Apple Stem Grooving Virus is a virus infecting apple plant material and can cause an undesirable reduction in the production of apple trees. Reliable methods of detection of ASGV are essential for proper control of this virus.
The present study therefore evaluated the current enzyme-linked immunoassay (ELISA) procedure performed at the South African Plant Improvement Organisation (SAPO). This was done by comparison to a multiplex reverse transcriptase polymerase chain reaction (RT-PCR) previously shown to be a sensitive, reliable and rapid approach for virus detection. Both ELISA and multiplex RT-PCR were performed on six apple plant samples from the SAPO for comparison between the two techniques. The ELISA procedure detected ASGV infection in three samples and the multiplex RT-PCR found infection in five samples. The multiplex RT-PCR procedure is concluded to be more reliable due to its ability to eliminate potential false-negative results. The multiplex RT-PCR procedure is additionally more sensitive and requires the use of less plant material and smaller volumes are assayed than the ELISA.
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Deeper insight into the origin of the virus and the relationships between local and international strains of ASGV is needed. Sequence determination and phylogenetic analysis was performed on five South African ASGV isolates. This was done after successful amplification of the ASGV coat protein (CP) gene by RT-PCR with designed primers, subsequent extraction and purification of RT-PCR products and cycle sequencing PCR. Phylogenetic analysis revealed high similarity between South African strains of ASGV and high likelihood of shared virus origin.
Apple (Malus x domestica Borkh.) is a commercially important fruit crop worldwide and in South Africa [1, 2]. Apple Stem Grooving Virus (ASGV) is an economically important and common pathogen occurring in apple plant material . Other frequently occurring apple viruses are Apple Chlorotic Leaf Spot Virus (ACLSV), Apple Stem Pitting Virus (ASPV) and Apple Mosaic Virus (AMV), but are not the main focus of this study. All four of these viruses can cause a significant reduction in fruit quality and yield [1, 3, 4], especially if they occur in mixed infections, as they often do [1, 3]. Except for AMV all of these viruses remain mostly symptomless and appropriate tests are necessary for their detection .
An increased occurrence of infected apple trees has highlighted the necessity to reevaluate current procedures in the detection of ASGV.
Enzyme-linked immunoassay (ELISA) is currently being used by the South African Plant Improvement Organisation (SAPO) for the detection of ASGV. The application of a more rapid and reliable method of detection is necessary for the appropriate certification of plant material . Studies have shown that reverse transcriptase polymerase chain reaction (RT-PCR) provides a more sensitive and rapid method of detection [1, 3, 5]. It is for this reason that comparison between these two procedures was performed and described in this study. Furthermore, the use of multiplex RT-PCR allows for the simultaneous detection of all four of these viruses in one reaction mixture [1, 3, 5]. To ensure the reliability and facilitate the interpretation of negative results, an internal control is present in this multiplex reaction. Menzel et al. designed a primer that amplifies apple mRNA, even in the presence of genomic DNA, and serves as the internal control. False-negative results due to RNA degradation or the presence of inhibitors of the reverse transcriptase or polymerase activity can thus be eliminated .
A mutation in the South African strains of ASGV could render present ELISA tests for ASGV ineffective. The possibility of mutation in the local strains of ASGV needs to be investigated by means of RT-PCR amplification and sequence determination of the coat protein (CP) gene of ASGV. Subsequent phylogenetic analysis is useful to determine the relationships between South African ASGV strains with those from locations abroad and to provide insight into the virus origin.
Pentaplex RT-PCR as a method of detection for apple viruses (including ASGV) was tested in a study by Hassan et al.  and results were confirmed by ELISA tests to determine the success of the procedure. The results obtained from the performed RT-PCR confirmed the positive results found by ELISA, but additional positive samples were found with RT-PCR where ELISA showed a negative result .
A comparative study between an immuno capture polymerase chain reaction (IC-PCR) and ELISA has been performed and it was concluded that IC-PCR was more reliable in the detection of ASGV than ELISA .
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Comparison between assays RT-PCR and ELISA for the detection of another virus, Tomato spotted wilt virus (TSWV) . It was found that RT-PCR procedures resulted in a higher number of positive tested samples than ELISA, but results were not determined to be significant.
A previous study reported the increased reliability and sensitivity of RT-PCR compared to ELISA in the detection of Prunus necrotic leaf spot virus (PNLSV) and Prune dwarf virus (PDV) .
It is thus indicative that ELISA, although a suitable and economical method of virus detection, is not the most sensitive procedure for virus detection.
The success in the use of multiplex RT-PCR procedures for simultaneous detection of viruses have been reported in several studies [1, 3, 5, 9].
This study describes both a multiplex RT-PCR procedure and an ELISA procedure performed on apple plant material received from SAPO. The same samples were screened using both procedures in order to determine the relative sensitivities of the two procedures.
The comparison of presently applied ELISA procedures for virus detection with a potentially more accurate method, RT-PCR, is valuable to the South African apple industry as this would indicate whether or not the current ELISA screening is sensitive enough for routine viral detection. The development of a multiplex RT-PCR assay for the detection of ASGV provides a rapid, sensitive and reliable diagnostic alternative and is additionally beneficial due to the ability to detect several viruses at the same time.
Additionally, sequence determination and phylogenetic analysis of the ASGV CP gene will provide insight as to possible mutation and relationships between various strains of ASGV virus locally and internationally.
Primer design - ASGV whole genome and ASGV CP sequences were obtained from Genbank. Alignments were done in BioEdit (v 184.108.40.206, Tom Hall) to determine where the CP gene lies within the ASGV whole genome sequence. An ASGV whole genome sequence (Genbank accession number: NC001749.2) was scanned using the LightCycler Probe Design Software 2.0 to generate possible primer sequences. The best set of primers was selected, with forward and reverse primers having identical TM (melting temperature) and GC contents of 50%.
Sample preparation - Leaf samples were obtained from the SAPO and are listed in Table 1. The leaf samples (0.1 g) were ground to a fine pulp using a mortar and pestle. Following this 2 mL grinding buffer (15 mM NaCO3, 35 mM NaHCO3, 2% (w/v) PVP40 (Sigma), 0.2% (w/v) BSA (Fluka), 0.05% (v/v) Tween 20, 1% (w/v) sodium metabisulphite, pH 9.6) was added as described by Visser et al. .
The ground sample was transferred to a microcentrifuge tube and centrifuged for 15 seconds at low-speed in a bench-top centrifuge (Picofuge). Four microlitres of the clear supernatant was added to 25 μL GES buffer (0.1 M glycine-NaOH, pH 9.0, 50 mM NaCl, 1 mM EDTA, pH 8.0, 0.5% (v/v) Triton X-100).
Samples were incubated in a thermal cycler at 95°C for 10 minutes, chilled on ice for 5 minutes and stored at -80°C for future analysis.
Gradient RT-PCR - RT-PCR mixture consisted of 14.875 μL Milli-Q® water, 2.5 μL 10 PCR buffer (Bioline, UK), 1.25 μL 0.1 M DTT, 1.5 μL 25 mM MgCl2, 1 μL 20 μM forward primer
(5'-GTCCCTCTCGGCTAGAATTGAAAGAT-3'), 1 μL 20 μM reverse primer (5'-GCGACCAAGTTTGCGGAATTTCACA-3'), 1 μL 5 mM dNTPs (Bioline, UK), 0.25 μL 5U/μL TaqTM DNA polymerase (Bioline, UK), 0.125 μL 200 U/μL SuperScriptTM III reverse transcriptase (Invitrogen, USA). Two microlitres of the homogenised sample/GES mixture was added to this reaction mixture. A TM gradient was performed to establish the optimal TA for the ASGV CP primers. The following program was used: reverse transcription at 48°C for 30 minutes; 35 cycles of denaturation at 94°C for 30 seconds, annealing gradient of 55°C to 60°C for 45 seconds and extension at 72°C for 60 seconds; final elongation at 72°C for 10 minutes and final hold at 15°C. RT-PCR products were electrophoresed in a 1% agarose gel (1 TAE buffer) containing ethidium bromide (1 μg/ml) for 70 minutes at 100V. A PstI digested λ-DNA marker was used for determination of fragment sizes. The gels were visualised using a transilluminator (Vilber Lourmat) at 321 nm.
RT-PCR amplification and sequencing of the ASGV CP gene - The RT-PCR mixture was identical to the mixture used to determine the optimal TA. The RT-PCR bands were excised and the cDNA cleaned from the gel band using the IllustraTM GFXTM PCR DNA and Gel Band Purification Kit.
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Two microliters of purified cDNA was used for cycle sequencing. The total reaction consisted of 5 μL Sequence dilution buffer, 1 μL 0.8 μM primer, 2 μL Terminator Dye. Each sample was sequenced twice, once with each primer. Cycle sequencing PCR followed (35 cycles of: 96°C for 10 seconds, 52°C for 30 seconds and 60°C for 4 minutes, 60°C for 10 minutes and final hold at 15°C). Analysis was done by means of an ABI® 3100 Genetic Analyzer (Applied Biosystems) at the University of Stellenbosch, Central Analytical Facility.
Sequence alignment and phylogenetic analysis - Sequence electropherograms were viewed and edited using Chromas (v 2.23, Technelysium, Pty., Ltd.) and subsequent alignment with published ASGV CP sequences obtained from Genbank was done using BioEdit (v 220.127.116.11, Tom Hall).
Phylogenetic analysis was done using parsimony in PAUP (4.0b10) , with Pear black necrotic leaf spot virus (PBNLSV) CP gene as an outgroup.
ELISA - Six samples were tested for ASGV and each was done in quadruplicate. An appropriate ELISA kit was used according to the manufacturer's instructions (Bioreba). IgG was diluted 1000- (1 μL per mL) after which 200 μL was added to each of the microtiter wells. Plates were covered and placed in a humid box after which incubation followed at 30°C for 4h or at 4 - 6°C overnight. The wells were emptied and washed 3 - 4 times with washing buffer (EASY WASH 2000, BIOREBA) and were patted dry on paper towels.
Fresh leaf samples (0.5g of each sample) were homogenised in 5 mL extraction buffer and 200 μL were added to each well. Six different plants were tested and samples were loaded in quadruplicate. A negative control and a positive control were included. Plates were covered, placed in humid box and incubated at 4 - 6°C overnight. The wells were then emptied and washed as before. Enzyme conjugate was diluted 1000- in conjugate buffer and 200 μL was added to each well. Plates were covered, placed in humid box and incubated at 30°C for 5h. The wells were subsequently emptied and washed as before.
One mg/mL p-nitrophenyl phosphate was dissolved in substrate buffer and 200 μL was to each well. Incubation at room temperature (18°C - 25°C) followed in a dark room. The reaction was observed and yellow colour development was read after 30 - 120 min spectrophotometrically at 405 nm.
One-step multiplex PCR - Samples were ground and prepared as previously done. Additionally, a ten-fold and hundred-fold dilution of prepared sample (constituting as one-fold) was done. Three μL of prepared 1- sample was added to 27 μL of GES buffer for the 10- dilution. Three μL of the sample diluted 10- was added to 27 μL of GES buffer for the 100- dilution. The multiplex PCR mixture contained 6.875 μL Milli-Q® water, 2.5 μL 10 PCR buffer (Bioline, UK), 1.25 μL 0.1 M DTT, 1 μL 25 mM MgCl2, 1 μL ACLSV forward primer (20 μM), 1 μL ACLSV reverse primer (20 μM), 1 μL ASPV forward primer (20 μM), 1 μL ASPV reverse primer (20 μM), 1 μL AMV forward primer (20 μM), 1 μL AMV reverse primer (20 μM), 1 μL ASGV forward primer (20 μM), 1 μL ASGV reverse primer (20 μM), 1 μL nad5 forward primer (20 μM), 1 μl nad5 reverse primer (20 μM), 1μL 5 mM dNTPs (Bioline, UK), 0.25 μL 5U/μL TaqTM DNA polymerase (Bioline, UK), 0.125 μL 200 U/μL SuperScriptTM III reverse transcriptase (Invitrogen, USA). Refer to Table 2 for the primer pair sequences.
To 23 μL of this reaction mixture, 2 μL of sample was added. Multiplex PCR conditions were applied as was described previously for RT-PCR as well as agarose gel electrophoresis and sample loading. Gels were visualised as before.
RT-PCR amplification of the ASGV CP gene - The designed primer pairs for ASGV was successful in its ability to amplify the ASGV CP gene (Fig. 1).
List of samples obtained from the SAPO
For RT-PCR and sequencing
For ELISA and Multiplex PCR
* Alphabetical and numerical designation assigned for
received samples for confidentiality purposes
Five amplification products were observed between 700 bp and 800 bp in size. These products (Sample A to Sample E) were extracted from the gel, purified and sequenced. Sequence data obtained from the RT-PCR products gave clear results. The sequences of these products were edited and a sequence alignment file was generated which included the retrieved ASGV CP sequences from Genbank. The procedure was performed on both plant sap and dry plant material that was stored at -80 °C. Amplification products were more distinct when prepared from dry plant material and were either faint or not observed when plant sap was used. Figure 1 is a digital image showing the successful amplification of Sample A through to Sample E and the products were obtained with dry apple plant as starting material. The bands were intense and clear.
Sequence alignment and phylogenetic analysis - Phylogenetic analysis was performed and a phylogenetic tree was created (Fig 2). PAUP (4.0b10)  determined two possible phylogenetic trees with the first one being the most likely and thus presented in Figure 2. The tree statistics for the parsimony analysis is shown in Table 3. Branch distances between South African strains (Sample A, B, C, D and E) were found to be very short and indicate high similarity between these strains. Sample A and B were identical. Sample C had eight differences relative to Samples A and B, five differences relative to Sample D and nine to Sample E. In the same way differences between the other strains can be determined from the tree. The largest difference between the South African strains of ASGV was 11. This is a very small value relative to the complete ASGV CP gene sequence of approximately 714 bp in length. The bootstrap value for the branch supporting the South African strains was found to be 100. Thereby the South African strains were found to be grouped separately from the various Chinese ASGV CP isolates. One Chinese ASGV strain was found to have relatively high similarity to the South African strains with distances between 50 and 59. The bootstrap value of this branch was found to be 76 and is well supported.
ELISA - Six plants were tested for ASGV. Three were found to be infected, and three tested negative for ASGV. Table 4 shows the absorbance values and respective positive or negative conclusions. It was determined that an absorbance value greater than 0.38 would be indicative of positive infection by ASGV. This cut-off point is equal to the mean absorbance value of the negative control multiplied by three. Sample 1, Sample 2 and Sample 3 tested positive for ASGV whereas Sample 4 and Sample 5 were just below the cut-off point and concluded to be negative according to the ELISA protocol. Sample 6 tested negative for ASGV.
1 2 3 4 5 6
Between 700 bp and 800 bp
FIGURE 1. A digital image of RT-PCR products on a 1% agarose gel, stained with ethidium bromide, under UV light. Lane 1: Sample A, Lane 2: Sample B, Lane 3: Sample C, Lane 4: Sample D, Lane 5: Sample E and Lane 6: 100 bp DNA ladder (Promega).
FIGURE 2. Phylogenetic tree with South African isolates (Sample A, B, C, D, E) and several isolates from other locations. Parsimony distances are given at the top part of the branch and bootstrap values are underneath it.
Multiplex RT-PCR - The same plant material used in the ELISA test, was used for the Multiplex RT-PCR procedure for comparative purposes. Sample 1, 2, 3, 4 and 5 tested positive for ASGV whereas Sample 6 was not infected according to the Multiplex RT-PCR results (Fig. 3). Amplification products were observed as a band on the gel between 200 bp and 300 bp at all samples infected with ASGV. Sample 4 did not have a band as clear as the other samples that tested positive, but an amplification product was still evident. No amplification product is observed between 200 bp and 300 bp for Sample 6. The nad5 primer amplified mRNA product in all the samples, but did so faintly with Sample 6. A dilution assay was performed with a 100 X and 1000 X dilution for each Sample. Bands cannot be clearly distinguished in the 100 X and 1000 X diluted sample lanes.
The ELISA test results on Sample 1 to Sample 6
at 405 nm
Negative Control 0.126
Positive Control 0.923
Sample 1* 1.090 +
Sample 2* 0.699 +
Sample 3* 0.941 +
Sample 4* 0.295 -
Sample 5* 0.309 -
Sample 6* 0.151 -
* The samples were concluded to be positive
or negative based on the cut-off point
The primers designed to amplify the ASGV CP gene in infected samples were successful. These primers were designed by scanning a published ASGV whole genome sequence (Genbank accession number: NC_001749.2) after determination of the relative position of the CP gene within this sequence. All of the CP gene sequences, regardless of origin were between 700 bp and 800 bp and this sequence is present at the end of the genome. In the event of successful amplification of the ASGV CP gene a RT-PCR product will be visible as a fragment on the gel and would have a position relative to a size between 700 and 800 bp. It is evident from the results (Fig. 1) that the RT-PCR products of Samples A, B, C, D and E were of expected size. Amplification was successful and these products were extracted from the gel and the ASGV CP gene of these samples could be cleaned, purified and sequenced. The five sequences obtained were aligned along with published ASGV CP sequences obtained from Genbank. The PBNLSV CP gene was chosen as an outgroup for phylogenetic analysis. A heuristic search was performed on the sequence alignment file and produced two possible phylogenetic trees of which the first one was the most reliable (Fig. 2). Only two trees were produced and therefore the likelihood of the tree presented in Figure 2 being correct is very great. It was seen from the phylogenetic analysis that the South African ASGV strains were highly similar. The largest difference
between the strains was observed between Sample D and Sample E and this difference was determined to be 11. Considering 11 nucleotide differences in the
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
FIGURE 3. A digital image of RT-PCR products on a 1% agarose gel, stained with ethidium bromide, under UV light. Lane 1: 100 bp DNA ladder (Promega), Lane 2: Sample 1, Lane 3: Sample 1 (100 X dilution), Lane 4: Sample 1 (1000 X dilution) Lane 5: Sample 2, Lane 6: Sample 2 (100 X dilution), Lane 7: Sample 2 (1000 X dilution), Lane 8: Sample 3, Lane 9: Sample 3 (100 X dilution), Lane 10: Sample 3 (1000 X dilution), Lane 11: Sample 4, Lane 12: Sample 4 (100 X dilution), Lane 13: Sample 4 (1000 X dilution), Lane 14: Sample 5, Lane 15: Sample 5 (100 X dilution), Lane 16: Sample 5 (1000 X dilution), Lane 17: Sample 6. Lane 18: Sample 6 (100 X dilution), Lane 19: Sample 6 (1000 X dilution), Lane 20: 100 bp DNA ladder (Promega).
ASGV CP gene sequence of approximately 714 bp in length, this difference is very small. Furthermore, the South African strains were supported by a branch with a bootstrap value of 100. A bootstrap value greater than 75 is considered to be a well-supported branch . It is therefore 100 % certain that the South African ASGV CP isolates are grouped together. Interestingly, two Indian strains of ASGV and one Brazilian ASGV strain were found to have high similarity with the South African strains and seem to fall within the same group. All the South African strains, the two Indian strains and the one Brazilian strain of ASGV are grouped separately from the Chinese isolates of ASGV.
It was found that RT-PCR products prepared from dry plant material were of higher quality than those from plant sap stored at -80 °C prior to performing the RT-PCR procedure. This is possibly due to ASGV being unstable in plant sap at this temperature. James et al. (1999) concluded that the virus is stable at -80 °C for a period of up to four months when stored as leaf tissue. Consistent with these findings, good results were obtained from samples prepared from leaf material stored at this temperature (Fig. 1), but cannot be applied to samples stored in liquid form.
An ELISA procedure was performed at the SAPO to test for ASGV in six apple plant samples. Three samples were concluded to be infected and three tested negative (Table 4).
Sample 4 and 5 were relatively close to the cut-off value, but were determined to be negative for ASGV nonetheless. A multiplex RT-PCR procedure was performed on the same plant material to test for infection by ASGV and the results were compared.
The multiplex RT-PCR assay determined five of the six samples infected by ASGV (Fig. 3). Thus Sample 4 and Sample 5 were concluded to be positive contrary to the negative result determined by the ELISA. The use of multiplex RT-PCR for routine viral detection can thus assist in determination of borderline negative or positively tested samples as was the case with Sample 4 and Sample 5 in this study.
Previous studies have shown that RT-PCR provides a more rapid and reliable method of detection [1, 3, 5]. The multiplex RT-PCR procedure applied in this study additionally eliminates the possibility of false-negative results due to RNA degradation through the inclusion of a nad5 primer as an internal control. The use of this primer leads to the amplification of a 181 bp product and its absence indicate that RNA degradation occurred. Sample 6 tested negative for ASGV with the ELISA procedure and this result was confirmed by the multiplex RT-PCR assay as the ASGV primer used did not amplify the 273 bp product as it did with the positively tested samples. In addition the nad5 primer successfully amplified its 181 bp product in all tested samples, although very faintly for Sample 6. The amplification product observed for Sample 4 was not as clear as the other samples that tested positive for ASGV, but was still present. This is possibly due to RNA degradation as the nad5 amplification product was also less clear in comparison.
The increased sensitivity of the multiplex RT-PCR procedure compared to ELISA is also evident. Multiplex RT-PCR makes use of much less plant material and uses smaller volumes for the assay than ELISA. Firstly, ELISA makes use of 0.5 g of plant material in 5 mL extraction buffer (0.1 g/mL) where the multiplex RT-PCR requires 0.1 g in 2 mL of grinding buffer (0.05 g/mL). Therefore, samples for multiplex RT-PCR are already twice as diluted as those for ELISA. 200 μL of the sample/extraction buffer mixture is transferred to the wells to be assayed in the ELISA procedure. Four microliters of the sample/grinding buffer mixture (50 X less compared to ELISA) during the RT-PCR procedure is further diluted in 25 μL GES buffer (6.25 X dilution). Of this, 2 μL is further added to 23 μL of PCR reaction mixture (11.5 X dilution). It could resultantly be reasoned that the multiplex RT-PCR procedure is about 7187 times more sensitive than the ELISA for virus detection.
The multiplex RT-PCR procedure is less time consuming than the ELISA and can be completed in less than a day.
The results of this study confirmed that multiplex RT-PCR offers a much more reliable, sensitive and rapid approach for the detection of ASGV in comparison to the ELISA procedures currently performed at the SAPO. Although the use of ELISA procedures have been valuable for certification of apple material in the past, multiplex RT-PCR can eliminate false-negative results with its inclusion of an appropriate internal control such as a nad5 primer .
Sequence determination and phylogenetic analysis of five South African strains of ASGV revealed that they have high sequence similarity. Phylogenetic analysis clearly indicated that the South African strains are clustered together by a very well supported branch and have similar origin. Compared to Chinese isolates, less similarity is seen and it is likely that they do not share the same origin. Two Indian and one Brazilian isolate were found to be very similar to the South African strains.