Comparative Evaluation of the LAMP Assay and PCR-Based Assays for the Rapid Detection of Alternaria solani

Abstract

Early blight (EB), caused by the pathogen Alternaria solani, is a major threat to global potato and tomato production. Early and accurate diagnosis of this disease is therefore important. In this study, we conducted a loop-mediated isothermal amplification (LAMP) assay, as well as conventional polymerase chain reaction (PCR), nested PCR, and quantitative real-time PCR (RT-qPCR) assays to determine which of these techniques was less time consuming, more sensitive, and more accurate. We based our assays on sequence-characterized amplified regions of the histidine kinase gene with an accession number (FJ424058). The LAMP assay provided more rapid and accurate results, amplifying the target pathogen in less than 60 min at 63°C, with 10-fold greater sensitivity than conventional PCR. Nested PCR was 100-fold more sensitive than the LAMP assay and 1000-fold more sensitive than conventional PCR. qPCR was the most sensitive among the assays evaluated, being 10-fold more sensitive than nested PCR for the least detectable genomic DNA concentration (100 fg). The LAMP assay was more sensitive than conventional PCR, but less sensitive than nested PCR and qPCR; however, it was simpler and faster than the other assays evaluated. Despite of the sensitivity, LAMP assay provided higher specificity than qPCR. The LAMP assay amplified A. solani artificially, allowing us to detect naturally infect young potato leaves, which produced early symptoms of EB. The LAMP assay also achieved positive amplification using diluted pure A. solani culture instead of genomic DNA. Hence, this technique has greater potential for developing quick and sensitive visual detection methods than do other conventional PCR strategies for detecting A. solani in infected plants and culture, permitting early prediction of disease and reducing the risk of epidemics.

Keywords: Alternaria solani; LAMP; early blight; histidine kinase gene; real-time PCR; sensitivity; specificity.

FIGURE 1

Sequence and location of the A. solani histidine kinase gene (HK1) used to design different polymerase chain reactions (PCRs) and loop-mediated isothermal amplification (LAMP) primers. Primer locations for different PCRs [Real-time PCR (qPCR F, qPCR R); Nested PCR (nPCR1 F, nPCR1 R, nPCR2 F, and nPCR2 R)], Duplex PCR (dpPCR F, dpPCR R), and LAMP assays [F3, B3, FIP (F1c-F2), BIP (B1c-B2)]. FIP is a hybrid primer consisting of the F1c and F2 sequences; BIP is a hybrid primer consisting of the B1c and B2 sequences. Primer sequence sites are colored and marked. Arrows indicate the extension direction.

FIGURE 2

Specificity of LAMP detection of A. solani. Assessment was based on (A) Calcein visualization of color change and (B) agarose gel electrophoresis analysis of the LAMP products. Lane 1: negative control; Lane 2–5: A. solani from different geographic areas; Lane 6: A. longipes; Lane 7: A. zinniae; Lane 8: A. porri; Lane 9: Colletotrichum gloeosporioides; Lane 10: Fusarium oxysporum; Lane 11: Pythium aphanidermatum; Lane M: DL2000 DNA markers. Similar results were observed in three repeat assessments.

FIGURE 3

Sensitivity of the LAMP assays. LAMP assay using 10-fold serial dilutions of purified target DNA from A. solani. (A) Detection of LAMP products by Calcein fluorescence dye. (B) Analysis of the LAMP products by agarose gel electrophoresis. Concentrations of template DNA were as follows: Lane 1: 1.36 × 10² ng μL^-1; Lane 2: 1.36 × 10¹ ng μL^-1; Lane 3: 1.36 ng μL^-1; Lane 4: 1.36 × 10^-1 ng μL^-1; Lane 5: 1.36 × 10^-2 ng μL^-1; Lane 6: 1.36 × 10^-3 ng μL^-1; Lane 7: 1.36 × 10^-4 ng μL^-1; Lane 8: negative control; Lane M: 2000-bp DNA marker. Similar results were observed in three repeat assessments.

FIGURE 4

LAMP assay tested for diluted Pure cultures. (A)Visual inspection of three different diluted pure cultures of A. solani was tested for LAMP assay without DNA extraction. (B) Analysis of the LAMP products by 2% agarose gel electrophoresis. Lane M: DL2000-bp DNA marker; Lane 1–3: different A. solani isolates from different geographic areas; Lane 4: positive control (A. solani genomic DNA); Lane 5: negative control. Similar results were observed in three repeat assessments.

FIGURE 5

Representative melting curves (A), amplification plot (B), and standard curve (C) of real-time PCR for detection of A. solani based on the histidine kinase gene (HK1). (A) Demonstrative melting curves using SYBR Green I for detection of A. solani. (B) A representative amplification plot for 10-fold serial dilution containing 100 ng to 1 pg of genomic DNA. (C) Standard curve derived from absolute quantification of 10-fold serially diluted DNA from a pure culture of A. solani.

FIGURE 6

LAMP detection of A. solani from infected potato field samples. (A)Visual inspection of LAMP assay using two different DNA extraction methods (rapid DNA extraction and CTAB DNA extraction) from potato plant tissues (leaf and stem). (B) Analysis of the LAMP products by 2% agarose gel electrophoresis. Lane 1: DNA extracted by rapid extraction from infected potato leaf; Lane 2: DNA extracted from infected stem by rapid DNA extraction; Lane 3: healthy plant tissue DNA extraction by rapid DNA extraction; Lane 4: DNA extracted from infected leaf by the CTAB method; Lane 5: DNA extracted from infected stem by the CTAB method; Lane 6: DNA extracted from healthy plant tissues by the CTAB method; Lane 7: positive control; Lane 8: negative control; Lane M: DL2000-bp DNA marker. Similar results were observed in three repeat assessments.