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	===LAMP Amplification===		===LAMP Amplification===

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Latest revision as of 16:00, 11 October 2022

R.solani AG-1 ITS (ITS1 & 5.8SrRNA) sequence

ITS (ITS1 and 5.8 s rRNA) sequence is a conservative sequence in the genomic sequence of Rhizoctonia solani, the pathogen of rice sheath blight. PCR reaction and LAMP reaction were performed to detect R.solani. Through the evaluation and comparison of different methods in each step of detection, we highlighted the advantages of LAMP amplification and demonstrated the rationality of our LAMP-LFD system.

Video: LAMP-LFD detection (by 2022 SZU-China) File:K4286001-005-LAMP-LFDdetection.mp4

Sequence and Features

LAMP Amplification

DNA extraction of Rhizoctonia solani (Evaluation of common laboratory extraction methods

We compared different labortory methods including crude extraction, protocols of three easy methods, CTAB protocol, TPS protocol, which were used to extract DNA from R.solani in our experiments. Fungal universal primers ITS5, ITS4 and ITS6 and ITS4 were used to amplify ITS sequences of R.solani, respectively (Fig. 1A, 1B), and each amplification product was running in 3% agarose gel. Using each method, R.solani ITS sequences (650~750bp) were amplified, with bands in the expected position (Fig. 1). Protocols of three easy methods could be used to extract DNA rapidly. The corresponding bands of methods III and IV were bright. CTAB and TPS bands were brighter. However, crude extraction is not ideal compared with other methods. ITS sequences of R.solani are unable to be amplified in some samples (Fig. 1C).

Figure 1. ITS sequence of R.solani AG-3 DNA samples extracted by different laboratory extraction methods. A. DNA templates amplified by fungal universal primers ITS5 & ITS4. B. DNA templates amplified by fungal universal primers ITS6 & ITS4. C. DNA templates amplified by fungal universal primers ITS6 & ITS4. EMII: DNA extracted by easy method II. EMIII: DNA extracted by easy method III. EMIV: DNA extracted by easy method IV. CTAB1, CTAB2: DNA extracted by CTAB protocol. TPS1, TPS2, TPS3, TPS4, TPS5: DNA extracted by TPS protocol. CE1~CE5: DNA extracted by crude extraction. +CE control: a positive control group of DNA extracted by crude extraction.

ITS sequence of R.solani and related PCR system

We use the following universal primers and related PCR system for fungi to amplify ITS sequences of R.solani though PCR reaction (Table 1). In our experiment, primers pair ITS1 & ITS4 was more suitable for R.solani AG-1, primers pairs ITS5 & ITS4 and ITS6 & ITS4 were more suitable for R.solani AG-3 (Figure 3,4).

Table 1. Primers for R.solani ITS sequences in PCR reaction

LAMP system

LAMP Primers

We use the following primers to amplify ITS sequences of R.solani though LAMP reaction (Table 2).

Table 2. Primers for R.solani ITS sequences in LAMP reaction

Optimization of Mg(2+) concentration

The effect of LAMP amplification reaction was affected by Mg(2+) concentration. We set a Mg(2+) concentration gradient (5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM), selected R.solani AG-3 genome DNA as templates, and used primers (AG-3: RSAGF3, RSAGB3, RSAG-3FIP, RSAGBIP) for LAMP reaction. The amplification was carried out at 65℃ for 60 min. Each amplification product was running in 1.3% agarose gel (Fig. 2). The bands were brightest at a Mg(2+) concentration of 7 mM (Fig. 2B). DNA extracted by each method was successfully amplified to present a ladder-like band within this concentration of Mg(2+). Therefore, LAMP Reactions were performed with 7 mM Mg(2+) in subsequent experiments.

Figure 2. Optimization of Mg(2+) concentration. A. DNA LAMP amplification products with Mg(2+) concentration of 5mM (left) and 6mM (left). B. DNA LAMP amplification products with Mg(2+) concentration of 7mM and 8mM. C. DNA LAMP amplification products with Mg(2+) concentration of 9mM and 10mM. Use Rhizoctonia solani AG-3 genome DNA as template. II: DNA extracted by easy method II. III: DNA extracted by easy method III. IV: DNA extracted by easy method IV. CTAB1, CTAB2: DNA extracted by CTAB protocol. TPS1, TPS2: DNA extracted by TPS protocol. +Control: An amplification product that is able to run out of the correct band. -Control: sterilized water was used instead of the DNA template.

Detection limit and sensitivity of LAMP protocol

We used R.solani AG-3 and AG-1 DNA extracted by different methods as templates, each template was serially diluted in sterile water to amplify its ITS sequence. For PCR reaction, we used primers (AG-3: ITS6 and ITS4; AG-1:ITS1 and ITS4) as well as corresponding reaction temperatures. For LAMP reaction, We used primers (AG-3: RSAGF3, RSAGB3, RSAG-3FIP, RSAGBIP; AG-1:RSAGF3, RSAGB3, RSAG-1FIP, RSAGBIP) with Mg(2+) concentration of 7mM and amplified for 60 mins at 65 ℃.

Table 3. Dilution gradient of DNA samples extracted by different methods and the quality of DNA in the amplification reaction.

DNA extracted by easy method III, easy method IV, CTAB protocol and TPS protocol were amplified by ordinary PCR and then detected by electrophoresis (Fig. 3-6). The detection limits of them were 1.1619ng, 165.54pg, 676.67fg, and 4.8918fg, respectively (Fig. 3). Our PCR system had the highest sensitivity to DNA extracted by TPS protocol (Fig. 3D). After LAMP amplification, Ladder-like bands can be observed even in samples diluted to the lowest concentration (Fig. 5). In addition, the LAMP reaction was more sensitive than the PCR reaction.

Figure 3. Detection limit and sensitivity of PCR protocol. A. DNA extracted by easy method III. B. DNA extracted by easy method IV. C. DNA extracted by CTAB protocol. D. DNA extracted by TPS protocol. Use Rhizoctonia solani AG-3 genome DNA as the template. The number above the loading well indicates the order of magnitude of the corresponding DNA template content in nanograms. Figure 4. Detection limit and sensitivity of PCR protocol. Use DNA extracted by TPS protocol. Use Rhizoctonia solani AG-1 genome DNA as the template. The number above the loading well indicates the order of magnitude of the corresponding DNA template content in nanograms. Figure 5. Detection limit and sensitivity of LAMP protocol. A. DNA extracted by easy method and easy method IV. B. DNA extracted by CTAB protocol and TPS protocol. C. DNA extracted by the four methods, corresponds to the lowest dilution. Use Rhizoctonia solani AG-3 genome DNA as the template. The number above the loading well indicates the order of magnitude of the corresponding DNA template content in nanograms. Figure 6. Detection limit and sensitivity of LAMP protocol. DNA extracted by CTAB protocol and TPS protocol. Use Rhizoctonia solani AG-1 genome DNA as the template. The number above the loading well indicates the order of magnitude of the corresponding DNA template content in nanograms.

Implementation of LAMP in LFD for the detection of R. solani

DNA crude extraction of isolated rice leaves infected with R.solani

Before LAMP-LFD detection, we collected 10 groups of isolated experimental rice leaves infected with R.solani, extracted DNA from each group of leaves by crude extraction method (Fig. 7), weighed the leaves and measured the extracted DNA concentration (Table 3).

Figure 7. Isolated rice leaves infected with R.solani. The figure shows the five experimental groups with the lightest collected leaf mass among all the experimental groups. Table 4. Weight of collected leaves and DNA concentration of each experimental groups

LAMP-LFD test

We used crude extraction of DNA from isolated rice leaves infected with R.solani, amplified DNA templates with biotin-labeled primers(each amplification product was running in 1.3% agarose gel), incubated the amplification products with probes labeled with fluorescein amidite, then loaded final products onto LFD (Fig. 8). For the isolated diseased rice leaves extracted in this experiment, ladders-like bands were found in experimental group CE1 and CE2. No ladders-like bands were found in other experimental groups, which may show rice DNA and primer dimers, and no ladders-like bands were found in the control group (Trichoderma DNA extract) (Fig. 8A). However, on lateral flow devices (LFD), all the experimental group showed positive results, and even a control group showed a false positive result (Fig. 8B).

Figure 8. LAMP-LFD test. CE1~CE10: crude extraction of DNA from isolated rice leaves infected with R.solani. T.a.III: genome DNA of Trichoderma atroviride extracted by easy method III, which is a negative control group. T.a.IV: genome DNA of Trichoderma atroviride extracted by easy method IV, which is a negative control group.

It is speculated that cross-contamination of laboratory samples has led to false positives. This can be avoided if researchers bring equipment to the field for testing. If LAMP-LFD test is performed in the laboratory, researchers should pay attention to conducting the experiment in different zones in order to avoid contamination among DNA samples. Final results should be analyzed combined with the control group, gel imaging, and LFD imaging. For instance, for the DNA of isolated rice leaves extracted this time, both CE1 and CE2 experimental groups showed ladders-like bands, and the LFD showed positive results, indicating that the corresponding rice leaves had been infected with R.solani.

References

Jaimin S. Patel, Mary S. Brennan, Aftab Khan & Gul Shad Ali (2015) Implementation of loop-mediated isothermal amplification methods in lateral flow devices for the detection of Rhizoctonia solani, Canadian Journal of Plant Pathology, 37:1, 118-129, DOI: 10.1080/07060661.2014.996610