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

Abstract

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.

Figure 1

Reaction temperature optimization. (a) Temperature optimization (ranged between 50 to 66 °C) for the LAMP assay with Ethidium bromide and basic fuchsin dyes. (b) Further, results were visualized on a 2% agarose gel. Where lanes 1 to 9 indicate the temperature in °C (50, 52, 54, 56, 58, 60, 62, 64, and 66), lane N indicates negative control, and Lane L indicates 1 kb ladder (Thermo Scientific, USA).

Figure 2

Reaction time optimization. Time optimization for the LAMP assay with Ethidium bromide and basic fuchsin (a) where NTC indicates non-template control. The results were visualized in 2% agarose gel (b), lane 1 to 3 indicates reaction time in min (10, 20, and 30), lane 4 indicates negative control, and the lane L indicates 1 kb ladder (Thermo Scientific, USA).

Figure 3

The specificity of LAMP assay. The specificity of the LAMP assay was validated for (a) Magnaporthe oryzae and (b) Sarocladium oryzae against Bipolaris oryzae, Rhizopus oryzae, Aspergillus flavus, Cladosporium fulvum, and Penicillium sp. using ethidium bromide and basic fuchsin. NTC indicates non-template control.

Figure 4

Comparative analysis of PCR, qPCR, LAMP assay, and HAD for their sensitivity using Sarocladium oryzae specific primers. (a) The sensitivity of the PCR assay, the result was visualized in a 1% agarose gel. Where lane L indicates 1 Kb ladder (Thermo Scientific), lanes 1–7 indicates 10 ng, 1 ng, 100 pg, 50 pg, 10 pg, 100 fg, 50 fg, and lane N indicates negative control. (b) The sensitivity of the qPCR assay. The standard curve represents log10 of DNA concentration in fg against Ct (cycle threshold) values for different DNA concentrations. (c) The sensitivity of the LAMP assay. The result was visualized in a 2% agarose gel. Lanes (1 to 7) indicates template DNA concentrations (10 ng, 1 ng, 100 pg, 50 pg, 10 pg, 100 fg, and 50 fg); lane N indicates negative control. (d) The sensitivity of the HDA assay. The result was visualized in a 1% agarose gel. Lanes (1 to 7) indicates template DNA concentrations (10 ng, 1 ng, 100 pg, 50 pg, 10 pg, 100 fg, and 50 fg), lane N indicates negative control, and lane L indicates 1 kb ladder (Thermo Scientific).

Figure 5

Reaction template-concentration optimization. Template-concentration optimization (10 ng to 50 fg) result was visualized using EtBr and basic fuchsin (a). Further, the result was confirmed on a 2% agarose gel (b), where the lanes (1 to 7) indicate template DNA concentrations (10 ng, 1 ng, 100 pg, 50 pg, 10 pg, 100 fg, and 50 fg), lane N indicates negative control, and the ane L indicates 1 kb ladder (Thermo Scientific).

Figure 6

Detection of Magnaporthe oryzae and Sarocladium oryzae on foldable membrane microdevice platform. The result was visualized using basic fuchsin dye.

Figure 7

Overview of the membrane-based microdevice design. (a) Sample chamber with nine absorption discs, (b) reaction chamber, and (c) detection chamber with fuchsin stained strips.