A quick and sensitive diagnostic tool for detection of Maize streak virus

Maize streak virus disease (MSVD), caused by Maize streak virus (MSV; genus Mastrevirus), is one of the most severe and widespread viral diseases that adversely reduces maize yield and threatens food security in Africa. An effective control and management of MSVD requires robust and sensitive diagnostic tests capable of rapid detection of MSV. In this study, a loop-mediated isothermal amplification (LAMP) assay was designed for the specific detection of MSV. This test has shown to be highly specific and reproducible and able to detect MSV in as little as 10 fg/µl of purified genomic DNA obtained from a MSV-infected maize plant, a sensitivity 105 times higher to that obtained with polymerase chain reaction (PCR) in current general use. The high degree of sequence identity between Zambian and other African MSV isolates indicate that this LAMP assay can be used for detecting MSV in maize samples from any region in Africa. Furthermore, this assay can be adopted in minimally equipped laboratories and with potential use in plant clinic laboratories across Africa strengthening diagnostic capacity in countries dealing with MSD.

www.nature.com/scientificreports/ alternative. Lately, numerous LAMP assays have led to the development of several phytopathological diagnostic protocols for the early detection of many diseases caused by viruses, however, none has been developed for the detection of MSV 18 . LAMP is a technique that uses six primers that recognise eight distinct regions in the target DNA, making it highly specific and with increased amplification efficiency than other DNA amplification methods 19,20 . LAMP assays run at a single temperature of around 65 °C and provide results in less than 30 min due to strand displacing DNA polymerases that display faster reaction kinetics 21 . Additionally, the LAMP chemistry exhibits tolerance of substances which are inhibitory to PCR, allowing versatility for on-site diagnostics of plant pathogens 21,22 .
Isothermal technologies are increasingly being developed particularly for low-resource regions where infrastructure, equipment, and skills to support the use of PCR as a diagnostic tool are lacking 23 . The current availability of several portable fluorescence-reading LAMP devices such as the Genie II (Optigene Ltd., West Sussex, UK) which offsets the need of a high-cost equipment used for thermal cycling, mean that this assay could be a promising alternative to conventional PCR in current general use and could increase diagnostic capacity in different maize-growing regions in Africa. In this study, the development and application of LAMP as a quick method for MSV detection are described.

Results
Specificity and sensitivity of LAMP method. To evaluate the suitability of LAMP for the detection of MSV, DNAs obtained from 26 symptomatic maize plants were screened by both the new LAMP assay and the PCR method in current general use. The LAMP assay was performed in a real-time thermal cycler set at 65 °C for 40 min (Fig. 1). All samples tested positive for MSV with results typically obtained within 10 min, suggesting a high virus titre in the samples tested. The same DNAs were analysed by PCR (Fig. 2) using primers designed by Martin et al 10 . All 26 samples tested positive for MSV by PCR ( Table 1).
The specificity of the LAMP assay was tested using DNAs obtained from asymptomatic maize plants as well as cassava plants infected with geminiviruses (cassava mosaic begomoviruses, CMBs). No cross reactions occurred for these samples, (Table 1; Fig. 1) and LAMP primers produced a single peak melt curve (Fig. 3) confirming the specificity of the amplified product. No melt peaks were detected for the negative controls.
To determine the detection limit of the LAMP assay, reactions were prepared with tenfold serially diluted DNA extracts obtained from a MSV-infected maize plant and by comparing results with PCR. Each dilution was tested in three independent assays. A representative assay is shown in Fig. 4. For the LAMP assay, positive results were consistently observed for samples containing DNA concentrations ranging from 100 ng/μl up to 10 fg/μl. None of the samples with a DNA concentration of 1 fg/μl were detected as positive (Fig. 4). The time required to detect MSV in the most diluted sample (i.e. containing a DNA concentration of 10 fg/μl) was around 25 min, suggesting that 40 min for data collection period is more than sufficient to detect MSV-positive samples.    www.nature.com/scientificreports/ In contrast, the detection limit for the conventional PCR was 1 ng/μl (Fig. 5) indicating that the LAMP assay developed in this study is at least 10 5 times more sensitive than PCR.
Sequence identities of MSV amplicons. PCR products of MSV-positive samples were sequenced and used for phylogenetic and diversity analysis. The mean pairwise nucleotide diversity between the MSV sequences obtained in this study was 97.6%. Nucleotide pairwise comparison of these sequences with MSV sequences deposited in GenBank revealed 93.9-99.7% nucleotide identities (see Supplementary Data S1 online).   (Fig. 6). The clustering of MSV Zambian sequences did not correlate with their country of origin. The Zambian MSV sequences obtained here are closely related to MSV sequences obtained from different countries in Africa. MSV-A_ZMB_Chie_36 (MT210148) had the highest nucleotide identity (99.7%) with MSV accessions HQ693362 and HQ693461 from Mozambique and Zambia, respectively whilst the lowest nucleotide sequence identity was obtained for MSV-A_ ZMB_Mpor_54 (MN562651) which had 93.9% identity to an MSV accession from Cameroon (HQ693327) (see Supplementary Sata S1 online).

Discussion
Maize streak virus disease (MSVD) is caused by Maize streak virus (MSV), a geminivirus that occurs in most sub-Saharan countries. MSVD is regarded as the third most devastating disease of maize worldwide after northern corn leaf blight (NCLB) and gray leaf spot (GLS) 24 . In Africa, however, where maize is a staple food, MSVD is the most important disease affecting maize crops and poses a big threat to food security in the region 5 . The MSV can cause up to 100% yield losses 25 and therefore has potential to cause a devastating effect not only on the maize crop and the livelihoods of the resource-poor farmers in Zambia, but also on other key actors in the maize seed/ grain value chain, especially seed companies and processors with consequent losses of sales. The development of robust, quick and efficient methods for the precisely diagnosis of MSVD is vital towards appropriate disease control and for increased food and income security.
In this study, a LAMP method was developed for the detection of MSV and compared to the standard PCR method for MSV diagnosis. The LAMP assay was 10 5 times more sensitive in detecting MSV than standard PCR and with high specificity as no cross-reactions with closely related geminiviruses were detected.
The detection of LAMP products was achieved using a laboratory real-time PCR instrument to measure fluorescence. Alternative detection methods such as agarose gel electrophoresis can be used 26 but these increase the risk of contamination through the opening of reaction tubes containing LAMP-amplified products 18 . LAMP reactions generate five to ten more amplicons than a standard PCR 27,28 . To avoid carry-over contamination issues it is very important not to open the tubes after a LAMP reaction. Although the real-time detection used in our study avoided any risk of contamination associated with post-amplification processes there are several low-cost portable fluorescence-reading instruments (e.g. Genie II (Optigene Ltd., West Sussex, UK), Bioranger (Diagenetix, Inc., Honolulu, HI, USA) and Genelyzer FIII (Canon Medical Systems, Tochigi, Japan)) available in the market that can be used to measure the fluorescence signal and thereby reducing the cost of the assay 18,30,31 . These simple battery-powered instruments offer a major advantage of the LAMP assay in places experiencing repeated power failures as reactions could proceed even without electricity.
The developed LAMP assay has more advantages compared to the standard PCR currently in general use for MSV diagnosis. In this study the reaction time of the LAMP assay was less than 40 min compared to > 90 min required for the PCR plus the time required for gel electrophoresis. Previous reports have described the use of LAMP in conjunction with crude plant extracts avoiding total DNA extractions 19,32 , reducing costs 18 , shortening the processing time and facilitating the use of the method in low-resource settings 32 . In contrast to LAMP, PCR needs more stringent conditions such as expensive equipment to perform thermal cycling steps at higher temperatures and highly specialised personnel. For these reasons, the LAMP developed here appears to be a promising alternative to PCR for testing for MSV.
Due to budget restrictions, only a small number of MSV sequences were obtained in this study. Nevertheless, the high degree of sequence identity between the Zambian and other African MSV isolates suggest that this LAMP assay can be used for detecting MSV in maize samples from any region in Africa. www.nature.com/scientificreports/ The developed LAMP will contribute not only to improve the diagnosis of MSV but also to strengthen the molecular diagnostic testing capability at the Zambia Agriculture Research Institute. It is paramount that the institute is equipped with appropriate diagnostic methods to detect the most important viruses present in maize to allow the development and application of appropriate control measures in maize cultivations in Zambia.
Future studies will investigate procedures to adapt the LAMP assay as a surveillance and early-warning tool for the presence of MSV and/or other pathogens as well as detection of MSV in leafhoppers to study the combined leafhopper/MSV diversity present in Zambian maize fields. In the short term, however, the LAMP assay developed in this study could be immediately employed for sanitary selection and in eradication programmes 18 .

Conclusion
In this study a specific and sensitive LAMP assay for the detection of MSV was developed. This assay was able to detect MSV from field maize plants and was 10 5 times more sensitive than conventional PCR currently in general use. The LAMP assay has the potential to be adopted for routine detection of MSV in maize breeding programs and/or certification laboratories. Most importantly, the developed method has the immediate impact of strengthening the molecular diagnostic testing capability at the Zambia Agriculture Research Institute.

Materials and methods
Plant material and DNA extraction. Maize leaf samples (n = 26) were obtained from MSV symptomatic maize plants from 13 districts (Chembe, Mansa, Chipili, Luwingu, Mwense, Mwansabombwe, Chienge, Kaputa, Mporokoso, Lunte, Kasama, Chinsali and Serenje) in Zambia. The districts are in the agro-ecological region III of Zambia which experiences the highest rainfall of 1000 mm and above per annum 33 with the potential for high maize production and at the same time regarded as a hotspot for MSV. Genomic DNA for the maize and cassava (used in the determination of LAMP specificity) leaf samples was extracted using a modified cetyltrimethylammonium bromide (CTAB) procedure as described by Lodhi et al. 34  Phylogenetic and sequencing analysis. The sequenced MSVs were submitted to GenBank using Bankit, a web-based data submission tool 35 . The quality of the virus sequences was processed by removing any low quality sequence using Chromas Software Version 2.6.6 (https ://techn elysi um.com.au/wp/chrom as/) 36 . Quality scores of 0.05 were used for trimming and sequences with scores below 50% were excluded from analyses. The single contiguous sequences (contigs) were generated from the two separate sequence files using the CAP3 sequence assembly program 37 . The sequences were analyzed against sequences available in GenBank using the basic local alignment search tool (BLASTn) 38 at the NCBI website (https ://www.ncbi.nlm.nih.gov/genba nk/). The MSV sequences from the database that had the highest similarity to each BLAST query sequence were selected for subsequent sequence similarity and phylogenetic analysis. Nucleotide sequences were aligned using the MAFFT v7.450 alignment program 39 with default option settings. The evolutionary history was inferred using the Neighbor-Joining method 40 . The evolutionary distances were computed using the Jukes-Cantor method 41 and in the units of the number of base substitutions per site. Evolutionary analyses were conducted in MEGA7 42 . The analyses included the MSV Zambian sequences obtained in this study (GenBank accession numbers MN562648, MN562649, MN562651, MN562652, MT210148 and MT210149) and published MSV full genome sequences comprising of 192 MSV isolates described 6 . The robustness of each tree was determined by generating a bootstrap consensus tree using 1000 replicates. Virus sequences obtained from GenBank were used for comparative analyses and accession numbers shown in the phylogenetic tree. Pairwise identity comparisons of nucleotide sequences were performed using the multiple alignment tool of the software GENEIOUS v11.1.5 43 .
LAMP primer design. LAMP primers ( Table 2) were designed to amplify a fragment between the MP and the coat protein (CP) genes of MSV. This was done through a multiple sequence alignment of complete MSV nucleotide sequences available in GenBank. Primers were designed using PrimerExplorer v5 software (available at https ://prime rexpl orer.jp/e/) with default settings. The targeted region of the LAMP primers has 70 bp overlap with the region covered by the PCR primers used in our study. Degenerate bases were incorporated into the LAMP primers to mitigate against any intraspecific variation between MSV isolates. BLAST analysis indicated that primers' regions selected for assay had high homology between MSV isolates and showed no putative crossreaction with other members of the family Geminiviridae.  Fig. S4 online).
To determine the detection limit of the LAMP assay and the PCR, serial dilutions of DNA obtained from MSV-infected plant was prepared. Dilutions were made with DNase-free water in a tenfold series with concentrations of 100 ng/μl, 10 ng/μl, 1 ng/μl, 100 pg/μl, 10 pg/μl, 1 pg/μl, 100 fg/μl, 10 fg/μl and 1 fg/μl of which 1 μl each was used as template for LAMP and PCR 44 . All reactions were performed in triplicates. LAMP reactions were incubated with fluorescence measurements as described above following the procedures described by Panek and Frąc 44 . To measure the melting temperature of the amplification products, the reactions were subjected to a slow annealing step from 65 to 95 °C (with 0.05 °C/s increments) with fluorescence monitoring 44 . The obtaining results were visualized as amplification plots (ΔR) expressed as the negative value of the change in RFU over the change in temperature (− dRFU/dT) versus temperature (degrees Celsius).
Received: 2 June 2020; Accepted: 29 October 2020 Table 2. LAMP primers designed in this study for the detection of Maize streak virus (MSV) using the Genbank accession AF003952 as reference (primers were designed using PrimerExplorer v5 software (available at https ://prime rexpl orer.jp/e/)).