Onsite detection of plant viruses using isothermal amplification assays

Abstract

Summary Plant diseases caused by viruses limit crop production and quality, resulting in significant losses. However, options for managing viruses are limited; for example, as systemic obligate parasites, they cannot be killed by chemicals. Sensitive, robust, affordable diagnostic assays are needed to detect the presence of viruses in plant materials such as seeds, vegetative parts, insect vectors, or alternative hosts and then prevent or limit their introduction into the field by destroying infected plant materials or controlling insect hosts. Diagnostics based on biological and physical properties are not very sensitive and are time‐consuming, but assays based on viral proteins and nucleic acids are more specific, sensitive, and rapid. However, most such assays require laboratories with sophisticated equipment and technical skills. By contrast, isothermal‐based assays such as loop‐mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) are simple, easy to perform, reliable, specific, and rapid and do not require specialized equipment or skills. Isothermal amplification assays can be performed using lateral flow devices, making them suitable for onsite detection or testing in the field. To overcome non‐specific amplification and cross‐contamination issues, isothermal amplification assays can be coupled with CRISPR/Cas technology. Indeed, the collateral activity associated with some CRISPR/Cas systems has been successfully harnessed for visual detection of plant viruses. Here, we briefly describe traditional methods for detecting viruses and then examine the various isothermal assays that are being harnessed to detect viruses.

Occurrence, Genetic Variability of Tomato Yellow Ring Orthotospovirus Population and the Development of Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Its Rapid Detection

“…Therefore, it can be carried out using lateral flow devices, making it suitable for onsite detection or testing in the field. The products of LAMP can be detected much faster than in standard techniques, sometimes only requiring analysis with the naked eye [ 87 ]. It has been reported that LAMP assay can be even 1000 times more sensitive than conventional PCR with equivalent specificity [ 88 ].…”

Advances in the differential molecular diagnosis of vesicular disease pathogens in swine

“…Loop-mediated isothermal amplification (LAMP) is an isothermal amplification approach developed in 2000, and has become the focus of point-of-care (POC) diagnostics due to its simple operation, rapid measurement and high sensitivity of this assay (Notomi et al, 2000). As shown in Figure 2 (Bhat et al, 2022), under the strand displacement activity of the Bst DNA polymerase from Bacillus stearothermophilus in 60-65 o C, the The labeled LAMP product is diluted and applied to the lateral flow strip. After 5 to 15 min of incubation, the appearance of bands on the test and control lines indicates the presence of the target nucleic acid (Bhat et al, 2022).…”

“…As shown in Figure 2 (Bhat et al, 2022), under the strand displacement activity of the Bst DNA polymerase from Bacillus stearothermophilus in 60-65 o C, the The labeled LAMP product is diluted and applied to the lateral flow strip. After 5 to 15 min of incubation, the appearance of bands on the test and control lines indicates the presence of the target nucleic acid (Bhat et al, 2022).…”

“…Then the stem-loop structure containing multiple initiation sites was used as the template of LAMP reaction for cyclic amplification, and many nucleotide chains with different lengths were generated finally. The results of LAMP assay can be visualized through the following ways (Nie, 2005;Almasi et al, 2014;Mansour et al, 2015;Peng et al, 2021;Bhat et al, 2022), ( 1 To date, several reverse transcription LAMP (RT-LAMP) assays have been developed that are capable of detecting all seven FMDV serotypes and serotype-specific detection of O, A, C and Asia 1 FMDVs (Lim et al, 2020). Timely diagnosis is essential for the control, monitoring and eradication of FMD.…”

“…The resulting amplicons are produced with FAM and biotin using a biotin-labeled reverse primer. The final RPA product is applied to the LFA strip, and the results are visualized after the appearance of matching lanes (Bhat et al, 2022(Bhat et al, ). 10.3389/fmicb.2022 Frontiers in Microbiology frontiersin.org simultaneous detection of 11 viral diarrhea pathogens (Shi et al, 2021).…”