Methods and systems for colorimetric detection of quiescent colletotrichum in harvested fruit

Abstract

The present invention is in the general area of post-harvest fruit disease detection, more pertinently the present invention is directed towards methods for colorimetric detection of pathogenic fungi in their latent phase in harvested fruit, using a paper / plate -based reverse transcription loop- mediated isothermal amplification (RT-LAMP). The present invention provides a paper-based device for performing reverse transcription loop-mediated isothermal amplification (RT-LAMP) with real-time simultaneous detection of C. gloeosporioides latent infections in tomatoes. RT- LAMP -based PAD platform comprises a paper substrate on which the RNA amplification reaction occurs. The assay provided by the present invention is highly specific for the latent stage of C. gloeosporioides with an analytical sensitivity of 0.5 pg of total extracted RNA, with results obtained within 40 min and hence can be efficiently employed for identifying C. gloeosporioides in resource-limited setting.

Description

[0001] METHODS AND SYSTEMS FOR COLORIMETRIC DETECTION OF QUIESCENT

[0002] COLLETOTRICHUM IN HARVESTED FRUIT

[0003] INVENTORS

[0004] Dr. Evgeni Eltzov and Dr. Noam Alkan

[0005] FIELD OF INVENTION

[0006] The invention is in the general area of post-harvest fruit disease detection, more pertinently the present invention is directed towards methods for colorimetric detection of pathogenic fungi in their latent phase in harvested fruit, using a paper / plate-based reverse transcription loop-mediated isothermal amplification (RT-LAMP).

[0007] BACKGROUND OF THE INVENTION

[0008] Postharvest food loss is a big threat to food safety and security, making the global food system vulnerable. According to the FAO-commissioned study, around one-third (1.3 billion tons) of food produced for human consumption is lost or wasted globally each year. Postharvest losses of fruits and vegetables are far higher than those of cereal crops and are estimated to be 40-50%. Decaying of postharvest fruits is a major cause of those losses and is primarily caused by fungal pathogens after fruit ripening. Further, the toxic compounds released by pathogens in the infected food also pose a serious health threat to both humans and livestock. Most fungal pathogens penetrate the agricultural produce in the field. Hence, to ensure food safety and alleviate food losses, it becomes vital to screen the fruits for the presence of the fungus after harvest and during the supply chain.

[0009] Fungi from several genera may cause postharvest decay of fresh produce (Feliziani and Romanazzi, 2016; Glos et al., 2022; Wenneker and Thomma, 2020). Colletotrichum gloeosporioid.es represents a common postharvest fungal pathogen that infects over 470 widely distributed host genera, including mango, avocado, yam, cassava, pomegranate, and strawberry (Alkan et al., 2013). On fruit, after conidial germination, C. gloeosporioides goes through three lifestyle stages: appressoria (penetration), quiescence (latent stage), and the necrotrophic stage characterized by anthracnose rot. Usually, after penetrating the unripe fruit, C. gloeosporioides remains quiescent until the fruit ripens. The pathogen swiftly switches to a necrotrophic stage at ripening due to the decline in the host defense responses. The quiescent infections are microscopic and cannot be visually detected during packaging or subsequent transport. Thus, there is a need to design assays that allows the identification of the fungi at an initial quiescent stage of infection in order to prevent potential decay in fruits and vegetables during the supply chain and consumer storage.

[0010] Traditionally, methods for detecting fungal pathogens were based on morphological characteristics, including the color of the colony, the shape, and size of conidia, the presence of setae, etc. (Photita et al., 2005). However, these identification methods are labor-intensive and time-consuming. Further, as morphological properties can change according to the environmental conditions, these methods are incompetent in identifying quiescent infection of fungal pathogens (Cai et al., 2009; Hyde et al., 2009; Phoulivong et al., 2010; Weir et al., 2012).

[0011] There is, therefore, a long-felt unmet need for improved methods of detecting fungal pathogens in their latent phase in harvested fruit.

[0012] BRIEF DESCRIPTION OF THE INVENTION

[0013] To overcome the restraints exhibited by traditional detection methods, several molecular methods (PCR, RT-PCR, ELISA) were characterized to detect fungal pathogens. These technologies provide reliable and sensitive results; however, they are time-consuming, expensive, require complicated methods, and require experienced personnel, making them less suitable for use in agricultural applications. Therefore, the industry did not adopt those methods, and there is a critical need to develop easy and less expensive methods to detect pathogenic fungi in the harvested produce.

[0014] Presently, the RT-LAMP assay is being considered more effective for detecting plant pathogens than PCR / RT-PCR as it is faster, easier, and does not require specialized laboratory equipment (Chandra et al., 2015; Hansen et al., 2016; Niessen and Vogel, 2010; Tomlinson et al., 2010). LAMP assay can be performed under isothermal conditions in a single tube using a dry heat block or a water bath, enabling the LAMP assay to be quicker, easier to use, and cost-effective. Further, the LAMP assay is highly specific owing to the use of several primers (four to six), which can anneal up to eight distinct sites on the DNA template, compared to only two in typical PCR (Foo et al., 2020; Soroka et al., 2021). Moreover, nucleic acid amplification during LAMP assay can be monitored by visual observation of the color change of a pH indicator dye (phenol-red) or DNA- intercalating dye (SYBR Green, calcein, or hydroxynaphthol blue). Although few studies have reported the use of LAMP for the detection of C. gloeosporioid.es (Lan et al., 2020; Wang et al., 2017; Wu et al., 2019), there are no reports about the use of LAMP to detect quiescent infection in fruit.

[0015] The present invention provides a novel paper or plate -based RT-LAMP assay for specific and sensitive detection of the latent infection of fungal pathogens in post-harvest fruit. Stagespecific transcript sequences of quiescence that are significantly upregulated during the latent stage of fungal pathogens are used as a target marker for the fungal presence at its latent stage.

[0016] Therefore, the present invention aims to provide a rapid, reliable, and easy-to-use analytical tool for the efficient management of postharvest decay of fruits. The present invention is directed towards a paper or plate-based LAMP assay for specific and sensitive detection of the latent infection of fungal pathogens in post-harvest fruit.

[0017] The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention wherein:

[0018] Figure 1 is a schematic representation of plate-based RT-LAMP assay and colorimetric detection of amplification products, according to the present invention.

[0019] Figures 2-6 refer to the experimental results of the RT-LAMP reaction optimization process:

[0020] Figure 2 refers to the optimization of DMSO concentration in RT-LAMP assay.

[0021] Figure 3 refers to RT-LAMP reaction time optimization

[0022] Figure 4 refers to RT-LAMP reaction temperature optimization

[0023] Figure 5 refers to the effect of heater type on reaction efficiency

[0024] Figure 6 and Figure 7 refer to RT-LAMP reaction specificity optimization

[0025] Figure 8 and Figure 9 refer to RT-LAMP reaction sensitivity optimization

[0026] Figure 10 Refers to the kinetics of RT-LAMP

[0027] Figure 11 is a schematic representation of Paper-based RT-LAMP assay and colorimetric detection of amplification products, according to the present invention.

[0028] Figures 7-10 refer to the experimental results of the Paper-based RT-LAMP reaction optimization process: Figure 12 refers to paper type selection

[0029] Figure 13 refers to paper-based RT-LAMP reaction time optimization

[0030] Figure 14 refers to paper-based RT-LAMP specificity optimization

[0031] Figure 15 refers to paper-based RT-LAMP sensitivity optimization

[0032] Figure 16 represents a comparative analysis of RT-LAMP reaction efficiency using different heat sources.

[0033] Figure 17 represents stabilized (i.e., heat-dried) vs. fresh RT-LAMP assay performance comparison.

[0034] Figure 18 represents experimental results of optimization of trehalose concentrations in heat-dry RT-LAMP reaction assay and its efficiency to detect latent Botrytis cinerea.

[0035] Figure 19 represents experimrntal results of assessing the sensitivity of the 43 °C heat-dried mixture in an optimized RT-LAMP Assay.

[0036] Figure 20 represents Detection of specific targets: Botrytis cinerea Polysaccharide Lyase family and Alternaria alternata (20A) and agarose gel electrophoresis presenting results of RT-LAMP reactions following heat drying at 43°C (20B).

[0037] Figure 21 represents experimental results of detection of latent Botrytis cinerea in infected unripe tomato fruit samples using heat-dry RT-LAMP.

[0038] Figure 22 Long-term stability of the dried LAMP mixture in a 384-well plate under different storage temperatures.

[0039] Figure 23 represents correlation between the decay rates in tomatoes (23A) and the responses of the LAMP-based setup to Botrytis cinerea conidia concentration (23B).

[0040] Figure 24 represents experimental results of LAMP results obtained from samples extracted using different in-house RNA extraction protocols compared to a commercial RNA extraction kit.

[0041] Figure 25 represents the effect of silica nanoparticle -based extraction approach on the efficiency of RNA extraction process. The objects of the invention are achieved by methods and systems characterized by what is stated in the independent claims. The preferred embodiments of the invention are disclosed in the dependent claims.

[0042] DETAILED DESCRIPTION OF THE INVENTION

[0043] In the present invention, a method for colorimetric detection of quiescent fungal pathogens in postharvest fruit is presented. According to the present invention, a novel paper or plate-based RT- LAMP assay was designed to allow specific detection of quiescent fungal pathogens as described in the following examples:

[0044] EXAMPLE 1

[0045] 1. Preparation of conidia suspensions of C. gloeosporioides

[0046] Single spore cultures collected from decayed avocado were cultured on potato dextrose broth media (Formedium, Tivan-Biotech Ltd, Israel) and incubated at 24 °C for 3 days in a shaking incubator at 150 rpm. The conidia were filtered through cheesecloth and collected in distilled water. The conidia were washed twice by resuspension in distilled water and centrifuged. The conidia suspensions were adjusted to 5x105 conidia / mL to be used as an inoculum.

[0047] 2. Fungal inoculation and RNA extraction

[0048] One set of mature green and ripe tomato (cv. Tory) fruits (n=30) were inoculated with 7 pL of the conidial suspensions of C. gloeosporioides (5xl05conidia / mL), and the green fruit was drop inoculated at 15 inoculation spots per fruit and the red fruit was wound inoculated (1mm deep) at 5 spots per fruit. Similarly, the second set of fruits (n=30) was wound inoculated (1mm deep) with 7 pL of the conidial suspensions of B. cinerea (5xl05conidia / mL). The inoculated fruits were incubated at 25°C. The infected tissue samples for the penetration and latent stages of fungi were taken after 72 h on green fruit and 48 h of infection on red fruit, respectively. Tissues (150 mg) collected from infected fruits were snap -frozen in liquid nitrogen and ground into a fine powder using mortar and pestle. Total RNA from the infected tissues was extracted using SpectrumTM Plant Total RNA Kit (STRN250-1 KT, Sigma Aldrich, Taufkirchen, Germany), according to the manufacturer's protocol. The RNA concentration and purity were determined on the Nanodrop™ Nanodrop 2000 UV-VIS Spectrophotometer (Thermo Scientific, Shoham, Israel). The integrity of RNA samples was checked by electrophoresis on 1.5% (w / v) agarose (Biomol, Sevilla, Spain) gels using IX TAE buffer (Thermo Fisher Scientific, Waltham, Massachusetts, United States) at 100 V for 35 min. 3. RT-LAMP primers

[0049] In one embodiment of the present invention, quintessence marker ECH transcripts are designed for pathogenic fungi, a quiescent stage- specific detection.

[0050] Accordingly, a set of primers for C. gloeosporioid.es quintessence marker enoyl CoA hydratase (ECH) was designed.

[0051] The RT-LAMP primer sets used against the ECH sequence were designed using Primer Explorer v5 (http: / / primerexplorer.jp / lampv5e / index.html). All the primers were purchased from Hylabs (Rehovot, Israel) and resuspended at a concentration of 100 pM in nuclease-free water (Invitrogen, United States). The sequences of each oligonucleotide primer used for the RT-LAMP assay can be found in Table 1. A synthetic DNA sequence sourced from Genewiz (South Plainfield, New Jersey) was employed as a positive control for the ECH gene.

[0052] 4. Optimization of RT-LAMP reaction

[0053] Before examining the amplification efficiency of the RT-LAMP assay in the presence of the paper membranes, preliminary experiments were conducted in test tubes to standardize the RT-LAMP assay.

[0054] 4.1 Colorimetric quantification of the RT-LAMP assay

[0055] For the colorimetric quantification, the RT-LAMP assay was conducted in a 20 uL reaction in 384- well plates in a heat block (AccuBlock™, D1301-230V, New Jersey, United States). To validate the colorimetric assay, a quantitative analysis was performed, and the colorimetric LAMP assay was monitored by visual observation of the color change of a pH indicator dye (phenol-red) for the detection of quiescent C. gloeosporioides. In the colorimetric assay, pyrophosphate and hydrogen ions released during amplification lower the pH of the reaction buffer containing phenol red indicator, resulting in an observable color change that can be quantified based on absorbance readings.

[0056] After the completion of the reaction, the plate was read in a plate reader at 434 nm and 560 nm (Synergy Hl — BioTek), which are the absorbance values for the two forms of the dye, i.e., yellow and pink, at pH 6 and 8, respectively. To interpret the color change, AOD was calculated by subtracting the absorbance at 560 nm from the one at 434 nm.

[0057] 4.2 Image analysis

[0058] The images of reaction tubes were captured after the completion of the LAMP reaction using a smartphone camera under ambient lighting conditions. The RGB color split function was used to divide the RGB images into their constituent red, green and blue channels using ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA). The mean green and red intensity of the selected colored zone of reaction tubes were measured to calculate the green / red ratio. The experiment was performed in triplicates.

[0059] 4.3 Optimization of DMSO concentration

[0060] The specificity optimization of the RT-LAMP assay was performed by adding three different concentrations of DMSO (50, 100, and 200 pg) to the reaction mix. Results showed that NTC also turned orange-yellow in reactions prepared without DMSO addition (Figure 2), which is attributed to non-specific amplification caused by hybridization among primers of LAMP. At 7% (v / v) DMSO, the positive samples presented yellow color while NTC remained pink after 23 min of incubation. At 8% (v / v) and 10% (v / v) DMSO, the samples with the target gene showed pinkishorange and dark-pink colors, respectively, after 23 min of incubation time (Figure 2). This is probably because a higher concentration of DMSO may decrease the rate of annealing of the template or cause inhibition of DNA polymerase activity, thereby resulting in slower amplification (Montgomery et al., 2014; Wang et al., 2015). Hence, 7% (v / v) DMSO concentration was selected as the optimal concentration to be used in further LAMP assays.

[0061] 4.3 Reaction time optimization

[0062] Determining the optimal reaction time for the RT-LAMP assay is crucial, as an extended incubation time could lead to non-specific amplification in NTC. Hence, the reaction plates were incubated for various lengths of time (0, 17, 23, and 25 min), and the optimal time was evaluated based on the colorimetric analysis and gel electrophoresis. At 17 and 20 min, the samples with the target gene showed light pink and pinkish-orange color, respectively, indicating that these incubation times are too short to allow appropriate amplification of the template (Figure 3A). At 23 min incubation time, the samples with higher concentrations (50 and lOOpg) turned yellow, while samples with a lower amount of target exhibited an orange color. While at the same incubation time, NTC remained pink. However, with a further increase in the reaction time to 25 min, NTC alone turned slightly orange, pointing to non-specific amplification, which is a known problem with RT-LAMP (Figure 3A).

[0063] For image analysis, the 24-bit colored image was first split into red-green-blue channels. The green channel images showed a clear difference between positive and negative samples, providing accurate image analysis. Hence, the green intensity was used to measure the progress of amplification, while the red intensity was used as a reference, and the image analysis data was plotted as a normalized green-to-red ratio (G / R). As shown in Figure 3B, the G / R ratio increases with the increase in the incubation time, thereby indicating the progression of amplification. The negative control also exhibited a gradual increase in the G / R ratio, owing to the color change of NTC from dark pink to light pink. However, a statistically significant difference (p < 0.01) in the G / R intensity was noted among the quiescent RNA sample (20pg) and NTC until 23 min of incubation. However, at 25 min, the difference was less significant (p < 0.05), owing to the color change of NTC from pink to orange.

[0064] Analysis of gel electrophoresis also showed a distinct band pattern for NTC at 25 min of incubation, while NTC showed no bands at incubation time lower than 25 min (Figure 3C). Hence, based on the observed results, an incubation time of 23 min was selected as the optimal reaction time for further RT-LAMP assays.

[0065] 4.4 Reaction Temperature Optimization

[0066] Initially, the optimal temperature for the RT-LAMP assay was selected by performing the reaction at three different temperatures, viz., 63 °C, 65 °C, and 68 °C. Results showed that at 63 °C, the non-template control (NTC) sample also exhibited color change indicating non-specific amplification, while no amplification was noted at 68 °C (Figure 4). At 65 °C, the target sample exhibited an efficient amplification, while the NTC sample presented dark pink color intensity showing no amplification. Therefore, 65 °C was selected as the optimum temperature for the paper-based RT-LAMP.

[0067] 4.6 The effect of heater type on reaction efficiency

[0068] The effect of the heater type (i.e., source) on the reaction efficiency was tested by heating at 65°C the 384 well plates with positive and negative samples with an oven, heat block and wet bath. Figure 5 demonstrates that the differences between the positive and negative samples were observed for all tested heating sources. However, the highest difference was observed in the samples heated in the oven. The possible reason for these results is that the plates are heated more homogeneously and faster in the oven, producing higher reaction efficiency.

[0069] 4.5 Specificity of the RT-LAMP assay

[0070] The detection specificity of the developed RT-LAMP assay was examined using other target markers. Three different samples were employed, including the RNA sample extracted from tomato tissue at the appressoria stage, a synthetic appressoria marker gene (SD), and a synthetic quiescent marker gene (HD AC), which is different from ECH. The synthetic mini-genes encoding for HDAC and SD were sourced from Genewiz (South Plainfield, New Jersey). The difference between the quiescent RNA samples of C. gloeosporioides in tomato fruit peel and other samples could be clearly seen by the naked eye as the quiescent stage samples turned yellow, while samples extracted from tomato fruit with C. gloeosporioides in appres soria stage and samples with synthetic appressoria marker gene (SD) remained pink (Figure 6 A-C). This clearly indicates that the assay did not cross-react with RNA from the appressorium stage of fungi. This also confirms that the ECH gene is highly expressed in C. gloeosporioides during the quiescent stage of its life cycle. Similarly, the color of the non-specific quiescent gene marker (HD AC) was similar to that of the NTC, which implies that the designed RT-LAMP primers and the developed RT-LAMP assay are specific to the ECH gene. The results of the colorimetric RT-LAMP assay correspond with the results recorded during gel electrophoresis, wherein quiescent stage samples exhibited the characteristic DNA laddering profile associated with RT-LAMP amplicons, while no bands were observed for appressoria stage samples. Along similar lines, both non-specific synthetic marker genes (SD, HDAC) were not amplified (Figure 6D). Therefore, in the present invention, designing RT-LAMP primers against the specific mRNA sequence (ECH) that is upregulated in the C. gloeosporioides during its quiescent stage in fruits, enhanced the overall specificity and sensitivity of the assay during quiescency.

[0071] The specificity of the assay and capability to test multiple fungi was determined by conducting the RT-LAMP assay using RNA extracted from the tomato infected during the appressorium stage of three different fungi strains (e.g., Botrytis Cinerea B.c), C. gloeosporioides (C.g) and Alternaria alternata (A.a)). The RT-LAMP assays were conducted in total reaction volumes of 25 pL containing 12.5 pL of WarmStart Colorimetric RT-LAMP 2X Master Mix (M1800, New England Biolabs), 2 pL of 10X primer mix, 2.5 pL (100 pg) of RNA and 8 pL of nuclease-free water. The 2X DNA & RNA WarmStart colorimetric LAMP master mix (New England Biolabs, Ipswich, MA, USA) consisted of Bst 2.0 WarmStart DNA Polymerase, WarmStart RTx, and phenol red as a pH indicator dye.

[0072] The 10X primer mix contained FIP (Forward Inner Primer) and BIP (Backward Inner Primer) at 1.6 pM each, F3 (Forward Outer Primer) and B3 (Backward Outer Primer) at 0.2 pM each, and LOOP F and LOOP B (Forward Loop and Backward Loop primers) at 0.4 pM each. As the RT- LAMP 2X master mix contains reverse transcriptase and therefore reverse transcription and LAMP reactions were conducted in a single step. The tubes were incubated at 65 °C for 23 min in a heat block (Model K30, Allsheng Zhejiang, China). Positive reactions presented a color change from pink to yellow, while the negative ones remained pink. After the completion of the reaction, the plate was read in a plate reader at 434 nm and 560 nm (Synergy Hl — BioTek), which are the absorbance values for the two forms of the dye, i.e., yellow and pink, at pH 6 and 8, respectively. To interpret the color change, AOD was calculated by subtracting the absorbance at 560 nm from the one at 434 nm.

[0073] Figure 7 represents not only the capability of the proposed system to detect different pathogenic fungi types but also demonstrates the system's specificity. Application constructed to determine Alternaria alternata in the extracts responded only to this fungus (Figure 7A). The system for the detection of Botrytis Cinerea also produced a positive reaction in the samples only with this strain.

[0074] 4.6 Sensitivity of the RT-LAMP assay

[0075] The assay's sensitivity was evaluated to determine the lower limit of detection of the RT-LAMP assay. The assay was conducted using different concentrations of RNA samples (ranging from Ipg to lOOpg) extracted from the infected tomato spots during the quiescent stage of C. gloeosporioides.

[0076] Results showed that after 23 min of incubation, samples with lower RNA concentrations (1 and 5pg) showed orange color, while samples carrying higher RNA concentrations (>10pg) exhibited yellow color (Figure 8A). In this experiment, a light orange color was noted in reactions wherein the initial amount of template was low. This is probably because samples with lower concentrations of template exhibit a lower amplification rate which eventually generates fewer pyrophosphate ions, thereby causing slight acidification of the sample. However, as the orange color could be easily distinguished from bright pink colored-NTC, colorimetric naked-eye sensing was observed predominantly even at low concentrations of the target gene.

[0077] The color image analysis results also showed an increasing trend in the G / R ratio with an increase in the concentration of quiescent RNA. A significant statistical difference (p < 0.01) was observed among the quiescent RNA samples and negative control (Figure 8B). The positive control (PTC) exhibited a higher G / R ratio than the quiescent RNA sample when employed at the same concentration (50pg). This is probably because the PTC is a synthetic ECH gene, while the quiescent RNA sample comprises the total RNA extracted from the fruit-infected tissue.

[0078] The amplification rate was also confirmed by agarose gel electrophoresis of LAMP amplicons, which showed that the intensity of bands was correlated with the initial target concentration (Figure 8C).

[0079] The results showed that the AOD values increased in a linear positive correlation as the RNA concentration increased, visible from the value of coefficient square, R2 = 0.9802 (Figure 9). This is because, at low RNA concentration, target samples exhibited an orange color owing to less amplification rate, which resulted in a lower absorbance value at nm. While samples with a high target concentration showed yellow color, leading to higher absorbance at 434 nm, which yielded higher AOD values. As in NTC samples, no amplification occurred; hence, it exhibited the highest negative OD values owing to higher absorbance recorded for the pink color at 560 nm. Hence, these results correspond with the sensitivity results, which showed that the designed RT-LAMP assay could enable a naked-eye detection of up to Ipg of quiescent RNA concentration.

[0080] 4.7 Kinetic changes of RT-LAMP reaction

[0081] Kinetic changes of the RT-LAMP through the reaction process were tested in the presence (100 pM, POS) or absence (NEG) of Botrytis Cinerea (Figure 10). Figure 9 demonstrates that the first color changes may be observed after 10 minutes, while the maximum differences were detected 24-26 minutes after sample addition and heating starting.

[0082] 5. Optimization of Paper-based RT-LAMP assay

[0083] Based on the experimental data obtained from the RT-LAMP assay optimization, a novel paperbased RT-LAMP assay was designed, as described in the following steps:

[0084] 5.1 Paper type optimization

[0085] In one embodiment of the present invention, a cellulose membrane substrate is used for the currently presented paper-based RT-LAMP assay.

[0086] The selection of paper type is crucial for developing a paper-based RT-LAMP assay. The paper should not inhibit the RT-LAMP components and should provide a large surface area for the RT- LAMP amplification.

[0087] Three different types of paper membranes, viz., nitrocellulose (NC) paper (pore size of 5pm) (Millipore, Burlington, MA, USA) and cellulose chromatography paper (Whatman, Pittsburgh, PA, USA) of grade 1 (pore size of 10pm) and grade 4 (pore size of 20-25 pm) were screened to carry out RT-LAMP reaction. To select the best suitable paper membrane compatible with the RT- LAMP reaction, all three matrices were first clipped into 5 mm x 5 mm circles with a fluid capacity of 15 pL. The membranes were placed in the 96-well plate, and two different concentrations (lOpg and 20pg) of extracted RNA samples were pipetted directly onto the surface of the studied paper types. While for the negative control, the paper membranes were added with nuclease -free water. As the RNA or water spread over the paper fibers via capillary action, the RT-LAMP reaction mixture consisting of primers and the master mix was added. The plate was then sealed with a sealing film to prevent evaporation and placed in the dry bath, pre-set to a temperature of 65 °C for 40 min. After completion of the reaction, the membranes were analyzed for color change. In one embodiment of the present invention, detecting the colorimetric change in the medium of assay wells is performed by visual inspection and calculating the colorimetric change is performed by photographing the assay module and analyzing the difference between green to red (G / R) ratio in control medium versus RNA-containing medium, using an image analyzer.

[0088] Results showed that the nitrocellulose membrane (NCM) showed yellow color for both extracted RNA samples and negative control, pointing to non-specific amplification (Figure 12, upper panel). This could be owing to the extremely small pore size (5 pm) of the NCM; the LAMP mixture did not get absorbed into the NCM, and the amplification occurred in the solution added on the surface of the NCM. Hence, an inefficient absorption of LAMP components could have facilitated improper annealing of primers, resulting in non-specific amplification. While the CM (Gl) membrane did not show any color change in the positive sample, as it presented pink color in both extracted RNA samples and NC. Unlike NCM, the RT-LAMP solution appeared to get absorbed into the CM (Gl) membrane; however, owing to its small pore size, CM (Gl) membrane did not provide adequate reaction space for the RT-LAMP reaction. On the contrary, the cellulose grade 4 membrane showed a distinct color transition from pink to yellow in positive samples, while the negative samples remained pink (Figure 12, upper panel). This suggests that the large pore size of the cellulose grade 4 membrane facilitated an efficient absorption of RNA and LAMP components, resulting in specific primer annealing.

[0089] After incubation, the amplicons were eluted out of the paper by submersion in 35 pL TE (Tris- HC1 and EDTA) buffer, followed by brief vortexing and centrifugation. Amplification was then confirmed with gel electrophoresis of eluted amplicons. According to the gel electrophoresis results (Figure 12, lower panel), the cellulose grade 4 membrane showed the best amplification result. Similar to colorimetric detection, amplicons on NCM showed smeared band patterns in the negative control, validating non-specific amplification.

[0090] In another embodiment of the present invention, assay module incubation is at 65°c for 40 minutes. The reaction temperature was optimized by first performing an RT-LAMP reaction in test tubes, as described above. In addition, optimization of reaction time for paper -based RT-LAMP assay was performed.

[0091] 5.2 Reaction time optimization

[0092] Determining the optimal reaction time for the RT-LAMP assay is crucial, as an extended incubation time could lead to non-specific amplification in the negative control. Hence, in the present invention, the reaction plates were incubated for various lengths of time (0, 20, 30, 40, and 50 min), and the optimal time was evaluated based on colorimetric analysis and gel electrophoresis. At 20 min, the samples with the target gene were retained with light pink color, indicating no amplification. At 30 min, the color of the paper disc impregnated with the target sample changed from pink to light orange, indicating the initiation of the amplification reaction, while the NC remained pink. After 40 min of incubation, the color of the paper disc with the target sample turned from orange to yellow, indicating an accurate and precise amplification of the target gene. While at the same incubation time, NC remained pink. However, with a further increase in the reaction time to 50 min, NC also turned slightly yellow, pointing to a non-specific amplification (Figure 13A, upper panel). The non-specific amplification is a known problem with RT-LAMP that has been reported in previous studies that prolonged reaction time induced the formation of spurious amplification products that result in pH-shift, thereby yielding false-positive results.

[0093] After the amplification, the paper images were captured using a smartphone camera (Xiaomi Redmi Note 7, China) under ambient lighting conditions. The RGB color split function was used to divide the RGB images into their constituent red, green and blue channels using ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA). The mean green and red intensity of the selected colored zone of membranes was measured to calculate the green / red ratio. The experiment was performed in triplicates.

[0094] The colorimetric detection results were found to be correlated with the Image J analysis that showed that until 20 min, there was no amplification, and no significant difference was noticed among the normalized green to red (G / R ratio) of extracted RNA sample and NC (Figure 13B). However, at 30 min, the RNA sample exhibited a slightly higher normalized G / R ratio as compared to the negative control (p<0.05). This could be attributed to the drop in red intensity value because of the transition of the membrane's color from pink to orange. At 40 min, a significant difference in the normalized G / R ratio of the real sample and NC was noticed along with the color change of the target sample from orange to yellow (p<0.01), while at incubation time of 50 min, both the target sample and negative control turned yellow, which leads to a non-significant change in the G / R ratio. Similarly, analysis of gel electrophoresis also showed a distinct but smeared band pattern for the negative control at 50 min of incubation, which indicates a non-specific amplification (Figure 13A, lower panel). Hence, based on the observed results, an incubation time of 40 min was selected as the optimal reaction time for further paper-based RT-LAMP assays.

[0095] Compared to the LAMP reaction conducted in tubes that allowed the detection of the latent RNA within 23 min, the paper-based LAMP assay exhibited a longer detection time. However, the tubebased LAMP is more prone to non-specific amplification and hence requires the addition of stabilizing agents such as DMSO and betaine to prevent the primer hybridization. On the contrary, paper-based LAMP was found to be more sensitive and specific as it was conducted without adding any stabilizing agent. This is probably because the large surface area of the cellulose matrix facilitated the uniform spreading of the LAMP components, thereby hindering the selfhybridization of primers.

[0096] 5.3 Specificity of Paper-based RT-LAMP reaction

[0097] The detection specificity of the paper-based LAMP assay was assessed using the non-specific RNA templates from the penetration stage of C. gloeosporioid.es and the latent stage of B. cinerea. The assay's specificity could be clearly recognized by the naked eye as the paper impregnated with different concentrations of latent stage samples of C. gloeosporioides turned yellow, while the samples extracted during the penetration stage of C. gloeosporioides and latent stage of B. cinerea remained pink (Figure 14A, upper panel). This indicates that the assay allowed the specific detection of the latent stage of C. gloeosporioides as it did not cross-react with the RNA template from the latent stage of other fungi (e.g., B. cinerea'). The inability of the assay to detect the penetration stage of C. gloeosporioides confirms that the ECH gene upregulates only during the latent stage of C. gloeosporioides.

[0098] The results of the colorimetric RT-LAMP assay correspond with the results recorded during image analysis which demonstrated that the G / R ratio of latent samples of C. gloeosporioides was significantly higher than the samples from the penetration stage of C. gloeosporioides and samples of the latent stage of B. cinerea (p<0.01) (Figure 14B).

[0099] Further, the results of gel electrophoresis also confirmed the specificity of the assay as the samples extracted from the latent stage of C. gloeosporioides samples exhibited the characteristic RNA profile associated with LAMP amplicons, while no bands were observed for penetration stage samples. Along similar lines, the RNA template from the latent stage of B. cinerea did not get amplified (Figure 14A, lower panel).

[0100] 5.3 Sensitivity of Paper-based RT-LAMP reaction

[0101] The assay's sensitivity was evaluated to determine the lower limit of detection of the paper -based RT-LAMP assay. The assay was conducted using different dilutions of RNA samples extracted from infected mature green tomato fruit during the latent stage of C. gloeosporioides fungi. Results showed that after 40 min of incubation, all tested concentrations of the latent fungi exhibited a visible color change from pink to yellow (Figure 15A, upper panel). The surface of the paper discs impregnated with a higher concentration of RNA sample exhibited a stronger yellow color, which resulted in a higher G / R ratio owing to a lower red intensity value. Contrarily, the paper discs subjected to lower concentrations (0.5-lpg) of the sample exhibited a partial pink color, causing an increase in red intensity value, thereby resulting in a comparatively lower G / R ratio (Figure 15B). Furthermore, a positive linear correlation was found between the higher RNA concentrations to the change of color from pink to yellow. The G / R ratio of all the studied concentrations of RNA templates exhibited a statistically significant difference as compared to the negative control (p<0.01), thereby allowing the detection of a significantly low level of 0.5pg of total RNA extracted from tomato fruit infected with latent fungi of C. gloeosporioides, which is at least 40- fold more sensitive than a regular PCR reaction targeting ITS or tubulin sequence. The positive control (PC) exhibited a slightly higher G / R ratio than the latent RNA sample when employed at the concentration of 10 pg. This is because PC comprises only from ECH gene, while the total extracted RNA from the latent sample represents all plant and fungal transcripts; hence the actual concentration of ECH is lower because it is diluted in the plant and fungal transcripts from the infected tissue.

[0102] The paper-based assay also exhibited lower LOD than LOD determined by the tube-based LAMP reaction (Ipg), thereby making it more sensitive than the tube -based LAMP assay. The amplification rate was also confirmed by agarose gel electrophoresis of LAMP amplicons, which showed that the intensity of bands was correlated with the initial target concentration (Figure 15A, lower panel).

[0103] Fungal Quiescent Marker Genes

[0104] Table 1- Table 5 demonstrates quiescent marker genes that are upregulated specifically during a quiescent stage in the different fungi and may be used in the proposed application.

[0105] Table 1 discloses the quiescence genes of Colletotrichum gloeosporioides.

[0106] Table 1 Table 1 (cont.)

[0107] Table 1 (cont.)

[0108] Table 1 (cont.) Table 1 (cont.) Table 1 (cont.)

[0109] Table 2 discloses the quiescence genes of Penicillium expansum.

[0110] Table 2 Table 2 (cont.)

[0111] Table 3 discloses the quiescence genes of Alternaria alternata.

[0112] Table 3 Table 4 discloses the quiescence genes of Botrytis cinerea.

[0113] Table 4

[0114] Table 5 discloses the quiescence genes of Botrytis cinerea.

[0115] Table 5

[0116] EXAMPLE 2

[0117] Optimization of RT-LAMP recation

[0118] Comparative analysis of RT-LAMP reaction efficiency using different heat sources was tested. Performance comparison of RT-LAMP reactions was conducted at 65°C using a wet bath, oven, and dry bath (heat chamber) for 30 minutes, 1 hour 15 minutes, and 45 minutes, respectively. Results are presented in Figure 16. A statistically significant difference between treatments is denoted by different letters, P value < 0.05, by one-way ANOVA.

[0119] Comparison of Stabilized (i.e., heat-dried) vs. Fresh RT-LAMP assay performance was performed.. A 50pg sample of the latent Polysaccharide Lyase family Botrytis cinerea marker was tested. The assay compared the performance of a 43 °C heat-dried RT-LAMP mixture against a fresh RT-LAMP mixture. The mixtures were incubated in multiwell plates at 65°C for 100 minutes for the heat-dried and 45 minutes for the fresh mixture. Results are presented in Figure 17. A statistically significant difference between treatments is denoted by different letters, P value < 0.05, by one-way ANOVA.

[0120] Optimization of trehalose concentrations in heat-dry RT-LAMP reaction assay was conducted and its efficiency to detect latent Botrytis cinerea was tested. Three different amplification times were tested. A) 1 hour 10 minutes, B) 1 hour 20 minutes, C) and 1 hour 40 minutes. Results are presented in Figure 18A-C. A statistically significant difference between treatments is denoted by different letters, P value < 0.05, by one-way ANOVA.

[0121] The sensitivity of the 43°C heat-dried mixture in an optimized RT-LAMP Assay was assessed. This assay evaluated a range of synthesized DNA target concentrations, from 0.0 Ipg to 50pg. A negative control without any DNA target was also incorporated into the assay. Results are presented in Figure 19.

[0122] EXAMPLE 3

[0123] Specificity of the RT-LAMP assay

[0124] The specificity of the RT-LAMP of the present invention was tested by evaluating the detection of specific targets: Botrytis cinerea Polysaccharide Lyase family (50pg) and Alternaria alternata (50pg), alongside a negative control. The results of RT-LAMP reactions following heat drying at 43°C were analyzed by agarose gel electrophoresis, featuring positive targets for Botrytis cinerea in wells 2-4, no amplification for Alternaria alternata in wells 5-7, and negative control in wells 8-10. Results are presented in Figure 20A and 20B, respectively. Detection of latent Botrytis cinerea in infected unripe tomato fruit samples was tested using heatdry RT-LAMP. Ing / pl of total RNA was used for both latent Alternaria alternata and Botrytis cinerea inoculating tomato fruit. The negative control contained no RNA or synthetic DNA of the non-target gene. Results are presented in Figure 21.

[0125] Long-term stability of the dried LAMP mixture was twstwd in a 384-well plate under different storage temperatures: 22°C (Figure 22A), 4°C (Figure 22B), -22°C (Figure 22C), and -80°C (Figure 22D). Figure 22 illustrates the impact of temperatures on the performance and stability of the mixtures over time, highlighting the optimal conditions for preserving the functionality of the LAMP reagents.

[0126] EXAMPLE 4

[0127] Correlation between decay rates and pathogenic fungi concentration as measured by RT-LAMP

[0128] Correlation between the decay rates in tomatoes (Figure 23A) and the responses of the LAMP- based setup to Botrytis cinerea conidia concentration (Figure 23B) was analyzed and analysis is presented in Figure 23.

[0129] Figure 23 illustrates that as the conidia concentration of pathogenic fungi as Botrytis cinerea (103, 104, 105, 106 CFU / mL) increases, the decay processes in tomatoes accelerate, and the LAMP setup shows a stronger O.D. response to the presence of the pathogens in the crops, indicating its effectiveness in detecting possible deterioration rates.

Claims

CLAIMS1. A method for colorimetric detection of quiescent pathogenic fungi in harvested fruit, comprising steps of: a. extracting RNA from cuticle layer of harvested fruit, b. carrying out a paper-based reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay on said RNA extract by;(i) adding RNA extracts of analyte, or RNA-free water for control, onto the surface of membrane substrate in wells of an assay module, for absorption of said RNA to said membrane substrate(ii) adding at least one LAMP assay primer set specific for fungal quiescence RNA sequence marker to said wells of assay module(iii) incubating said assay module in a pre-heated heating means c. detecting colorimetric change or flourocentic signal in medium of said assay wells by visual inspection, in that a change in medium color or a flourocentic signal indicates the amplification of quiescence sequence markers in analyzed RNA extracts d. calculating colorimetric change in medium of said assay wells by photographing said assay module and analyzing the difference between green to red (G / R) ratio in control assay medium and RNA-containing assay medium, using an image analyzer.

2. The method of claiml wherein said membrane substrate is selected from the polymeric porous membranes group consisting of: Polyethersulfone (PES), Polyvinilidene fluoride (PVDF), Polysulfone (PS) and Polyacrylonilitrile (PAN), and / or paper -based membranes group consisting of: Cellulose paper, Nitrocellulose paper, Glass fiber paper, Chromatogrphy paper and filter paper.

3. The method of claim 1 wherein said assay module is a 3D-printed assay module made of plastic, silicone, or any convenient material4. The method of claim 1 wherein said assay module incubation is at 63°C-68°C for 40-50 minutes5. A kit for colorimetric detection of quiescent fungi in harvested fruit, the kit comprising:(i) An assay module constructed with assay wells(ii) Cellulose membrane(iii) At least one LAMP assay primer set specific for fungal quiescence RNA sequence marker6. The kit of claim 5 wherein said assay module is a 3D-printed assay module made of plastic, silicone, or any convenient material7. The kit of claim 5 wherein said cellulose membrane is selected from the polymeric porous membranes group consisting of: Polyethersulfone (PES), Polyvinilidene fluoride (PVDF), Polysulfone (PS) and Polyacrylonilitrile (PAN), and / or paper-based membranes group consisting of: Cellulose paper, Nitrocellulose paper, Glass fiber paper, Chromatogrphy paper and filter paper.

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