Plant materials
For antigen preparation, hybridoma screening and MAb characterization
Cassava plants with CMD symptoms were collected from cassava production fields in Sisaket and Prachin Buri provinces in 2018. SLCMV-infected samples were identified by PCR using previously published SLCMV-specific primers [26] and confirmed by DNA sequence analysis. These SLCMV-infected samples were used as source materials for antigen preparation, hybridoma screening, antibody characterization and TAS-ELISA development. The presence of ICMV in these samples was also ruled out by PCR using ICMV-specific primers. Details of all the primers used in this study are summarized in Additional file 1: Table S1.
For specificity analysis
Commercial SLCMV positive control (PC-0424, DSMZ, Germany), SLCMV-infected and healthy cassava plants, as well as plants previously diagnosed for other begomoviruses by PCR and DNA sequencing, including Ageratum yellow vein virus (AYVV), Pepper yellow leaf curl Indonesia virus (PepYLCIV), Pepper yellow leaf curl Thailand virus (PepYLCTHV), Squash leaf curl China virus (SLCCNV), Tomato leaf curl New Delhi virus (ToLCNDV), Tomato yellow leaf curl Kanchanaburi virus (TYLCKaV) and Tomato yellow leaf curl Thailand virus (TYLCTHV) were used for specificity analyses of the newly developed MAbs and TAS-ELISA.
For assay validation
Leaves from the top canopy of cassava plants with or without typical CMD symptoms (n = 114) were collected from cassava production fields in Buriram (n = 7), Nakhon Ratchasima (n = 80), Sa Kaeo, and Sisaket (n = 27) provinces between 2018 and 2020. Cassava stems of the shoots that bore symptomatic or asymptomatic leaves in Nakhon Ratchasima province (n = 77) were also collected for testing. Some cassava stems were also purchased from local farmers (n = 275). For each cassava stem, a 30–45 cm long stem cutting was prepared and maintained in soil inside an insect-proof greenhouse to allow sprouting of young leaves (7–14 days). These young leaf sprouts (0.5–2 cm in size) were used as samples for indirect detection of SLCMV in the original cassava stems. Where applicable, green barks from cassava stem tips were also collected and used as samples for direct detection of SLCMV in the stems. Leaves of chaya (Cnidoscolus aconitifolius) and a coral plant (Jatropha multifida) found bordering CMD-affected cassava fields in Nakhon Ratchasima province were also collected and used for evaluation of the developed TAS-ELISA in comparison with PCR (Fig. 1).
Cloning and expression of recombinant SLCMV and ICMV coat proteins
The coat protein (CP) gene of SLCMV was amplified from DNA extracts of an SLCMV-infected cassava sample from Sisaket province (SSK3-14) by PCR using primers SLCMV-CPF (5′ GGA TCC ATG TCG AAG CGA CCA GCA 3′) and SLCMV-CPR (5′ AAG CTT TTA ATT GCT CAC TGA ATC 3′). The PCR product was cloned in-frame into the expression vector pQE-80L (QIAGEN, Germany) at the HindIII and BamH1 restriction sites and transformed into Escherichia coli (E. coli) DH5α cells. Transformants with the correct insert size were selected by colony PCR using the SLCMV-CPF and SLCMV-CPR primers and grown overnight for plasmid preparation according to the manufacturer’s protocol (QIAPrep Spin Miniprep Kit, Germany). The identity and orientation of the SLCMV-CP gene in the resulting plasmid was confirmed by DNA sequence analysis. The recombinant SLCMV-CP was expressed, purified and analyzed as previously described [31]. Briefly, a bacterial clone containing the correct recombinant plasmid was grown at 37 °C in LB medium containing 100 µg/ml ampicillin to an absorbance at 600 nm (OD600) of 0.6. Protein expression was induced by addition of 1 mM isopropyl-1-thio-β-D galactoside (IPTG) for 5 h at 37 °C. Recombinant SLCMV-CP was purified using a Ni–NTA agarose resin column (QIAGEN, Germany) under denaturing conditions following the manufacturer’s recommendations. Purified proteins were analyzed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and confirmed by western blotting using a commercial MAb to SLCMV (DSMZ, Germany) and MAb D2, which broadly reacts with begomoviruses [31]. The purified SLCMV-CP was dialyzed against phosphate-buffered saline (PBS), pH 7.4, before being used for mouse immunization.
The recombinant CP of ICMV was generated for cross-reactivity testing. ICMV CP ORF was synthesized based on the nucleotide sequence of ICMV isolate Maharashtra (GenBank accession no. AJ314739) and cloned into the pUC57 vector to generate the recombinant plasmid pUC57-ICMV-CP (GenScript, USA). To produce the recombinant ICMV-CP protein, the ICMV-CP gene in pUC57-ICMV-CP was subcloned into an expression vector (pQE-80L, QIAGEN) at the HindIII and BamHI sites to generate the recombinant expression plasmid pQE-80L-ICMV-CP. The recombinant ICMV-CP protein was expressed and purified as described for SLCMV-CP.
Preparation of MAbs
The production of MAbs was performed as described previously by our group [31]. Briefly, 6-week-old BALB/c mice were immunized with purified recombinant SLCMV-CP protein by intraperitoneal injection. Sera from immunized mice drawn 7 days after immunization (starting from the third immunization) were tested for antibodies against recombinant SLCMV-CP by PTA-ELISA. After the fifth immunization, the mouse with the highest antibody titer to recombinant SLCMV-CP was boosted with the purified recombinant SLCMV-CP in PBS (pH 7.4) and sacrificed 4 days later for hybridoma preparation. Hybridomas producing antibodies against recombinant SLCMV-CP were screened by PTA-ELISA as previously described. Hybridoma cells found to produce antibodies against recombinant SLCMV-CP with OD405 higher than 1.0 were subsequently tested for their reactivity against sap extracts of SLCMV-infected and healthy cassava by western blot analysis as follows. Briefly, leaves of SLCMV-infected or healthy cassava (0.4 g fresh leaf tissue or 0.025 g dried leaf tissue) were homogenized in 1 ml of extraction buffer (0.05 M Tris–HCl; 0.06 M sodium sulfite, pH 8.5). After centrifugation of the samples at 10,000 × g for 5 min, supernatants were collected. Two hundred and fifty microliters of protein extracts from SLCMV-infected or healthy plant sap were subjected to SDS-PAGE in one combined well. Separated proteins were electro-transferred to 0.45 µm nitrocellulose membranes (Protran™ GE Healthcare Life Science, Germany) and the blotted membranes were air-dried. Membranes were soaked in Tris-buffered saline plus Tween-20 (TBST; 10 mM Tris pH 8.0; 150 mM NaCl, 0.05% Tween 20) for 10 min and subsequently blocked with 5% (w/v) skim milk in TBST for 1 h. Approximately 3 mm-wide strips were then cut from both sides of the membrane and probed with prefusion sera to locate the position of the virus coat protein band on the membrane. Virus coat protein bands on the membrane (around 31 kDa) were then cut into 2 × 6 mm strips, each of which was transferred to 96-well microtiter plate and incubated with different hybridoma cells previously found to produce antibodies against recombinant SLCMV-CP by PTA-ELISA. After three successive washes with TBST, the membranes were incubated with alkaline phosphatase-conjugated goat anti-mouse polyvalent immunoglobulin (G, A, M) for 1 h. The membranes were washed as described above and incubated with BCIP/NBT substrate solution (Invitrogen, USA) for 5–10 min. Color development was stopped by adding distilled water.
Hybridoma cultures capable of differentiating between SLCMV-infected and healthy cassava were subcultured at limiting dilutions to produce monoclonal cultures of antibody-producing hybridoma cells.
Characterization of MAbs
The reactivity of MAbs was initially analyzed against recombinant SLCMV-CP, partially purified SLCMV, and sap extract of SLCMV-infected and healthy cassava plants by PTA-ELISA. Specificity of a selected MAb was then determined against sap extracts of SLCMV-infected and healthy cassava as well as sap extracts of plants infected with other begomoviruses by western blot analysis. The isotypes of MAbs were determined using a mouse immunoglobulin isotyping ELISA kit (BD Biosciences, USA). PTA-ELISA and western blot analysis were performed as described previously [31].
TAS-ELISA for SLCMV detection
Plant sap preparation
For field-collected cassava leaf and young leaf sprout samples, sap extracts were prepared by grinding cassava leaf tissues in extraction buffer containing 0.05 M Tris–HCl and 0.06 M sodium sulfite (pH 8.5) at a ratio of 1:5 (w/v, g/mL). For green bark samples, green bark tissues from cassava stem tips were ground in extraction buffer containing 2% PVP, 0.2% Na-DIECA, 0.05 M Tris–HCl, and 0.06 M sodium sulphite (pH 8.5) at a ratio of 1:2.5 (w/v, g/mL). After centrifugation, supernatants were collected for SLCMV detection.
TAS-ELISA
TAS-ELISA for SLCMV detection was developed using a previously established rabbit polyclonal antibody against begomovirus (PAb PK) as the capture antibody, and the newly prepared MAb 29B3 as the detection antibody. TAS-ELISA was conducted according to Seepiban et al. [31] with some modifications. Briefly, ELISA plates were coated with PAb PK diluted 1:5000 (v/v) in coating buffer (0.05 M carbonate buffer, pH 9.6) and incubated at 37 °C for 3 h. Plates were washed with phosphate buffered saline plus 0.05% Tween 20 (PBST) and blocked with 2% (w/v) BSA in PBST as previously described. After three washes with PBST, plant sap extracts were added to the wells (in duplicate) and the plates were incubated at 4 °C for overnight. After three washes, MAb 29B3 diluted to 1 µg/ml in 0.5% (w/v) BSA in PBST was added, and the plates were incubated at room temperature for 2 h. After three washes, alkaline phosphatase-conjugated goat anti-mouse IgG (whole molecule) antibody (Sigma-Aldrich, USA) diluted 1:5000 in 0.5% (w/v) BSA in PBST were added, and the plates were incubated at room temperature for 1 h. After the final wash step, p-nitrophenyl phosphate substrate (Life Technologies, USA) diluted in diethanolamine buffer (1 mg/ml) were added, and the plates were incubated at room temperature for 30 min. The absorbance values at 405 nm were measured using a microplate reader (Multiskan FC, Thermo Fisher Scientific, Finland). Reactions were considered positive when the mean absorbance values were at least two-fold higher than that of the healthy control.
PCR and DNA sequence analysis
Leaf or green bark samples were subjected to DNA extraction using the DNeasy Plant Mini Kit (QIAGEN, Germany) according to the manufacturer’s instruction. SLCMV detection was performed by PCR using SLCMV-specific primers (5′-GAAGGGAGACACATATACCTCG-3′ and 5′-CACATATATATTGTCTCCAATTCAC-3′) targeting the replication initiation protein (Rep) gene on SLCMV DNA-A [26]. The PCR products were cloned into the pGEM-T Easy Vector (Promega, USA), and sequenced by 1st BASE Laboratories (Selangor, Malaysia). Nucleotide sequence comparison was performed using the nucleotide BLAST program (http://blast.ncbi.nlm.nih.gov/Blast).
Preparation of plasmid standard and determination of SLCMV DNA-A genome copy number by quantitative real-time PCR (qPCR)
To quantify the virus copy number, serial tenfold dilutions of a plasmid standard carrying a 2.5-kb SLCMV DNA-A fragment was prepared and used for quantitative analyses by qPCR. Briefly, a 2.5-kb fragment of SLCMV-DNA-A was amplified from DNA extracts of SLCMV-infected plants using primers SLCMV2.5-F: 5′ GCA AGG AAC AGG CTT TAG T 3′ and SLCMV2.5-R: 5′ GGA CTT AAC GCA AAA CCT CT 3′ (targeting the SLCMV DNA-A genome at positions 875-893 and 636-617, respectively). The PCR product was purified and cloned into pGEMT-Easy (Promega, USA) according to the manufacturer’s protocol. The resulting recombinant plasmid was confirmed for its identity by DNA sequence analysis. The plasmid construct harboring the correct SLCMV-DNA-A sequence was linearized by digestion with the SalI restriction enzyme (Thermo Fisher Scientific, USA), purified using the PCR purification kit (QIAGEN, Germany), and quantified using the Qubit 2.0 Fluorometer and the dsDNA BR Assay Kit (Thermo Fisher Scientific, USA). Serial tenfold dilutions of the linearized plasmid was then prepared at copy numbers ranging from 3 × 108 to 30 copies per 5 μl. Conversion from the copy number of interest to the mass of recombinant plasmid was done considering the size of recombinant plasmid (5521 bp), the average molecular weight of one double-stranded DNA molecule (660 g/mole), and the Avogadro’s constant (6.023 × 1023 molecule/mole).
To determine the sensitivity of the developed TAS-ELISA in terms of DNA content, serial two-fold dilutions of SLCMV-infected plant extracts were prepared from 3 SLCMV-infected leaf samples and subjected to TAS-ELISA as previously described. For each set of serial dilutions, total DNA was isolated from 100 µl of sap extract from the maximum dilution showing positive results by TAS-ELISA and used as a template for qPCR. Serial ten-fold dilutions of the plasmid standard were also assayed in the same run to generate a standard curve for virus copy number quantification. The qPCR was performed in 20 µl reactions containing 10 µl of 2 × SsoAdvanced™ Universal SYBR® Green Supermix (BIO-RAD, USA), 0.2 µM each of forward and reverse primers (SLCMV-Q4F2377: 5′ GAA TTG CCG ATT GTT TGT GAT TGT G 3′ and SLCMV-Q4R2512: 5′ AGC TTG AGT CTT CCG ACA AAC ATC 3′ newly designed to amplify a 136-bp fragment from the replication initiation protein (Rep) gene on SLCMV DNA-A) and 5 µl of DNA template. The qPCR was carried out on the CFX96 Touch Real-Time PCR Detection System (BIO-RAD, USA) as follows: 1 cycle of 98 °C for 3 min, 40 cycles of 98 °C for 10 s and 60 °C for 30 s. Triplicate reactions were performed for each sample and each standard dilution.
A standard curve plotting the mean Cq values (y-axis) against the log plasmid copy number for each standard dilution (x-axis) was constructed. The equation of the linear regression line, y = m(x) + b, or Cq = slope (log quantity) + y-intercept, was then obtained and used to interpolate the starting copy number of SLCMV DNA-A in test samples. Since only 5 µl of DNA extract (1/10 of the total volume of DNA extract) were used as template for qPCR, all the results were multiplied by ten to determine the copy number of SLCMV DNA-A genomes in sap extracts in the dilution at the TAS-ELISA limit of detection. Quantification of virus copy number by qPCR was considered valid when (1) PCR amplification efficiency was between 90 and 110%, (2) the correlation coefficient was > 0.98, (3) the Cq values of test samples fell within the linear dynamic range of the standards, and (4) No Template Control wells were negative. All data and statistical analyses were done using the CFX Manager Software version 3.1 (BIO-RAD, USA).
Validation of the newly developed TAS-ELISA for detection of SLCMV in field samples
To validate the newly developed TAS-ELISA for the detection of SLCMV, a total of 490 field samples (483 cassava samples, six chaya samples and one coral plant sample) were tested for SLCMV by the TAS-ELISA and PCR, with the latter serving as the gold standard for this study. The number of positive agreements (PA), positive deviations (PD), negative agreements (NA), and negative deviations (ND) were determined and used to calculate the relative accuracy, relative specificity and relative sensitivity as previously described (relative accuracy = [(PA + NA)/N] × 100%; relative specificity = [NA/(PD + NA)] × 100%; relative sensitivity = [PA/(PA + ND)] × 100%, N = total number of samples [PA + PD + NA + ND]) [32].