Advanced DNA-Based Point-of-Care Diagnostic Methods for Plant Diseases Detection

Abstract

Diagnostic technologies for the detection of plant pathogens with point-of-care capability and high multiplexing ability are an essential tool in the fight to reduce the large agricultural production losses caused by plant diseases. The main desirable characteristics for such diagnostic assays are high specificity, sensitivity, reproducibility, quickness, cost efficiency and high-throughput multiplex detection capability. This article describes and discusses various DNA-based point-of care diagnostic methods for applications in plant disease detection. Polymerase chain reaction (PCR) is the most common DNA amplification technology used for detecting various plant and animal pathogens. However, subsequent to PCR based assays, several types of nucleic acid amplification technologies have been developed to achieve higher sensitivity, rapid detection as well as suitable for field applications such as loop-mediated isothermal amplification, helicase-dependent amplification, rolling circle amplification, recombinase polymerase amplification, and molecular inversion probe. The principle behind these technologies has been thoroughly discussed in several review papers; herein we emphasize the application of these technologies to detect plant pathogens by outlining the advantages and disadvantages of each technology in detail.

Keywords: HDA; LAMP; RCA; RPA; isothermal amplification; point-of-care diagnostic.

FIGURE 1

Schematic outline of loop-mediated isothermal amplification (LAMP) (Tomita et al., 2008). (A) LAMP involves two sets of primers to target six distinct regions. (B) The inner primer containing sense and antisense sequences of the target DNA hybridizes to the targeted sequence and initiates DNA synthesis. The outer primer carries out the strand-displacement DNA synthesis and produces a single stranded DNA which works as a template for second inner and outer primers for DNA synthesis that hybridize to the other end of the target to form a DNA loop structure. (C) From the double stem-loop structure, the inner primer binds to the loop and synthesizes a new strand. The extension of the primer opens the loop at the 5′ end and again the outer primer strand displaces the newly form longer DNA to produce ssDNA to form a DNA loop. LAMP produces loop structure DNA in various sizes.

FIGURE 2

Schematic outline of helicase dependent amplification (HDA). (A) Helicase opens the dsDNA. (B) The primers anneal to the target sequences. (C) Primer extension by DNA polymerase. The newly formed dsDNAs are opened by helicase and the process starts again. (D) The newly formed dsDNAs are opened by helicase and the process starts again.

FIGURE 3

Schematic outline of rolling circle amplification (RCA). (A) A primer complementary to a region of a circular probe anneals to the circular template. (B) DNA polymerase initiates the DNA synthesis. (C) Strand displacement allows the continuation of DNA synthesis along the circular template. (D) DNA synthesis continues to generate a long ssDNA product.

FIGURE 4

Padlock probe assay with RCA. (A) Linear ssDNA probes contain two binding sites at both 3′ and 5′ ends to target the specific sequences. (B) Denaturation of dsDNA target sequence and hybridization of ssDNA probe toward the target region forming a loop. (C) After the ligation to form a circular probe, RCA primer binds to the primer target region and starts the RCA.

FIGURE 5

Schematic outline of the recombinase polymerase amplification (RPA). (A) Recombinase integrates with primers to form recombinase-primer complexes and target specific DNA sequences. (B) Strand exchange occurs and single stranded binding proteins (SSB) bind to the DNA to form a D-loop. (C) DNA polymerase initiates DNA amplification. (D) Displaced D-loop stabilized by SSB as amplification continues. (E) Two dsDNA molecules form and the entire cycle start again.

FIGURE 6

Schematic outline of MIP assay (Lau et al., 2014). MIP consists of two binding sites at 3′ and 5′ ends (B1 and B2) which are complementary to target sequences, and two universal primer sites (P1 and P2). B1 and B2 hybridize to specific sequences on the target with single stranded gap between two binding regions. (A) Hybridization: B1 and B2 bind to specific sequences on the target DNA creating single stranded gap between the binding domains of the MIP. (B) Gap filling: A DNA polymerase that lacks exonuclease and strand displacement activities synthesizes DNA from 3′ end of the MIP to 5′ end until the single stranded gap is filled. (C) Ligation: A DNA ligase ligates the 3′ end the 5′ end of the MIP creating a circular DNA. (D) Digestion: exonucleases I and III digest the linear MIPs and the DNA target in the reaction mixture leaving the circularized MIPs for amplification. (E) Polymerase chain reaction (PCR): A pair of universal primers (P1 and P2) amplifies the circularized MIP using the universal primer binding domains to generate PCR amplicons.