LAMP-based foldable microdevice platform for the rapid detection of Magnaporthe oryzae and Sarocladium oryzae in rice seed

Rice blast (caused by Magnaporthe oryzae) and sheath rot diseases (caused by Sarocladium oryzae) are the most predominant seed-borne pathogens of rice. The detection of both pathogens in rice seed is essential to avoid production losses. In the present study, a microdevice platform was designed, which works on the principles of loop-mediated isothermal amplification (LAMP) to detect M. oryzae and S. oryzae in rice seeds. Initially, a LAMP, polymerase chain reaction (PCR), quantitative PCR (qPCR), and helicase dependent amplification (HDA) assays were developed with primers, specifically targeting M. oryzae and S. oryzae genome. The LAMP assay was highly efficient and could detect the presence of M. oryzae and S. oryzae genome at a concentration down to 100 fg within 20 min at 60 °C. Further, the sensitivity of the LAMP, HDA, PCR, and qPCR assays were compared wherein; the LAMP assay was highly sensitive up to 100 fg of template DNA. Using the optimized LAMP assay conditions, a portable foldable microdevice platform was developed to detect M. oryzae and S. oryzae in rice seeds. The foldable microdevice assay was similar to that of conventional LAMP assay with respect to its sensitivity (up to 100 fg), rapidity (30 min), and specificity. This platform could serve as a prototype for developing on-field diagnostic kits to be used at the point of care centers for the rapid diagnosis of M. oryzae and S. oryzae in rice seeds. This is the first study to report a LAMP-based foldable microdevice platform to detect any plant pathogens.


Scientific Reports
| (2021) 11:178 | https://doi.org/10.1038/s41598-020-80644-z www.nature.com/scientificreports/ which encounter many problems like the low quality of template DNA due to the presence of PCR inhibitors in seeds during the assay 8 . In addition to the above, the conventional PCR techniques also have other drawbacks such as the requirement of sophisticated instruments like thermocycler and gel documentation systems.
The LAMP assay has been widely used to detect viruses 17 , bacteria 18,19 , and fungal pathogens 20 due to its high sensitivity and specificity compared to other conventional PCR methods 19 . The HDA assay involves in vivo DNA replication using helicase enzyme 14,16,21,22 and does not require initial heat denaturation and subsequent thermocycling steps as required by PCR. Based on the principles of the LAMP assay, several microdevices have been developed integrating foldable technology [23][24][25] . A foldable LAMP microdevice for fuchsin-based colorimetric detection of multiple food-borne pathogens has been developed by 24 , where a foldable membrane fully integrating DNA purification, amplification, and detection processes for detecting multiple food-borne pathogens has been standardized. Although the LAMP and other isothermal assays have been developed to detect several plant pathogens, the foldable microdevices are not available for any plant pathogen. Foldable microdevice can be effectively utilized at the point-of-care centers for the rapid diagnosis of the plant pathogen without using any sophisticated instruments.
This study has developed two isothermal amplification techniques for the rapid, sensitive, specific, and costeffective detection of M. oryzae and S. oryzae in the rice seeds. The relative sensitivity of isothermal amplification methods and PCR based techniques were compared. Further, a portable, foldable membrane-based microdevice platform has been developed using the basic fuchsin dye. It can be effectively utilized for performing the LAMP assay in the place of polypropylene tubes. The membrane microdevice platform designed in this study can be a prototype for developing point-of-care diagnostic kits used by the field extension service persons.

Results
PCR and qPCR assay. The PCR and qPCR assay for the M. oryzae and S. oryzae using pathogen-specific primers showed positive results when the respective pathogen's DNA template was used. Similarly, both assays detected both M. oryzae and S. oryzae when template DNA from the infected rice seeds was used. No amplification was detected when template DNA from the healthy rice seeds was used. The PCR and qPCR primers of M. oryzae and S. oryzae yielded negative results when DNA template of Bipolaris oryzae, Rhizopus oryzae, Aspergillus flavus, Cladosporium fulvum, and Penicillium sp. was used, which confirmed the pathogen-specificity of the primers and the assays. Further, the PCR amplification was confirmed on a 1.5% agarose gel.
The HDA assay. For the HDA assay, the thermal stability was determined by incubating the helicase at 65 °C at different times. The assay for the ATPase activity was carried out with substrates at the experimentally determined optimal temperature of 55 °C for 10 min. The assay was carried out separately for M. oryzae and S. oryzae detection. The amplified products were analyzed on a 1% agarose gel where amplification was observed when DNA template from the fungal cultures (M. oryzae and S. oryzae) or infected seeds was used, whereas; no amplification was detected when DNA template of healthy seeds or B. oryzae was used. Similarly, HDA assay was carried out for DNA template of B. oryzae, R. oryzae, A. flavus, C. fulvum, and Penicillium sp, which resulted in the negative amplification, thus confirmed the primer and assay specificity.
Optimization of the LAMP assay. The reaction was optimized based on three key criteria, i.e., time, temperature, and target DNA template concentration. Template DNA of both M. oryzae and S. oryzae was used to optimize the LAMP assay. Colour differentiation between positive and negative results for detecting M. oryzae and S. oryzae was the primary criterion for selecting dyes. Both dyes (EtBr and basic fuchsin) used in the study were sensitive for the detection. EtBr showed fluorescence under UV light for positive amplification, whereas basic fuchsin showed purple color for a positive result and colorless for a negative result. To determine the optimum temperature and reaction time for the assay, a short-range of temperatures (56 °C to 60 °C) ( Fig. 1) was used to analyze the highest fluorescence intensity and minimum time (15- Fig. 3b). All the LAMP assay result was reconfirmed by 2% agarose gel electrophoresis, and the absorbance intensity for the dye was also validated.
The sensitivity of different nucleic acid amplification methods. The sensitivity of PCR methods (conventional PCR and qPCR) and isothermal amplification methods (the LAMP and the HDA assays) was analyzed using different DNA template concentrations ranging from 10 ng to 50 fg (of M. oryzae and S. oryzae).
Scientific Reports | (2021) 11:178 | https://doi.org/10.1038/s41598-020-80644-z www.nature.com/scientificreports/ Conventional PCR showed positive results down to 1 ng, whereas the qPCR showed a positive result down to 10 pg. Among the isothermal assays, the HDA assay showed positive results down to 50 pg, whereas the LAMP assay showed positive down to 100 fg ( Fig. 4). Therefore, the LAMP assay was the most sensitive among the different methods tested in our study for the positive detection of M. oryzae and S. oryzae infection in rice seeds (Fig. 5).
Foldable membrane microdevice. The LAMP assay for M. oryzae and S. oryzae detection was carried out on a newly developed foldable membrane-based microdevice. The assay was performed separately for M. oryzae and S. oryzae, and the result was observed at the detection chamber. The foldable microdevice detected M. oryzae and S. oryzae genome when the DNA template from the pure cultures or the infected seeds was loaded in the sample chamber, whereas no amplification was observed for the DNA template of healthy seeds. The fuchsin coated membrane at the detection chamber was turned into a purple color for the positive amplification, whereas it turns magenta to colorless for negative results (Fig. 6). In the foldable microdevice-LAMP assay, the pathogen-specific LAMP primers showed positive amplification only when the respective pathogen template DNA was present in the reaction chamber, whereas; negative results were observed when template DNA from either healthy seeds or nonspecific pathogen (B. oryzae, R. oryzae, A. flavus, Penicillium and C. fulvum) was added, indicating its specificity as that of the LAMP assay carried out in polypropylene tubes. The assay was optimized for different incubation times, temperatures, and template concentrations. Like the LAMP assay, in the foldable microdevice, the genome of target pathogens can be detected at the optimized conditions of 56 °C for 20 min using a minimum of 100 fg of DNA template.

Discussions
Among the forty-one seed-borne pathogens of rice, M. oryzae and S. oryzae are responsible for destructive diseases such as blast and sheath rot, respectively 3,4,6 . Therefore, it is essential to detect the seed-borne inoculum so that an early control measure such as seed treatment, seed certification, and quarantine legislation can be undertaken. Though conventional methods such as visual observations and incubation methods are available, improved methods such as PCR based assays (conventional PCR, qPCR, multiplex PCR, Bio-PCR, nested PCR, etc.) are widely used for routine diagnosis of multiple seed-borne pathogens due to their rapidity, sensitivity, and www.nature.com/scientificreports/ robustness as compared to the conventional methods 6,8,9,12,26,27 . However, PCR methods require a sophisticated apparatus for thermocycling at different stages of the amplification, which significantly limits more extensive application in under-resourced settings. Therefore, many isothermal amplification methods, such as the LAMP and HDA, which do not require thermocycling, are becoming increasingly popular among the seed pathology laboratories. The LAMP assay 13 possesses characteristics such as the non-requirement for a thermocycler, rapid amplification, and high specificity and sensitivity well suited for facilitating point-of-care field applications 19 . Therefore, in the present study, we investigated the applicability of isothermal amplification methods (LAMP and HDA) for sensitive and specific diagnosis of M. oryzae and S. oryzae, two predominant seed-borne pathogens of rice. The LAMP and HDA assays were optimized for incubation time and template DNA concentration. Both methods were found efficient in detecting the seed-borne M. oryzae and S. oryzae in seeds. The HDA assay has been widely used to detect viruses and bacteria in the animal system21. Here we report its applicability in the routine diagnosis of the seed-borne pathogens of rice. The Lamp assay results were visualized using EtBr and basic fuchsin dyes, and both were able to distinguish the positive amplification from the healthy control.
Among the different nucleic acid-based techniques tested, the LAMP assay was highly sensitive (up to 100 fg of template DNA) compared to HDA, PCR, and qPCR ( Supplementary Fig. 1). Although the HDA assay was less sensitive and required longer assay-time than the LAMP assay, its sensitivity was comparable with qPCR and hence, can be the next option after LAMP assay in the resource-poor settings. The LAMP and HDA assays were highly specific to their target pathogens (M. oryzae and S. oryzae), as evident from the specificity assays using the template DNA of commonly ice seed-associated fungi such as B. oryzae, R. oryzae, A. flavus, Penicillium sp. and C. fulvum. Therefore, either the LAMP or HDA assays can be used for diagnosing seed-borne M. oryzae and S. oryzae pathogens in seeds, and wherein, the LAMP assay was more sensitive and rapid than HDA.
There are many reports where the LAMP assay has been modified into portable devices for better applicability in the resource-poor settings and the point-of-care facilities 23,25 . In this study, we have developed a portable  www.nature.com/scientificreports/ foldable microdevice platform for performing the LAMP assay for the rapid diagnosis of seed-borne M. oryzae and S. oryzae in rice seeds. This platform was based on the principles developed previously for diagnosing foodborne pathogens 24 . The assay conditions (incubation at 60 °C for 30 min using 100 fg of template DNA) were optimized on the microdevice platform for both M. oryzae and S. oryzae pathogens. In the newly developed microdevice, the sensitivity, specificity, and assay conditions were similar to conventional LAMP assay. Therefore,  For result visualization, we have tested two colorimetric dyes, and both the dyes showed similar results. However, basic fuchsin was selected for result visualization in the foldable microdevice as it is very safe to use and does not require any sophisticated instruments for result visualization. Previous studies have also reported the feasibility of basic fuchsin dye in the foldable isothermal amplification microdevice for colorimetric detection of multiple food-borne pathogens 24 . The use of basic fuchsin dye also eliminates the limitations of the conventional colorimetric detection methods where pyrophosphate, the end product of LAMP assay, acts as a polymerase inhibitor and interferes with amplification of the target site in the template DNA 28,29 .
The portable, foldable LAMP-based microdevice developed in this study is highly sensitive, specific, cheap, and robust for detecting seed-borne M. oryzae and S. oryzae inoculum in the rice seeds and, therefore, can be used as a prototype for the development of fabricated diagnostic kits. Early detection of seed-borne pathogens enables taking appropriate precautionary control measures, ensuring high-quality seed standards, and avoiding potential field epidemics.

Materials and methods
Source of the fungal culture and rice seeds. Pure cultures of M. oryzae strain MG01, S. oryzae strain Saro-13, R. oryzae, A. flavus, Penicillium sp., and C. fulvum were obtained from the Endophytes project, Department of Plant Pathology, University of Agricultural Sciences, Bangalore, India. Pure culture of B. oryzae strain Bo-BLR1 (GenBank Accession No.MH481660) was obtained from the Rice Pathology Laboratory, All India Coordinated Rice Improvement Programme, Gangavathi, India. The rice seeds were collected from the blast and sheath rot infected plants (cv. BPT5204), and seeds were collected from the disease-free plants of cv. BPT5204 were served as negative control. Healthy seeds were again confirmed for the absence of seed-borne inoculum through a blotter test as described previously 30 . Isolation of fungal genomic DNA from the fungi. The genomic DNA from M. oryzae strain MG01, S. oryzae strain Saro-13, R. oryzae, A. flavus, Penicillium sp., B. oryzae, and C. fulvum were isolated using the CTAB method 31 . RNA contamination was removed by digesting the obtained genomic DNA sample with 10 µg/ mL RNase. The isolated genomic DNA was quantified using Nanodrop (Model-DS-11 FX + , DeNovix, USA).
Genomic DNA isolation from rice seeds. Genomic DNA was isolated from rice seeds by the CTAB method. RNA contamination was removed by digesting the obtained genomic DNA sample with 10 µg/mL RNase. Similarly, DNA from the rice seeds was also extracted using a high-throughput DNA extraction method 32 . The extracted DNA quality was assessed using Nanodrop (Model-DS-11 FX + , DeNovix, USA).

Primer design.
Complete details of the different primers designed have been described in Table 1. The PCR and HDA primers were designed using the Primer3.4.1 program (http:// prime r3. ut. ee). For qPCR, forward and reverse primers were designed using IDT analyzer software (https:// www. idtdna. com). Four sets of loop primers www.nature.com/scientificreports/ for the LAMP assay were designed using Primer Explorer V5 (https:// prime rexpl orer. jp/e/). The loop primers included two inner primers, a forward inner primer (FIP) and a backward inner primer (BIP), and two outer primers F3 and B3. FIP consisted of F1c and F2 regions, and BIP consisted of B1c and B2 regions (Supplementary Fig. 2 and 3).
The PCR assay. The PCR assay was conducted in a 10 µl reaction mixture containing 5 µl of 2 × master mix (Takara, Japan), 3 µl of nuclease-free water, 10 pmol/µleach of forward and reverse primers, 50 ng of template DNA. The assay was performed in a thermal cycler (Eppendorf-vapo. protect, Germany The qPCR assay. The assay was performed as described previously 33,34 . Briefly, the reaction mixture contained 10 μl of SYBR Green I, 1 μl of each (2 pmol/µl) forward and reverse primers along with 50 ng of template DNA, and finally, nuclease-free water was used to make up the final volume.  Table 2). The LAMP reaction mixture was optimized for reaction temperature (from 50 to 66 °C) and reaction time (from 10 to 90 min). The optimized time and temperature were further used to assess the LAMP assay's sensitivity at different template DNA dilutions (10 ng to 100 fg). The reaction was also terminated by heat inactivation at 80 °C for 10 min in a water bath (KEMI, India). The assay results were visualized using ethidium bromide (EtBr) and basic fuchsin dyes. About 1 µl EtBr dye (10 mg/mL) was added post-reaction, whereas 1 µl of basic fuchsin (9 μM) was added post the reaction (Supplementary Table 3). The products were visualized directly after the completion of the process. All reactions were performed in triplicates.
The specificity of the LAMP assay. The LAMP assay's specificity for detecting M. oryzae and S. oryzae was evaluated using template DNA of other rice-seed associated fungi such as B. oryzae, R. oryzae, A. flavus, Penicillium sp., and C. fulvum.
The sensitivity of different nucleic acid amplification techniques. The sensitivity of each nucleic acid amplification technique (PCR, qPCR, HDA, and LAMP) was tested at different DNA template concentrations viz., 10 ng, 1 ng, 100 pg, 50 pg, 10 pg, 100 fg, and 50 fg. Pathogen-specific (M. oryzae and S. oryzae) primers of PCR, qPCR, HDA, and LAMP assays were tested against different template concentrations using optimized amplification conditions.
Foldable membrane microdevice platform. A PCR sealing film (40 × 90 mm) was used as a bottom layer to develop a portable, foldable membrane-based microdevice for performing the LAMP assay. The PCR film was divided into three chambers viz., sample chamber, reaction chamber, and a detection chamber as reported previously 24 . The reaction chamber was covered with polycarbonate (PC) film (40 × 30 mm), and nine reaction wells (2.5 mm radius each) were cut out of the PC film. The detection chamber was coated with basic fuchsin dye for result visualization. The basic step involved in designing a membrane microdevice is illustrated in Fig. 7. In the sample chamber, 10μL of template DNA (≈100 fg) was added and allowed for 2 min at room temperature. Similarly, in the reaction chamber, 10μL of LAMP reaction mixture (buffer, enzyme, and primers) was added to each reaction well and allowed for 2 min at room temperature. Later, the sample chamber was folded onto the reaction chamber and was finger pressed firmly for a min and then separated. The reaction chamber was then covered with a sealing film to avoid the reagents evaporation and incubated at 60 °C for 30 min. After incubation, the sealing film was removed, and about 1μL of HCl was added to each reaction wells, and again incubated at 58 °C for 5 min. To the incubated reaction chamber, 2 μL of Sodium sulfite was added and folded onto the detection chamber to visualize the result. The results were inferred based on the color change either from magenta to colorless (for negative amplification) or magenta to purple (for positive amplification). This method was standardized separately for the detection of M. oryzae and S. oryzae.