Development of a multiplex RT-PCR assay for simultaneous detection of Cucumber green mottle mosaic virus and Acidovorax citrulli in watermelon

Viruses and bacteria

The viruses CGMMV (CGMMV-lnxg; GenBank ID: KY040049.1), Cucumber mosaic virus (CMV) and the bacteria A. citrulli (JZ17; GenBank ID: MG655621), Pseudomonas syringae pv. lachrymans and Xanthomonas campestris pv. cucurbitae were stored in the Plant Virology laboratory at the College of Plant Protection, Shenyang Agricultural University. Other microorganisms, such as Watermelon mosaic virus (WMV), Zucchini yellow mosaic virus (ZYMV), and Squash mosaic virus (SqMV), were kindly provided by Dr. Qinsheng Gu (Zhengzhou Fruit Research Institute, CAAS).

Plant material

Watermelon plants (Citrullus lanatus cv. Qilin) cultivated in greenhouse were mechanically inoculated with different kinds of viral and bacterial pathogens, and the samples were collected, immediately frozen in liquid nitrogen and stored at −80 °C until use. The leaves from field-grown watermelons used for disease diagnosis were collected from two crop fields: Xinmin city (42°03′22.21″N, 122°38′19.17″E) and Qingyuan city (42°05′10.70″N, 124°55′28.16″E), Liaoning Province, China. The seeds of cucurbitaceous crops used for pathogen detection were purchased randomly and the information was listed in Table S1.

Design of primers

According to the genomic sequences of the CGMMV-lnxg isolate (GenBank ID: KY040049.1), the A. citrulli internal transcribed spacer (ITS; GenBank ID: MG655621), and the watermelon transcriptional elongation factor-1α (EF-1α; accession number: Cla010539, CuGenDB, http://www.cucurbitgenomics.org/), three pairs of specific primers, CGMMV-F/CGMMV-R, Ac-ITS-F/Ac-ITS-R, and SlEF-1α-F/SlEF-1α-R, were designed using Primer Premier 5.0 (Premier Biosoft International) and shown in Table 1. Their specificities were tested using NCBI Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/), respectively. The PCR amplification products of CGMMV and A. citrulli were 756 and 248 bp in size, respectively. In order to avoid false negative results, watermelon EF-1α was chosen as an internal control gene. Due to a 569-bp intron in DNA of EF-1α, the PCR amplification products of EF-1α were 540-bp by cDNA templates and 1,109-bp by DNA templates, which facilitates the visualization of multiple RT-PCR. All of primers were synthesized by Sangon Biotech Co. Ltd., Shanghai, China.

Optimization of multiplex RT-PCR system

For total nucleic acid extraction, about 100 mg of samples were ground and homogenized in two mL of nucleic acid extraction solution A (each 100 mL of the extraction solution contained two g sodium dodecyl sulfate (SDS), 1.5 g PVP-40, 0.9 g NaCl, 6.2 g NaAc · 3H₂O, five mL HAc, and four mL of 0.5 mol L⁻¹ Methylenediaminetetraacetic acid; the solution was incubated at 35 °C in a water bath for 15 min and shaken well before use), followed by 0.1 g sample and one g quartz sand. The samples were ground into a homogeneous mixture, and 500 μL of the mixture was transferred into a 1.5 mL centrifuge tube and incubated in a water bath at 65 °C for 30 min. Next, 200 μL nucleic acid extraction solution B (each 100 mL solution contained 29.4 g KAc and 11.5 mL HAc) was added, and the solution was mixed and incubated at −20 °C for 5 min. Subsequently, the solution was shaken by inverting, and 750 μL chloroform was added. The solution was shaken and centrifuged at 4 °C at 12,000 rpm for 20 min. Next, 500 μL supernatant was obtained, and 400 μL chloroform and 400 μL water-saturated phenol were added. The solution was shaken well and then centrifuged at 4 °C at 12,000 rpm for 15 min. The supernatant was collected, and an equal volume of isopropanol was added. The solution was well mixed and then incubated at −20 °C for 20 min. Subsequently, the solution was centrifuged at 4 °C at 12,000 rpm for 15 min. The supernatant was discarded, and the pellet was rinsed using one mL anhydrous ethanol. After the ethanol was removed, the pellet was dried at room temperature, and the nucleic acid was dissolved in 20 μL diethyl pyrocarbonate-treated water. Using a micro-volume spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), the A260/A280 values of total DNA and RNA were 1.99–2.03 and 2.01–2.05, respectively; and the A260/A230 values of total DNA and RNA were 1.79–1.94 and 1.79–1.92, respectively. Two microliters of total nucleic acid (approximately 1.5 μg RNA) was collected as the template, to which 0.5 μL of Oligo dT (0.5 μg μL⁻¹; Sangon Biotech, Shanghai, China), 0.5 μL random primers (0.5 μg μL⁻¹; Sangon Biotech, Shanghai, China), and one μL dNTPs (10 mmol L⁻¹; Sangon Biotech, Shanghai, China) were added. DEPC-water was added until the total volume reached 14.5 μL. The solution was incubated at 70 °C for 5 min and then placed on ice for 3–5 min. Four microliters of 5× reverse transcription buffer (Sangon Biotech, Shanghai, China), 0.5 μL ribonuclease inhibitor (40 U μL⁻¹; Sangon Biotech, Shanghai, China), and one μL M-MLV reverse transcriptase (200 U μL⁻¹; Sangon Biotech, Shanghai, China) were added. The solution was mixed well and then placed in a water bath at 42 °C for 1 h. The obtained cDNA was stored at –20 °C.

The total volume of multiplex RT-PCR reaction was 25 μL, including one μL of template (reverse transcription products of total nucleic acids), upstream and downstream primers (Table 2), 12.5 μL of 2× TaKaRa Premix (Ex Taq, 1.25 U/25 μL; dNTP Mixture, each 0.4 mmol L⁻¹; Mg²⁺, 4 mmol L⁻¹), and appropriate ddH₂O. The PCR procedures were as follows: pre-denaturation at 94 °C for 5 min; 15, 20, 25, 30, 35, or 45 cycles of denaturation at 94 °C for 30 s, annealing at varying temperatures (48.2, 48.9, 50.1, 51.6, 53.6, 55.9, 58.0, 59.6, 60.9, or 61.6 °C) for 30 s, and extension at 72 °C for various durations (15, 25, 35, 45, 60, or 80 s); and a final extension at 72 °C for 10 min.

The amplification products of multiplex RT-PCR were obtained using a TIANgel Midi Purification Kit (Tiangen Biotech, Beijing, China). Purified PCR products were cloned and constructed pEASY-T1-CGMMV, pEASY-T1-A. citrulli, and pEASY-T1-EF-1α (540-bp) plasmid by using a pEASY-T1 Simple Cloning Kit (TransGen Biotech, Beijing, China), respectively. Positive clones were further verified using colony PCR and the plasmids were submitted for sequencing (Sangon Biotech, Shanghai, China).

Specificity and sensitivity of the multiplex RT-PCR

Watermelon leaves infected by various viruses, including CMV, WMV, ZYMV, and SqMV, and by two bacteria, including P. syringae pv. lachrymans, and X. campestris pv. cucurbitae, were examined to verify the specificity of the multiplex RT-PCR system. Amplified reaction was conducted using the optimized multiplex PCR system and conditions. Furthermore, a series of 10-fold dilutions from 1 × 10¹⁰ to 1 × 10⁰ copies was performed by using the templates (mixture of pEASY-T1-CGMMV, pEASY-T1-A. citrulli and pEASY-T1-EF-1α plasmids, with the concentration of 50.6 ng μL⁻¹, 48.4 ng μL⁻¹ and 45.1 ng μL⁻¹, respectively) to determine the lowest detection limit. PCR products were separated by 1.5% agarose gel electrophoresis, and a gel imaging system (Bio-Rad, Hercules, CA, USA) was employed to observe the electrophoresis results.

Enzyme-linked immunosorbent detection

Dot enzyme-linked immunosorbent assay (Dot-ELISA) and double antibody sandwich enzyme-linked immunosorbent assay (Das-ELISA) were performed to detect CGMMV and A. citrulli in these seed samples of cucurbitaceous crops, respectively. The seeds were ground in the TBS buffer (20 mM Tris-HCl, 0.15 M NaCl, pH 8.0) by a ratio of 1:6 (w/v). For Dot-ELISA, monoclonal antibody of CGMMV was used as previously reported (Shang et al., 2011). For Das-ELISA, the detection kit (Agdia, Elkhart, IN, USA) for A. citrulli was used.

Specificity of multiplex RT-PCR

Total nucleic acids extracted from the watermelon leaves infected by CMV, WMV, ZYMV, SqMV, P. syringae pv. lachrymans, and X. campestris pv. cucurbitae were performed to assess the specificity of the primers used for multiplex RT-PCR (Fig. 1). Three amplified products (756, 246, and 540 bp) were detected in the sample mixture of watermelon leaves infected by CGMMV or A. citrulli. Moreover, the corresponding amplification segment of 756 or 246 bp in length was showed in watermelon samples infected by CGMMV or A. citrulli, while the segments of watermelon EF-1α were observed only in the watermelon leaves infected by other pathogens (e.g., CMV, WMV, ZYMV, SqMV, P. syringae pv. lachrymans, and X. campestris pv. cucurbitae) or healthy watermelon leaves (Fig. 1). Unexpectedly, the 1,109-bp segment of EF-1α was weak or undetectable in the presence of CGMMV or A. citrulli or both, which had no effects on the results of pathogen detection. These results demonstrated that the developed multiplex RT-PCR system was highly specific to simultaneously detect CGMMV and A. citrulli.

The amplification products of CGMMV, A. citrulli ITS and watermelon EF-1α (540 bp) generated by multiplex RT-PCR were cloned and sequenced. The results showed that the obtained fragments had over 99% nucleotide sequence identity (CGMMV, identity 99.63%; A. citrulli ITS, identity 100%; watermelon EF-1α, identity 100%) with the known sequences, suggesting that the primers were highly specific and the detection results of multiplex RT-PCR were reliable.

Optimization of reaction condition in multiplex RT-PCR

The optimization of the primer concentrations showed in Table 2 was performed. The results showed that high amplification efficiency and low primer dimers were observed in line 4 (Fig. 2A), of which the final concentrations of primers for CGMMV, A. citrulli ITS and watermelon EF-1α were determined as 0.1, 0.1, and 0.2 μmol μL⁻¹, respectively. The results of annealing temperature optimization demonstrated that the amplification product of A. citrulli was weak and unclear when the annealing temperature was higher than 55.9 °C (Fig. 2B). To ensure the amplification efficiency, the annealing temperature at 54 °C was finally selected for the multiplex RT-PCR. In the assay of extension time optimization, 45 or 60 s as extension time permitted effective detection of the target segments of CGMMV, A. citrulli ITS, and watermelon EF-1α (Fig. 2C). In consideration of time saving, 45 s was chosen as the extension time. For cycle number optimization, effective detection of target segments of CGMMV, A. citrulli ITS, and watermelon EF-1α was observed when the cycle number was 35 or 40 (Fig. 2D). Thus, to make the assay time-saving and more efficient, we chose 35 cycles for the multiplex RT-PCR. In conclusion, the optimized multiplex RT-PCR was carried out with appropriate primer concentration, annealing temperature at 54 °C, extension time for 45 s with 35 cycles.

Sensitivity of multiplex RT-PCR

To determine the sensitivity of multiplex RT-PCR, a series of 10-fold dilutions of the mixture contained three kinds of plasmids (pEASY-T1-CGMMV, pEASY-T1-A. citrulli and pEASY-T1-EF-1α) were tested by the optimized system of multiplex RT-PCR. The results showed that the developed multiplex RT-PCR method could simultaneously detect CGMMV and A. citrulli as little as both 10² copies (0.51 and 0.48 fg uL⁻¹, respectively) of plasmid DNA, and EF-1α up to 10³ copies (4.51 fg uL⁻¹) (Fig. 3), indicating that the developed multiplex RT-PCR method in this study was highly sensitive for simultaneous detection of CGMMV and A. citrulli.

Disease diagnosis of field-grown samples and pathogen detection of seeds by multiplex RT-PCR

To evaluate the feasibility of the established multiplex RT-PCR for simultaneous detection of CGMMV and A. citrulli in the field, multiplex and single RT-PCR were performed using the watermelon samples with the symptoms of CGMMV or A. citrulli-like (Figs. 4A–4D). The results of multiplex RT-PCR assessment revealed that CGMMV was detected in the watermelon leaves from Xinmin city, whilst A. citrulli was identified in the samples collected from Qingyuan city (Fig. 4E). However, neither CGMMV nor A. citrulli was found in other five watermelon leaves and seeds sampled randomly. Additionally, the data of single RT-PCR provided strong support for the results of multiplex RT-PCR (Fig. 4E). These results demonstrated that the multiplex RT-PCR method was successfully applied for the detection of these two quarantine pathogens in the field, which contributed to the diagnosis and monitoring epidemic pattern of these two watermelon diseases.

Considering the seed transmission of both CGMMV and A. citrulli, 184 varieties of seeds of cucurbitaceous crops from Liaoning province were collected and tested by multiplex RT-PCR (Table S1). The results showed that CGMMV was detected in 17 varieties of seeds (Fig. 5), which were mainly from Cucurbita moschata (Duch. ex Lam.) and Citrullus lanatus (Thunb.), and distributed in Xinmin city (Table S1). Meanwhile, 18 varieties of seeds carrying A. citrulli were detected (Fig. 5), including Citrullus lanatus (Thunb.) and Cucumis melo Linn., which were mainly distributed in Shuncheng and Qingyuan city (Table S1). Both CGMMV and A. citrulli were detected in the regions of Shuncheng, Jiangping, Tai’an and Xinmin city (Table S1). Moreover, the detection results of multiplex RT-PCR were verified by Dot-ELISA for CGMMV (Fig. S1) and Das-ELISA for A. citrulli (Table S2). The results also revealed that multiplex RT-PCR was more sensitive than Dot-ELISA and similar with Das-ELISA (Table S1). These results demonstrated that the established multiplex RT-PCR can be used for high-throughput screening for seeds of cucurbitaceous crops.