Multiplex PCR for Identification and Detection of Cassava Mosaic Begomoviruses in Togo ()
1. Introduction
Cassava (Manihot esculenta Crantz) is a vegetatively propagated staple crop of great economic importance. Due to its inherent tolerance to drought and inadequate soil nutrients, cassava produces some storage roots where other food crops would fail; hence, it is considered a food security crop [1] [2] . It provides food security in several African countries [3] , as it is easy to grow and its yields help to survive during famine [4] . The food security and livelihood benefits of cassava in Africa are, however, constrained by cassava mosaic disease (CMD). It is caused by Cassava Mosaic Begomoviruses (CMBs) transmitted by the whitefly vector Bemisia tabaci [5] and are also disseminated through infected stem cuttings used for new plantings, since cassava is a crop propagated vegetatively. Several cassava infecting Begomoviruses have been reported occurring in Sub-Saharan Africa [6] but only three are highlighted in this study. They are: African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV), East African cassava mosaic Cameroon virus (EACMV/CM). ACMV and EACMV has been associated with a pandemic in East Africa [7] [8] . CMBs can occur in an infected plant either alone or as mixed infections of different combinations [9] [10] [11] .
CMBs were detected by uniplex PCR, the usual method, using species-specific primers. Uniplex PCR are disadvantage of cost for a large-scale diagnosis of plants viruses. In recent years multiplex-PCR assay was developed by some authors [12] [13] using primers targeting replicase and coat protein regions of DNA component for specific and simultaneous detection of CMBs. To overcome the inherent disadvantage of cost, as well as to improve diagnostic capacity, multiplex PCR, a PCR variant in which more than one target sequence is amplified using more than one pair of primers, is an interesting alternative [14] to facilitate high throughput diagnosis of CMBs in epidemiological studies. The aim of this study is to develop a multiplex PCR approach for the detection of ACMV, EACMV and EACMV/CM in Togo CMD-infected cassava leaves.
2. Material and Methods
2.1. Collection of Virus-Infected Cassava Leaves
In August-September 2020, cassava leaves with characteristic CMD symptoms and CMD-free were collected from farmers’ fields located at intervals of between 10 km in five (05) agroecological zones of Togo. The latitude and longitude of each farm were recorded using Global Positioning System equipment (Garmin GPSMAP 64 s). Symptomatic and asymptomatic samples were collected and kept in the envelopes and dried at 25˚C.
2.2. Extraction of Total DNA
Total DNAs were extracted from 100 mg of leaf tissue of infected plants. Total DNA was extracted from leaf samples using InnuPREP Plant DNA Kit (Endress + hauser Compagny, Germany) according to the manufacturer’s instructions. The extracted DNA was stored at −20˚C for analysis. The concentration and purity of extracted DNA samples were determined using a Nano Drop spectrophotometer (Thermo Scientific, Nano drop-2000C, Germany).
2.3. Primer Design Used in the PCR
The primers JSP001/JSP002 [8] identifying ACMV amplify a 783 bp fragment; for EACMV primers CMBRep F/EACMVRep R [12] amplify a 650 bp fragment; and for EACMV/CM primers VNF031/VNF032 [15] amplify a 560 bp fragment (Table 1).
2.4. Single PCR Performance
The single (uniplex) assays were performed to detect each virus using the primer pair in the Table 1 designed to detect ACMV and EACMV. The samples were subjected to another round of PCR using specific primers for the detection of EACMV/CM (VNF031/VNF032). The reaction mixtures (25 µl) contained 12.5 µL Master Mix (New England Biolab, NEB), 1.25 µL each of forward and reverse primers (10 µM), 8 µL nuclease free PCR water (Inqaba Biotech West Africa Ltd) and 2 µL DNA. The PCR conditions used are those described by the various authors (Table 1).
2.5. Multiplex PCR Performance
The multiplex PCR for simultaneous detection ACMV, EACMV and EACMV/CM reaction conditions were optimized by using different primers concentrations, annealing temperatures, extension times to achieve optimum conditions (Table 2). The multiplex PCR reaction was set up in one tube as a 25 µl mixture containing 1 µl each of forward and reverse primers (10 µM), 5 µL nuclease free PCR water (Inqaba Biotech West Africa Ltd.), 12 µL Master Mix (New England Biolab, NEB), and 2 µL DNA.
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Table 1. Oligonucleotide primer sequences for the single PCR.
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Table 2. Oligonucleotide primer sequences for the multiplex PCR.
The PCR protocol started at 94˚C for 4 min followed by 35 cycles of amplification (94˚C for 1 min, 52˚C for 1 min, 72˚C for 1 min) and a final extension at 72˚C for 10 min. The DNA amplification was carried out in the Applied Bio systems Thermal Cycler. Amplicons were separated by electrophoresis on 1% agarose gels (which was stained with ethidium bromide) for 100 V for 35 min for uniplex PCR and 45 min for multiplex. Bands were viewed under UV light using and the images saved using a gel documentation system (MultiDoc-It Digital Imaging System).
3. Results
3.1. Single PCR for ACMV, EACMV, EACMV/CM
Uniplex amplification bands with expected sizes of 560 bp, 650 bp and 783 bp were obtained from the cassava samples with VNF031/VNF032, CMBRepF/EACMVRepR and JSP001/JSP002 respectively (Figure 1). ACMV were detected on 14/38, EACMV on 31/38 and EACMV/CM on 37/38 (Table 3). The volume of consumables used was 1710 µL (except DNA and nuclease free water) and the number of reactions to detect each of the viruses in uniplex is 114 reactions.
3.2. Multiplex PCR for ACMV, EACMV, EACMV/CM
Three amplification bands with expected sizes were obtained from the mixture of PCR products (Figure 2). ACMV were detected on 12/38, EACMV on 31/38, EACMV/CM on 37/38 (Table 3). The volume of consumables used was 684 µL (except DNA and nuclease free water) or 40% of the uniplex PCR and the number of reactions to detect each of the viruses in multiplex is 38 reactions.
The primers detected the three viruses in 12 of the 38 samples and EACMV, EACMV/CM were detected in 31 samples and none in one sample.
3.3. Comparison between Single and Multiplex PCR
Single and multiplex PCR detected ACMV, EACMV and EACMV/CM in plant DNA extracts prepared from cassava leaves infected with either two or three viruses. The primers JSP001/JSP002 amplified 783 bp specific to ACMV, primers CMBRepF/EACMVRepR amplified 650 bp specific to EACMV and primers VNF031/VNF032 amplified 560 bp specific to EACMCV/CM in both uniplex
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Table 3. Results obtained by uniplex and multiplex PCR.
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Figure 1. (a) Detection of EACMV/CM virus by PCR using specific primer VNF031/VNF032. Virus-specific amplification products correspond to 560 bp for EACMV/CM virus (Lane 1, 2, 3, 4, 5, 6 and 7). Lane M: 100 bp or 1 kb DNA ladder; (b) Detection of EACMV virus by PCR using specific primer CMBRepF/EACMVRepR. Virus-specific amplification products correspond to 650 bp for EACMV virus (Lane 1, 3, 4, 6, 7). Lane M: 100 bp or 1 kb DNA ladder; (c) Detection of ACMV virus by PCR using specific primer JSP001/JSP002. Virus-specific amplification products correspond to 783 bp for ACMV virus (Lane 1, 2, 3, 4, 5, 6, 7, 8). Lane M: 100 bp or 1 kb DNA ladder.
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Figure 2. Simultaneous detection of all possible combinations of three viruses by multiplex PCR. Lane M: 100 bp or 1 kb DNA ladder; Lane 1, 2 and 4: for EACMV, ACMV and EACMV/CM respectivily; Lane 3, 5, 6 and 7: 650 and 560 for ACMV and EACMV/CM respectively.
and multiplex formats. Distinct sizes enabled the differentiation of virus-specific DNA bands by agarose from all samples Multiplex and uniplex obtained consistent results, except for ACMV where two less than those positive for primers JSP001/JSP002 using multiplex. Multiplex and uniplex results indicated that the former was relatively reliable for detecting mixed infection in the samples.
4. Discussion
In this study, a multiplex PCR was performed in order to simultaneously detect and identify three different begomoviruses (ACMV, EACMV, EACMV/CM) in cassava field. These viruses are responsible for Cassava Mosaic Disease that is the most devastating disease of Cassava in Africa. For individual virus identification, specific primers were used using the uniplex PCR method. The process used primers JSP001/JSP002, CMBRepF/EACMVRepR, and VNF031/VNF032 for individual identification of ACMV, EACMV and EACMV/CM viruses respectively. This method is very costly and time-consuming for the diagnosis of several viruses in a very large number of samples.
Multiplex PCR has been developed in this study for the simultaneous detection of mixed infections of ACMV, EACMV and EACMV/CM. Different conditions were optimized by using different primers concentrations, annealing temperatures, extension times to achieve optimum conditions for the multiplex PCR. Multiplex PCR enabled for the first time the simultaneous detection of only two CMBs, ACMV and EACMV [12] and it is followed by other research on the detection of more than two viruses [13] [16] . The Mixed infection of ACMV, EACMV [17] [18] [19] and EACMV/CM [11] [20] [21] as revealed in the present study concurs with that of other parts of Africa in which more the one CMB species were reported from CMD-affected cassava plants. On the other hand, these low observed bands could also be explained by the fact that multiplex PCRs using capsid protein-specific primers do not necessarily give better results with a balanced intensity of amplified DNA fragments for each virus compared with multiplex PCRs based on Rep-specific primers [12] . Overall, the results showed that there was no significant difference between simple PCR and multiplex PCR, with both procedures generating amplicons of the targeted product. When the two processes were compared, they are distinct of volume of consumables and number of reactions used. Multiplex PCR used a quarter of consumables used in the uniplex PCR. The number of reactions to detect each of the viruses in uniplex is 114 compared to 38 in multiplex reactions. The intensities of the PCR amplicons in multiplex PCR were relatively weak for each virus while the intensities of the PCR amplicons in uniplex PCR were strong, presumably due to the number of primers used.
It allowed to confirm the results with those of the uniplex PCR and gives similar results. The sensitivity and efficiency of multiplex PCR assays is greatly influenced by a number of factors, including primer concentration, annealing temperatures, as well as competition between the primers [22] [23] . These protocols minimize sample handling and can markedly reduce the time, labor, and costs of large-scale diagnosis of cassava viruses because this allows to go faster and reduce the use of consumables. The multiplex PCR test developed in this study provides a faster and more efficient way to detect these viruses and could contribute greatly to virus surveillance.
5. Conclusion
Cassava Mosaïc Disease continues to be the major constraint to cassava production and a serious cause of yield reduction of cassava in Togo. The use of multiplex PCR made it possible to detect ACMV, EACMV and EACMV/CM in mixed infections and can also be used for studies of the relation between mixed infections of viruses responsible for CMD in Africa. This method is cost-effective, rapid, sensitive, specific and reliable for the diagnosis of CMB in cassava plants. Also, Multiplex PCR is an excellent diagnostic tool for Cassava mosaic disease surveillance and for disease outbreak prevention or disease effective control.
Acknowledgements
We thank the Central and West African Virus Epidemiology for Togo (WAVE-UL) project funded by the Bill and Melinda Gates Foundation (Grant number OPP1082413) and the Foreign, Commonwealth & Development Office.