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Article

Loop-Mediated Isothermal Amplification for the Rapid Detection of the Mutation of Carbendazim-Resistant Isolates in Didymella bryoniae

by
Lina Shen
,
Mengyu Huang
,
Anfei Fang
,
Yuheng Yang
,
Yang Yu
and
Chaowei Bi
*
College of Plant Protection, Southwest University, Chongqing 400715, China
*
Author to whom correspondence should be addressed.
Agronomy 2022, 12(9), 2057; https://doi.org/10.3390/agronomy12092057
Submission received: 22 July 2022 / Revised: 26 August 2022 / Accepted: 27 August 2022 / Published: 29 August 2022
(This article belongs to the Section Pest and Disease Management)
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Abstract

:
Gummy stem blight (GSB) caused by Didymella bryoniae (D. bryoniae) is a worldwide fungal soil-borne disease that can cause severe yield reduction of watermelon. To shorten the monitoring time of carbendazim-resistant strains of D. bryoniae in the field, in this study, we developed a loop-mediated isothermal amplification (LAMP) assay for rapid detection of carbendazim-resistant strains of D. bryoniae. The β-tubulin gene of carbendazim-resistant strains was selected as the target for primer design. Based on the color change of hydroxy naphthol blue (HNB) and gel electrophoresis, the optimal reaction conditions for LAMP were determined at 65 °C for 50 min. In specificity tests, the LAMP assay was able to distinguish between carbendazim-resistant and sensitive strains of D. bryoniae. Moreover, in sensitivity tests, the detection limit was 1 ng/μL D. bryoniae DNA of the carbendazim-resistant strain. In addition, the LAMP method was successfully applied to detect carbendazim-resistant strains in D. bryoniae-infested samples. Therefore, the developed LAMP assay provides a new method for the rapid detection of carbendazim-resistant strains of D. bryoniae.

1. Introduction

Watermelon (Citrullus lanatus (Thunb.) Matsum. et Nakai) production regions are mainly concentrated in Asia. The Americas, Africa, and Europe have comparable production, and Oceania has the least. China is the world’s number one watermelon producing country, with the world’s largest watermelon growing area and production. According to the Ministry of Agriculture, China’s watermelon cultivation area reached 1,890,800 hectares in 2016, and watermelon production reached more than 79.4 million tons. With the expansion of the watermelon cultivation area, the types of pests and diseases in the process of watermelon cultivation are increasing each year, which will seriously threaten the production of watermelons and bring economic losses to farmers. Gummy stem blight (GSB) is a worldwide soil-borne disease that can cause a pathogenic infestation in all reproductive stages of watermelons [1]. In common planting plots, the incidence of the plant is 15–25%, which can lead to a yield reduction of about 30%. For perennial continuous crop plots, the incidence rate can reach 80%, which can cause all the leaves and vines to die within a few days, bringing serious economic losses [2]. The sexual form of the GSB fungus is Didymella bryoniae (Auersw) Rehm and the asexual form is Phoma cucurbitacearum (Fr.) Sacc [3,4,5]. There are many methods of GSB control, such as selecting and breeding disease-resistant varieties, strengthening agricultural control, and using biological control. Chemical control has the advantages of good effects and quick results; thus, among the many control methods, the use of fungicides is still one of the methods used to control GSB. However, due to the long-term single use of fungicides, the development of resistance in plant pathogenic fungi has become an important problem [6]. Keinath showed that GSB in the New York and South Carolina areas has developed resistance to benomyl and thiophanate-methyl [7]. In addition, several reports have indicated that GSB has developed resistance to carbendazim, azoxystrobin, pyraclostrobin, pristine, and difenoconazole fungicides [8,9]. Fungicide resistance is mediated by various mechanisms [10,11,12]. Previous studies have shown that changes at codons 6th, 50th, 198th, and 240th in the β-tubulin gene could result in the development of carbendazim-resistant isolates in the field [13], and mutations in different codons may lead to different levels of resistance. D. bryoniae can produce high levels of resistance when the 198th codon mutates from GAG to GCG; this mutation has become dominant in the field [14].
The conventional methods for detecting resistant isolates are based on the use of different concentrations of fungicides to inhibit mycelial growth and spore germination [15]; however, these procedures are labor-intensive and time-consuming. With the development of molecular biology technology, there are now new methods for efficiently detecting fungicide-resistant isolates since the mechanisms are at the molecular level. For example, PCR, PCR-RFLP, and Allele-Specific Real-time PCR have been successfully used to detect mutations [16,17]. However, these molecular techniques have several disadvantages, such as expensive instruments and complicated processes. Here, a simpler assay was developed for the detection of D. bryoniae mutants resistant to carbendazim by loop-mediated isothermal amplification (LAMP).
Loop-mediated isothermal amplification (LAMP) is a rapid nucleic acid-based technique with high specificity, sensitivity, and convenience under isothermal conditions. The technology was invented by Notomi et al. in Japan in 2000 [18]. It designs four primers for six specific regions of the target gene and amplifies the target fragment in large quantities in a constant temperature water bath at 60–65 °C using strand-substituted DNA polymerase (Bst 2.0 DNA Polymerase). The results can be observed with the naked eye by adding dyes to the reaction, and the entire amplification process does not require the use of expensive testing instruments. The LAMP assay not only avoids the time loss caused by the need for denaturation and annealing for conventional PCR amplification, but it is also fast and easy without relying on a PCR instrument. Based on the above advantages, recently, LAMP has been widely applied in the detection of pathogens, such as Phialophora gregatef sp. sojae [19], Sclerotinia sclerotiorum [20], Erwinia amylovora [21], and the potato virus Y [22]. Additionally, LAMP has been applied in the detection of fungicide resistance in the field [23,24]. However, this method has not been applied to detect D. bryoniae mutants resistant to carbendazim. In this study, a LAMP system for detecting D. bryoniae mutants resistant to carbendazim was established, which can help guide the management of carbendazim-resistance and provide a vital reference for the control of D. bryoniae.

2. Materials and Methods

2.1. Fungal Isolates and Reagents

In this study, we used WL7 and RC11, which were highly resistant to carbendazim due to the 198th amino acid locus mutating from glutamic acid (Glu-E) to alanine (Ala-A) of β-tubulin. LZ2 and BB13 were carbendazim-sensitive wild type strains. These strains were identified prior to this study and stored at 4 °C.
Bst 2.0 DNA polymerase was procured from NEB; betaine and hydroxy naphthol blue (HNB) were procured from Solarbio, and the dNTPs and MgSO4 were procured from Takara.

2.2. DNA Extraction

These fungi were cultured on potato dextrose agar (PDA, 200 g peeled potatoes were grated, boiled, and filtered to create potato infusion, which was mixed with 20 g dextrose and 18 g agar to create 1 L of media) medium for 7 days at 25 °C.
The genomic DNA of D. bryoniae was isolated from mycelia grown on a PDA medium, using the cetyltrimethylammonium bromide (CTAB) method [25,26] and the extracted DNA was stored at −20 °C. Thereafter, the genomic DNA of D. bryoniae from tissue was extracted according to the manufacturer’s instructions in the DNA secure Plant Kit procured from TIANGEN.

2.3. LAMP Primer Design

On the basis of the sequence uniqueness, the β-tubulin gene of the carbendazim-resistant strain of D. bryoniae was selected as the target for the LAMP primer design (GenBank accession number KX032515, KX032516). Four LAMP primers (F3, B3, FIP, and BIP) were designed using the PrimerExplorer V5 software program (http://primerexplorer.jp/lampv5e/index.html) (accessed on 16 July 2018) and screened through a series of specificity and sensitivity tests. In order to increase the specificity of the primers, one or two artificial mismatch bases were introduced at the end of the inner primer. Sequences of the LAMP primers are shown in Table 1.

2.4. Specificity of LAMP Primer

To distinguish the mutant genotype from the wild type, the primers in Table 1 were used. The LAMP reaction was performed in a total volume of 10 μL, including: 1 μL of 10 × isothermal amplification buffer, 0.4 μL of Bst 2.0 DNA polymerase (0.16 U/μL), 0.8 μL of dNTP mix (10 mM), 0.4 μL of MgSO4 (25 mM), 0.8 μL of betaine (8M), 0.4 μL of HNB (2.5 mM), 0.4 μL of FIP/BIP (40 μM), 0.4 μL of F3/B3 (10 μM), and 0.5 μL of target DNA. The reaction mixture was incubated in a water bath at 65 °C and different incubation times (15–80 min) were evaluated to optimize the reaction conditions.
After the reaction, the LAMP amplification products were analyzed by a 3% agarose gel electrophoresis (1 × TAE) and the color change of the metal ion indicator (HNB). The positive products were indicated by a color change from violet to sky blue and showed a ladder-like pattern in 3% agarose gel, while the negative products remained violet and had no bands in the agarose gel.

2.5. Optimization of LAMP Reaction Time

To define the optimal reaction time, the LAMP reaction mixture was incubated at 65 °C for 15, 30, 45, 50, 60, 70, and 80 min.

2.6. Sensitivity of LAMP Assay

The sensitivity of the LAMP assay was assessed using a 10-fold gradient dilution of DNA (10 ng/μL to 1 pg/μL) extracted from the resistant strain.

2.7. Confirmation of the LAMP Assay

To further confirm the reliability of LAMP, on the one hand, PCR amplification was performed with partial sequences of β-micro-tubulin genes of strains RC11 and LZ2 (the primers used were GK-F1: 5′-TGGTGCTGGTAACAACTG-3′, GK-R1: 5′-GTCCTCGACCTCCTTCAT-3′). The sequences of genes and amino acids were compared with the NCBI database by sequencing. The PCR reaction was performed in a total volume of 50 μL, including: 5.0 μL of 10×Taq Buffer; 4.0 μL of dNTP Mixture (2.5 mmol/mL); 1.0 μL of Primer1 (10 μmol/mL); 1.0 μL of Primer2 (10 μmol/mL); 0.5 μL of DNA Polymerase (2.5 mmol/μL); and 3 μL of DNA Template (6 μg/mL); we added ddH2O to 50 μL. The PCR assay was performed with an initial denaturation at 95 °C for 3 min, followed by 30 cycles of denaturation (1 min at 95 °C), annealing (30 s at 55 °C), and extension (1 min at 72 °C), with a final extension for 10 min at 72 °C.
On the other hand, the isolates of RC11 and LZ2 were compared by the minimum fungicidal concentration (MFC) method. The strains were inoculated on the PDA medium amended with 100 μg/mL of carbendazim at 25 °C for 48 h. The resistant strains could grow normally, while sensitive strains could not.

2.8. Application of LAMP on Monitoring Carbendazim-Resistance of D. bryoniae in Diseased Watermelon Samples

To assess the feasibility of using LAMP to monitor the carbendazim-resistance of D. bryoniae in diseased watermelon samples, on the one hand, resistant and sensitive strains were artificially inoculated on watermelon leaves separately, and DNA of the diseased tissue was extracted as a template for LAMP. On the other hand, 25 watermelon disease samples infested by D. bryoniae were collected in Chongqing, and DNA was extracted from the diseased tissues as a template for LAMP.

3. Results

3.1. Selection and Specificity of LAMP Primer

In this study, artificially mismatched bases were added to the 3’ end of the FIP to increase the possibility of distinguishing carbendazim-resistant isolates from wild type. A total of five FIPs were designed, as shown in Table 1. To screen the specific primers, LAMP was performed using DNA extracted from WL7 (mutant strains) and BB13 (wild strains) as templates and ddH2O as the blank control. The different reaction combinations are shown in Table 2. When primers of R1 or R2 were used, the ladder-like pattern could be amplified on WL7 and BB13. When primers of R3 or R5 were used, the bands could not be amplified on both WL7 and BB13. This indicates that none of the above four primer combinations were specific. The ladder-shaped bands were only observed on WL7 but not on BB13 when using the primers in the R4 combination (Figure 1). Therefore, primers from R4 were used in the LAMP assay.
Based on the R4 primers, the mutant genotype (RC11) and wild type (LZ2) were used as templates for the LAMP assay. The results showed that (Figure 2), in the positive reaction, the color of the HNB changed from violet to sky blue, and ladder-like bands were observed in the agarose gel. However, in the negative reaction, the color of HNB remained violet and no bands could be observed in the agarose gel. The blank control with ddH2O as the template was the same as the negative reaction.

3.2. Optimization of LAMP Reaction Time

The reaction time was optimized to improve the efficiency of the LAMP detection. According to the reaction time, it can be seen from Figure 3 that within 30 min, the color of HNB did not change, and there was no typical ladder-like pattern in the agarose gel. When the reaction time was 45 min, the color of the HNB change was not obvious and the band was weak. When the reaction time reached 50 min, the reaction showed a distinct band and the color of HNB changed. Therefore, the optimal LAMP reaction time was 50 min.

3.3. The Sensitivity of LAMP Primer

To determine the sensitivity, LAMP was performed with 10-fold serial dilutions of DNA extracted from the resistant strain as a template. The results showed that (Figure 4) when the DNA concentration in the reaction system was 10 ng/μL or 1 ng/μL, a significant change in the color of HNB and a clear band on the agarose gel could be observed after the reaction. When the DNA concentration was below 1 ng/μL, neither of these changes could be produced. Therefore the detection limit of LAMP is 1 ng/μL of resistant strains DNA of D. bryoniae.

3.4. Confirmation of the LAMP Assay

On the one hand, partial β-tubulin genes of RC11 and LZ2 were amplified using common PCR. After comparison sequences of genes and amino acids through the NCBI database (Figure 5 and Figure 6), there was a difference between RC11 and LZ2. The point mutation A→C resulting in the 198th amino acid locus mutating from glutamic acid (Glu-E) to alanine (Ala-A). The sequence of RC11 was identical to the resistant strain in the database, and the sequence of LZ2 was identical to the sensitive strain in the database. This result indicates that the LAMP primers could accurately detect resistant strains.
On the other hand, isolates of RC11 and LZ2 were tested by the MFC method. As shown in Figure 7, the sensitive isolates could not grow normally on the PDA containing carbendazim, while the resistant isolates grew normally on the PDA plates containing carbendazim after 48 h. This result illustrates the reliability of LAMP detection.

3.5. Application of LAMP on Monitoring Carbendazim-Resistance of D. bryoniae in Diseased Watermelon Samples

DNA was extracted from artificially inoculated lesion tissues for the LAMP assay. After the reaction of tissues inoculated with resistant strains, the color of HNB changed from violet to sky blue, and there were bands in the agarose gel. However, after inoculating the tissue reaction with sensitive strains, the color of HNB was unchanged and there was no band in the agarose gel (Figure 8A,C). When the MFC method was used to differentiate between resistant and sensitive strains, the resistant strains could grow on PDA containing 100 μg/mL carbendazim, while the sensitive strains could not (Figure 8B).
In addition, DNA was extracted from 25 morbid tissues collected for the LAMP assay. A total of 13 samples had color changes of HNB from violet to sky blue and ladder-like bands in the agarose gel (Figure 9), suggesting that these samples were infected by resistant isolates. In summary, the above-established LAMP system could detect the carbendazim-resistant strains of D. bryoniae from the diseased tissues of watermelon.

4. Discussion

LAMP is a novel gene amplification method performed without the thermal cycler that enables the synthesis of larger amounts of DNA; the result can be visually assessed by the turbidity [27] or color change after adding an intercalating dye to the reaction solution [28,29]. There are various LAMP detection methods; the common ones are turbidity detection, fluorescence detection, metal indicator method, gel electrophoresis, etc. Since the turbidity detection method requires real-time quantitative testing, it is not suitable for grassroots field testing [30]. The fluorescent dye used in the fluorometric assay needs to be added with the lid open at the end of the reaction, which can easily cause contamination and produce false positive results [31]. Therefore, in this study, the metal indicator method was used, as well as the addition of the HNB indicator before the LAMP reaction could reduce the contamination during the reaction. At the end of the reaction, the HNB color changed from violet to sky blue, thus visualizing the results.
LAMP technology has been widely used in the field of plant–pathogen detection [32,33,34] because of its specificity, high sensitivity, simplicity, rapidity, and absence of expensive instruments. The use of LAMP for the detection of D. bryoniae has also been reported [35]. However, this method has not been applied to detect D. bryoniae mutants resistant to carbendazim. In this study, a LAMP system was established for the detection of carbendazim-resistant isolates by screening specific primers, optimizing reaction conditions, and detecting sensitivity. The optimal reaction conditions for LAMP were determined at 65 °C for 50 min, and the LAMP assay was able to distinguish between carbendazim-resistant and sensitive strains of D. bryoniae. In sensitivity tests, the detection limit was 1 ng/μL D. bryoniae DNA of a carbendazim-resistant strain, which was less sensitive compared to others [36]. This might be related to the reaction system and the amplified gene. Our next study will focus more on increasing the sensitivity of the LAMP reaction.
Using the traditional method of MFC to detect the carbendazim-resistant mutants of D. bryoniae requires about 4 days; however, the LAMP assay takes only about 3 h from DNA extraction to the result. The primer design is the most critical step for the LAMP assay. A LAMP assay requires at least four highly specific primers to distinguish six distinct regions on the target DNA. In this study, the LAMP primer set (external primers: RF3 and RB3, internal primers: RBIP and RFIP) was designed based on the β-tubulin gene of the D. bryoniae resistant strain to carbendazim. The LAMP reaction could be accelerated by adding the LooF or LooB primers [37]. In this study, we designed the LooF primer, but this primer was not added to the reaction because the addition failed to detect resistant strains. The LAMP results could be visually assessed by the color change of HNB. This would greatly decrease the detection time and it does not require expensive equipment. In summary, the LAMP assay is useful for the detection and identification of D. bryoniae mutants during field testing.

Author Contributions

Conceptualization, L.S., M.H., and C.B.; writing—original draft, M.H.; writing—review and editing, L.S.; methodology, A.F. and Y.Y. (Yuheng Yang); resources, Y.Y. (Yang Yu) and C.B.; supervision, Y.Y. (Yang Yu) and C.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Science Foundation of China (grant 31871990).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflict of interest.

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Agronomy 12 02057 g001 550
Figure 1. Specificity of LAMP primers CK: blank control; 1–5: the carbendazim-sensitive strain BB13; 6–10: the carbendazim-resistant strain WL7.
Figure 1. Specificity of LAMP primers CK: blank control; 1–5: the carbendazim-sensitive strain BB13; 6–10: the carbendazim-resistant strain WL7.
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Figure 2. The detection of the carbendazim-resistant mutant in D. bryoniae. (A): LAMP detection mutant using the visualization of HNB color change; (1) blank control; (2) the carbendazim-sensitive strain LZ2; (3) the carbendazim-resistant strain RC11; (B) the LAMP detection mutant using gel electrophoresis. M: DL2000 DNA marker: (1) blank control; (2) the carbendazim-sensitive strain LZ2; (3) the carbendazim-resistant strain RC11.
Figure 2. The detection of the carbendazim-resistant mutant in D. bryoniae. (A): LAMP detection mutant using the visualization of HNB color change; (1) blank control; (2) the carbendazim-sensitive strain LZ2; (3) the carbendazim-resistant strain RC11; (B) the LAMP detection mutant using gel electrophoresis. M: DL2000 DNA marker: (1) blank control; (2) the carbendazim-sensitive strain LZ2; (3) the carbendazim-resistant strain RC11.
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Figure 3. Optimization of the reaction time. (A) Visualization of the color change based on the reaction time; (1) 15 min; (2) 30 min; (3) 45 min; (4) 50 min; (5) 60 min; (6) 70 min; (7) 80 min. (B) Gel electrophoresis analysis based on the reaction time. M: DL2000 DNA marker; (1) 15 min; (2) 30 min; (3) 45 min; (4) 50 min; (5) 60 min; (6) 70 min; (7) 80 min.
Figure 3. Optimization of the reaction time. (A) Visualization of the color change based on the reaction time; (1) 15 min; (2) 30 min; (3) 45 min; (4) 50 min; (5) 60 min; (6) 70 min; (7) 80 min. (B) Gel electrophoresis analysis based on the reaction time. M: DL2000 DNA marker; (1) 15 min; (2) 30 min; (3) 45 min; (4) 50 min; (5) 60 min; (6) 70 min; (7) 80 min.
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Figure 4. Sensitivity test of LAMP. (A) Visualization of the color change based on the sensitivity test of LAMP; 1. 10 ng/μL; 1 ng/μL; 100 pg/μL; 10 pg/μL; 1 pg/μL. (B) Gel electrophoresis analysis based on the sensitivity test of LAMP. M: DL2000 DNA marker; 1. 10 ng/μL; 2. 1 ng/μL; 3. 100 pg/μL; 4. 10 pg/μL; 5. 1 pg/μL.
Figure 4. Sensitivity test of LAMP. (A) Visualization of the color change based on the sensitivity test of LAMP; 1. 10 ng/μL; 1 ng/μL; 100 pg/μL; 10 pg/μL; 1 pg/μL. (B) Gel electrophoresis analysis based on the sensitivity test of LAMP. M: DL2000 DNA marker; 1. 10 ng/μL; 2. 1 ng/μL; 3. 100 pg/μL; 4. 10 pg/μL; 5. 1 pg/μL.
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Figure 5. Partial comparison of β-tubulin gene in D.bryoniae.
Figure 5. Partial comparison of β-tubulin gene in D.bryoniae.
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Figure 6. Partial comparison of β-tubulin amino acids in D. bryoniae.
Figure 6. Partial comparison of β-tubulin amino acids in D. bryoniae.
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Figure 7. Growth of sensitive and resistant strains on PDA. (A) Growth of D. bryoniae on PDA amended with 100 μg/mL of carbendazim after 24 h. (B) Growth of D. bryoniae on PDA amended with 100 μg/mL of carbendazim after 48 h.
Figure 7. Growth of sensitive and resistant strains on PDA. (A) Growth of D. bryoniae on PDA amended with 100 μg/mL of carbendazim after 24 h. (B) Growth of D. bryoniae on PDA amended with 100 μg/mL of carbendazim after 48 h.
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Figure 8. LAMP detection of resistant mutant in D. bryoniae. (A) HNB visualization color change of detecting the resistant and sensitive types; 1–4: diseased tissues infested with sensitive strains; 5–8: diseased tissues infested with resistant strains. (B) Growth status of resistant and sensitive strains on PDA amended with 100 μg/mL of carbendazim. (C) Agarose gel electrophoresis of LAMP detecting the resistant and sensitive types; 1–4: diseased tissues infested with sensitive strains; 5–8: diseased tissues infested with resistant strains.
Figure 8. LAMP detection of resistant mutant in D. bryoniae. (A) HNB visualization color change of detecting the resistant and sensitive types; 1–4: diseased tissues infested with sensitive strains; 5–8: diseased tissues infested with resistant strains. (B) Growth status of resistant and sensitive strains on PDA amended with 100 μg/mL of carbendazim. (C) Agarose gel electrophoresis of LAMP detecting the resistant and sensitive types; 1–4: diseased tissues infested with sensitive strains; 5–8: diseased tissues infested with resistant strains.
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Figure 9. Application of LAMP for the detection of carbendazim-resistant strains of D. bryoniae in the field. (A,C) HNB visualization color change for detecting the resistant and sensitive strains in the field. (B,D) Agarose gel electrophoresis of LAMP detecting the resistant and sensitive strains in the field. Note: Lane M: DL2000 DNA marker; Lane 1–25: the diseased tissues collected from the field; Lane 26: watermelon healthy tissue.
Figure 9. Application of LAMP for the detection of carbendazim-resistant strains of D. bryoniae in the field. (A,C) HNB visualization color change for detecting the resistant and sensitive strains in the field. (B,D) Agarose gel electrophoresis of LAMP detecting the resistant and sensitive strains in the field. Note: Lane M: DL2000 DNA marker; Lane 1–25: the diseased tissues collected from the field; Lane 26: watermelon healthy tissue.
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Table
Table 1. LAMP primers used in this study.
Table 1. LAMP primers used in this study.
PrimerSequence
RF35′-TACAACGCCACCCTCTCC-3′
RB35′-TGAGCTGACCGGGGAAAC-3′
RBIP5′-ACAACCCCTCTTACGGTGACCT-GCAGGTGGTTACACCAGAC-3′
RFIP-15′-TGTCGTAGAGGGCCTCGTTGTC-TTGTCGAGAACTCTGACGC-3′
RFIP-25′-TGTCGTAGAGGGCCTCGTTGTC-TTGTCGAGAACTCTGACCC-3′
RFIP-35′-TGTCGTAGAGGGCCTCGTTGTC-TTGTCGAGAACTCTGAGGC-3′
RFIP-45′-TGTCGTAGAGGGCCTCGTTGTC-TTGTCGAGAACTCTGACGCC-3′
RFIP-55′-TGTCGTAGAGGGCCTCGTTGTC-TTGTCGAGAACTCTGACGCGT-3′
Note: The yellow colors are the mutated bases and the red colors are the artificially mismatched bases.
Table
Table 2. Different reaction combinations for LAMP assays.
Table 2. Different reaction combinations for LAMP assays.
Primer SetR1 R2 R3 R4 R5
PrimersRF3 + RB3 +
RBIP + RFIP-1		RF3 + RB3 +
RBIP + RFIP-2		RF3 + RB3 +
RBIP + RFIP-3		RF3 + RB3 +
RBIP + RFIP-4		RF3 + RB3 +
RBIP + RFIP-5
Template DNAS/RS/RS/RS/RS/R
Oder1/62/73/84/95/10
Note: S: wild type; R: mutant genotype.
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Shen, L.; Huang, M.; Fang, A.; Yang, Y.; Yu, Y.; Bi, C. Loop-Mediated Isothermal Amplification for the Rapid Detection of the Mutation of Carbendazim-Resistant Isolates in Didymella bryoniae. Agronomy 2022, 12, 2057. https://doi.org/10.3390/agronomy12092057

AMA Style

Shen L, Huang M, Fang A, Yang Y, Yu Y, Bi C. Loop-Mediated Isothermal Amplification for the Rapid Detection of the Mutation of Carbendazim-Resistant Isolates in Didymella bryoniae. Agronomy. 2022; 12(9):2057. https://doi.org/10.3390/agronomy12092057

Chicago/Turabian Style

Shen, Lina, Mengyu Huang, Anfei Fang, Yuheng Yang, Yang Yu, and Chaowei Bi. 2022. "Loop-Mediated Isothermal Amplification for the Rapid Detection of the Mutation of Carbendazim-Resistant Isolates in Didymella bryoniae" Agronomy 12, no. 9: 2057. https://doi.org/10.3390/agronomy12092057

APA Style

Shen, L., Huang, M., Fang, A., Yang, Y., Yu, Y., & Bi, C. (2022). Loop-Mediated Isothermal Amplification for the Rapid Detection of the Mutation of Carbendazim-Resistant Isolates in Didymella bryoniae. Agronomy, 12(9), 2057. https://doi.org/10.3390/agronomy12092057

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