Isothermal Amplification and Lateral-Flow Assay for Detecting Crown-Gall-Causing Agrobacterium spp.

Abstract

Agrobacterium is a genus of soilborne gram-negative bacteria. Members carrying oncogenic plasmids can cause crown gall disease, which has significant economic costs, especially for the orchard and nursery industries. Early and rapid detection of pathogenic Agrobacterium spp. is key to the management of crown gall disease. To this end, we designed oligonucleotide primers and probes to target virD2 for use in a molecular diagnostic tool that relies on isothermal amplification and lateral-flow-based detection. The oligonucleotide tools were tested in the assay and evaluated for detection limit and specificity in detecting alleles of virD2. One set of primers that successfully amplified virD2 when used with an isothermal recombinase was selected. Both tested probes had detection limits in picogram amounts of DNA. Probe 1 could detect all tested pathogenic isolates that represented most of the diversity of virD2. Finally, the coupling of lateral-flow detection to the use of these oligonucleotide primers in isothermal amplification helped to reduce the onerousness of the process, and alleviated reliance on specialized tools necessary for molecular diagnostics. The assay is an advancement for the rapid molecular detection of pathogenic Agrobacterium spp.

Review: a comprehensive summary of a decade development of the recombinase polymerase amplification

“…Shorter PCR primers (typically between 18 and 25 nucleotides) can also be used in the RPA reaction but may decrease the reaction speed and sensitivity. 42 Application of short PCR primers in RPA has been demonstrated by Mayboroda et al, 43 Martorell et al, 37 Wang et al 44 and Fuller et al 45 The latter two authors have shown that the PCR primers used in RPA resulted higher analytical sensitivity of detection compared to their usage in PCR: RPA detected 100 DNA copies of genetically modified GTS 40-3-2 soybean and 3.5 pg of genomic DNA of Agrobacterium spp. respectively, while the benchmark method PCR detected 1000 DNA copies and 350 pg of genomic DNA respectively.…”

“…respectively, while the benchmark method PCR detected 1000 DNA copies and 350 pg of genomic DNA respectively. 44,45 The company that sells the commercialised RPA reagents, TwistDx™ Ltd (see section 2.4 and Tables 2 and 3 for more details) provides additional probes that can be incorporated during the RPA reaction. The TwistAmp™ exo probe (typically between 46 and 52 nucleotides) and the TwistAmp® fpg probe (typically between 32 and 35 nucleotides) are used for fluorogenic real-time detection (Fig.…”

“…However, several research groups have studied RPA reaction temperatures that lie outside of the recommended range. 38,44,45,60,[62][63][64][65][66][67][68][69][70][71][72][73][74][75][76][77][78] The largest temperature range was tested between 15°C and 50°C; 62,64,69,70,76 and results indicated the marginal reaction temperature to produce a positive result should be greater than 30°C. [62][63][64]66,67,69,71,74,76,77 However, Sun et al 65 and Poulton and Webster 60 showed that temperature as low as 25°C could still generate a positive signal after RPA amplification and subsequent lateral flow strip detection.…”

“…1/100 dilution) before running on the strip to (1) improve its wicking performance 114 and (2) avoid "ghost band" effects. 45,54,[115][116][117] Notably, Powell and co-workers pointed out that the viscous wicking problem on the lateral flow strip can be mitigated by replacing the high molecular weight PEG (5.5% 35 kDa; see also section 2.1) with the low molecular weight PEG (6.5% 3 kDa) in the RPA reagent formulae. 114 Moreover, Powell and co-workers also developed methodology to alleviate the dilution step for lateral flow strip detection.…”

Mathematical modeling of a real‐time isothermal amplification assay for Erwinia amylovora

“…As part of ongoing research, we have evaluated the model of Equation for a real‐time RPA protocol, which uses dual‐labeled (fluorophore/quencher) oligonucleotide probes for generating a fluorescent signal. An RPA‐based assay for Agrobacterium spp . served as the basis for the real‐time protocol; Figure illustrates replicate measurements for a positive Agrobacterium control sample.…”