) and a control line (C). The pad and nitrocellulose film were dried at 37°C for 2 h. The sample pad, conjugate pad, nitrocellulose film, and absorbent pad were pasted successively on a polyvinyl chloride base plate (Figure 1(A)). Strips were cut according to 3.5 mm/strip, sealed, and stored at room temperature.

Figure 1. Schematic illustration of the TRFIA system. (a) Sample pretreatment and components of a TRFIA test strip. Tobacco leaves were roughly cut and mixed with extraction buffer. One-hundred microliters of supernatant were applied onto the sample pad, and the fluid flowed along the strip driven by capillary action. (b) In the absence of TMV, Eu³⁺-detection mAb only accumulated on the C line, resulting in a signal on the C line. (c) Detection mode in the presence of TMV, Eu³⁺-detection mAb accumulated on the T line and C line, both lines showed signals. (d) The test strip was incubated for 10 min at room temperature, until when the fluorescent signal was read using a portable fluorometer.

Application of TMV strips in spiked and real samples

The standard curve was established by plotting the T/C value against a series of TMV concentrations. To evaluate the performance of TRFIA test strips with the presence of matrix interference, 0.5 g healthy tobacco leaf sample was roughly cut and placed in a 1.5 mL tube. The sample was spiked with TMV virions at three concentrations and mixed thoroughly with 1 mL extraction buffer (containing 10 mM PBS and 1% Tween 20). Subsequently, 100 μL supernatant was transferred to the sample pad, and the T/C value was read after 10 min incubation at room temperature. TMV concentration was determined according to the standard curve.

The performance of TMV strips was further evaluated using tobacco leaves sampled from five tobacco-growing fields (Banqiao, Lufu, Dake, Xijiekou, and Shilin, Kunming, China) during July 2021 and transferred to a local resource-limited laboratory to quantify TMV. The sample size was 2000 for each location. Test procedure was carried out in accordance with the measurement of spiked samples.

Detection of TMV using RT–PCR

A TMV-infected tobacco leaf sample was homogenised with grinding in liquid nitrogen. The powder was equally divided into two (each weighed 60 mg). One portion was analysed using TRFIA strips, whilst the other portion was analysed using RT–PCR. Total RNA was extracted using the Genepure RNA Extraction Kit (Codonx, Beijing, China), followed by cDNA synthesis using the PrimeScript RT Reagent Kit (Takara Bio, Kusatsu, Shiga, Japan), as per manufactures’ instructions. Primers were designed targeting the CP gene within TMV sequences (Aman et al., 2020; Zhao et al., 2012). The primers were forward, 5′-CTATTTCTAGTGTCAAATGCACCTA-3′, and reverse, 5′-CAACAAGCTCGAACTGTCGT-3′. The internal control primers targeting the tobacco housekeeping gene (NtActin) were forward, 5′-TATTCCCTAGTATTGTTGGC-3′, and reverse, 5′-CTGGGGTATTAAAAGTCTCA-3′. For comparative evaluation with the TMV TRFIA system, viral cDNA solutions were diluted up to 10⁻⁷, which were used as templates for nucleic acid amplification. PCR cycling parameters were: one cycle at 95°C for 3 min; 30 cycles of denaturation at 95°C for 30 s, annealing at 54°C for 30 s, elongation at 72°C for 30s; and one cycle at 72°C for 5 min. PCR products were subjected to a 1% agarose gel.

TRFIA principle

TRFIA test strips were constructed based on double-antibody sandwich immunoassay (Figure 1(A)). In the absence of TMV, Eu³⁺ nanosphere-labelled detection mAb in the conjugate pad was captured by GAM secondary antibody (Figure 1(B)). Consequently, a fluorescent C line was formed, otherwise the result was considered as invalid. In the presence of TMV, TMV was recognised by Eu³⁺-labelled detection mAb, succeeded by reaction with capture mAb on the T line (Figure 1(C)). Excess unbound detection mAb reacted with GAM secondary antibody. Thereby, fluorescent signals were formed on the T line and the C line. The fluorescent intensity was read using a time-resolved fluorometer at 340 nm excitation wavelength and 610 nm emission wavelength (Figure 1(D), FD-10A, Zuoan-Tech, Zhengzhou, China). The fluorometer provides a T/C value, which is positively correlated with TMV concentration.

Results and discussion

Characterisation of mAbs

BALB/c mice were immunised with purified TMV virions prepared in Freund's complete adjuvant. Mice showing good antiserum titrations were chosen to generate hybridomas, succeed by injecting positive clones into mice intraperitoneally to produce antibodies. Indirect competitive ELISA showed that purified mAbs detected homogenised TMV-infected tobacco leaves at a titration of 1:51200 (dilution of homogenised TMV-infected tobacco leaves) and below (Figure 2(A)). Furthermore, the optimal mAb pair was determined between colloidal gold-conjugated mAb 1, mAb 2, and mAb 3 (Table 1). All mAb pairs showed a negative T line with blank extraction buffer, suggesting no pairs exhibited non-specific binding. mAb pairs 1-1, 1-3, and 3-3 showed good response to TMV virions, whereas mAb pair 1-2, 2-1, 2-2, 2-3, and 3-2 showed weak or no signal. Thus, mAb pair 1-3 was selected for further test strip construction.

Figure 2. Titration of mAb 1, 2, and 3 against homogenised TMV-infected tobacco leaves was detected using indirect competitive ELISA.

Table 1. mAb pair screening.

Capture mAb	Detection mAb	TMV virions	Blank
mAb 1	mAb 1	+	−
	mAb 2	−	−
	mAb 3	+	−
mAb 2	mAb 1	−	−
	mAb 2	−	−
	mAb 3	−	−
mAb 3	mAb 1	+	−
	mAb 2	−	−
	mAb 3	+	−

Immunological kinetics analysis

The reaction time was determined based on the immunological kinetics analysis. TMV virions were diluted to 1 ng/mL in extraction buffer, and the T/C value of the test strip was monitored for 30 min (Figure 3(A)). The value increased and reached the plateau at 10 min. Therefore, 10 min was decided as the optimal reaction time.

Construction of the standard curve

For quantitative analysis, a standard curve for TMV concentration was established. TMV virions were serial diluted in extraction buffer. The linear relationship between T/C value and TMV concentration was y = 0.0329x – 0.0049, with R² = 0.9965 (Figure 3(B)). The detection limit was 0.24 ng/mL, defined as the concentration corresponding to three times the standard deviation (SD) of the blank T/C value. In addition, a standard curve covering the lower concentration range was also constructed, y = 0.0404x – 0.025, R²= 0.9996 (Figure 3(C)).

Figure 3. Performance and evaluation of TMV TRFIA. (a) Immunological kinetics of the TRFIA test strip. (b) Standard curve of T/C value versus TMV concentration in the range of 0∼317 ng/mL. (c) Standard curve of T/C values versus TMV concentrations in the range of 0∼35 ng/mL. Data are presented as mean ± SD from three measurements. (d) Specificity of test strips. Test strips from left to right characterise the detection results of TMV, CMV, Poty, PVX, PVY, TEV, TRSV, TSWV, A. longipes, P. nicotianae, and R. solanacearum.

Cross-reactivity of test strips

To evaluate the specificity, TRFIA test strips were tested with cucumber mosaic virus (CMV), potyvirus (Poty), potato virus X (PVX), potato virus Y (PVY), tobacco etch virus (TEV), tobacco ringspot virus (TRSV), tomato spotted wilt virus (TSWV) (Agdia, Inc., Elkhart, IN, USA), as well as Alternaria longipes, Phytophthora nicotianae, and Ralstonia solanacearum (Zhengzhou Tobacco Research Institute, Zhengzhou, China). As shown in Figure 3D, all strips apart from the one tested TMV showed negative results, indicating high specificity towards TMV.

Precision of TRFIA

Intra-day and inter-day precision were tested at three TMV virion concentrations to evaluate the repeatability of test strips (Table 2). For the intra-day precision, 10 repeated measurements were performed on the same day. For the inter-day precision, each concentration level was tested in triplicate for five consecutive days. The coefficient of variation (CV) ranged from 9.5% to 16.5% for the intra-day precision, and 9.8∼16.6% for the inter-day precision. These results suggest high precision of test strips.

Table 2. Intra-day and inter-day precision.

Samples	Intra-day precision (n = 10)			Inter-day precision (n = 15)
	Mean (ng/mL)	SD	CV %	Mean (ng/mL)	SD	CV %
1	3.3	0.4	12.3	3.07	0.4	13.5
2	9.6	1.6	16.5	8.3	1.4	16.6
3	35.3	3.4	9.5	25.8	2.5	9.8

Spike-and-recovery analysis

Tobacco leaf extract was spiked with TMV virions at three concentrations (3, 9, and 27 ng/mL). As shown in Table 3, percentage recovery of TMV ranges from 90.7% to 108.1%, with a CV% ranging from 6.9% to 9.8%. These results demonstrate the present method has satisfactory accuracy and repeatability which could meet the in-field detection requirement.

Table 3. Recovery of TMV in spiked tobacco samples.

Spiked sample (ng/mL)	Mean ± SD	CV % (n = 3)	Recovery %
3	3.2 ± 0.3	9.8	108.1
9	8.2 ± 0.6	6.9	90.7
27	28.1 ± 2.0	7.2	104.0

TMV detection in naturally infected tobacco samples

The in-field applicability of TRFIA strips was carried out by technicians from a company that offers testing services. In the preliminary testing, 28 samples were tested side by side using TRFIA strips and colloidal gold strips (Agdia, Inc). Several samples reported positive with TRFIA strips, whereas they showed negative results with colloidal gold strips (Figure 4(A)). To rule out the possibility of false positive events, a TMV-infected tobacco leaf was divided into two equal parts and analysed by TRFIA strips and RT–PCR in parallel. TMV detected by TRFIA was quantified as 16.24, 2.86, and 1.66 ng/mL at 10⁻⁴, 10⁻⁵, and 10⁻⁶ dilutions, respectively. A primer set specific for the TMV-CP gene amplified a fragment of 164 bp, whilst a primer set specific for the NtActin gene amplified a 284-bp segment as internal control (Figure 4(B)). The primers detected up to 10⁻⁵ dilution of total synthesised TMV-CP cDNA, providing further validation on the proposed TRFIA system.

Figure 4. Comparative evaluation of TRFIA with colloidal gold LFA and RT-PCR, and assessment of field applicability. (a) Representative results of a TRFIA test strip and a colloidal gold strip. (b) Sensitivity of RT-PCR in the detection of TMV. The assay included duplicate reactions for each dilution. NC was the non-template control without cDNA.

To match with results of colloidal gold strips, TMV concentrations below 3.00 ng/mL were considered as TMV-negative in leaf samples from five tobacco growing fields. This suggests that TRFIA test strips outperformed colloidal gold LFA strips by showing at least two orders of magnitude improvement in detection limits, 0.24 ng/mL versus 3.00 ng/mL of Agdia strips, or 200 ng/mL triplex immunostrips (Guo et al., 2022). Compared to the others, Banqiao has the highest number of positive samples (Table 4). In contrast, although Xijiekou had 33 positive results, the mean and median values of Xijiekou was the highest, with a mean value significantly different than that of Banqiao and Shilin (statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test).

Table 4. Descriptive statistics of field-collected samples.

	Banqiao	Lufu	Dake	Xijiekou	Shilin
Total number of values	643	96	55	33	15
Positive rate %	32.1	4.8	2.7	1.6	0.7
Median (ng/mL)	6.1	6.5	6.1	15.4	5.0
Mean (ng/mL)	21.2	32.1	21.9	53.4	5.1

Conclusions

TMV is a highly infectious and persistent virus that has a major economic impact on a wide range of hosts. Proactive testing is an effective and eco-friendly strategy for plant disease surveillance, not only preventing extensive use of pesticides, but contributing to crop yield and quality as well. Herein, mAbs against TMV were developed and labelled with Eu³⁺ nanospheres to fabricate a TRFIA test strip that achieves quantitative TMV detection in tobacco. To our knowledge, this is the first report that constructed a TRFIA platform for rapid TMV onsite detection in tobacco, providing advantages including high accuracy, specificity, sensitivity, and in-field applicability. Equipped with a portable time-resolved fluorometer, the proposed TRFIA system proves its usability in disease early warning and allows end users to screen for TMV infection routinely. By doing so, we expect this technique that will contribute to TMV disease management and reduce the reliance on pesticides to protect quality and yield of tobacco.

Acknowledgements

We thank the field crew at Shilin Tobacco Station for maintaining the tobacco fields from which our samples were collected, and technicians from Best Science & Technology for research support.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the Guizhou Branch of CNTC under Grant (2022XM08); China National Tobacco Quality Supervision and Test Center under Grant (562021CR0010).