The Potential Use of Isothermal Amplification Assays for In-Field Diagnostics of Plant Pathogens

Abstract

Rapid, sensitive, and timely diagnostics are essential for protecting plants from pathogens. Commonly, PCR techniques are used in laboratories for highly sensitive detection of DNA/RNA from viral, viroid, bacterial, and fungal pathogens of plants. However, using PCR-based methods for in-field diagnostics is a challenge and sometimes nearly impossible. With the advent of isothermal amplification methods, which provide amplification of nucleic acids at a certain temperature and do not require thermocyclic equipment, going beyond the laboratory has become a reality for molecular diagnostics. The amplification stage ceases to be limited by time and instruments. Challenges to solve involve finding suitable approaches for rapid and user-friendly plant preparation and detection of amplicons after amplification. Here, we summarize approaches for in-field diagnostics of phytopathogens based on different types of isothermal amplification and discuss their advantages and disadvantages. In this review, we consider a combination of isothermal amplification methods with extraction and detection methods compatible with in-field phytodiagnostics. Molecular diagnostics in out-of-lab conditions are of particular importance for protecting against viral, bacterial, and fungal phytopathogens in order to quickly prevent and control the spread of disease. We believe that the development of rapid, sensitive, and equipment-free nucleic acid detection methods is the future of phytodiagnostics, and its benefits are already visible.

Loop-Mediated Isothermal Amplification for Detection of Plant Pathogens in Wheat (Triticum aestivum)

“…A drawback is the cost of some lab-on-a-chip devices, which require the use of unique and sophisticated equipment for their manufacture or signal interpretation. To date, most existing microfluidic systems are complex and expensive to integrate into a functional system ( Zhao et al, 2019 ; Ivanov et al, 2021 ).…”

“…However, the lower reaction temperatures (between 30°C and 55°C) make RPA more prone to non-specific primer binding compared to other isothermal amplification techniques, which can cause amplification of non-target templates ( Oliveira et al, 2021 ). Other limitations of RPA include amplification of only small DNA fragments of less than 1,500 bp with long primers (30–50 nt), which can yield non-specific amplification and a highly variable sensitivity ( Ivanov et al, 2021 ).…”

“…It uses a DNA oligonucleotide containing T7 promoter sequence at the 5′ end that anneals with target RNA and a T7 DNA polymerase. The reaction is conducted at 65°C, and the products are detected by quantification of fluorescently labeled probes ( Ivanov et al, 2021 ).…”

“…LAMP can be coupled with a Lateral Flow Dipstick (LFD) device, which can detect biotin-labeled amplicons upon hybridization to a fluorescein-labeled DNA probe complexed with a gold-labeled anti-fluorescein antibody ( Rigano et al, 2014 ; Ivanov et al, 2021 ). LAMP reaction is carried out for 30 min at 65°C, then a specific fluorescence-labeled probe is added to the reaction mixture and incubated for another 10 min.…”

“…The resulting complex moves by capillarity and is trapped by a biotin ligand at the test zone. The local gold concentration increases, and the positive reaction can be observed as a reddish-brown color line that develops on the test zone ( Ivanov et al, 2021 ). This has the potential to replace visualization methods that are not compatible with field applications, such as gel electrophoresis ( Rigano et al, 2014 ).…”