Dolgotrajna stabilnost predpripravljenih reagentov omogoča poenostavitev izvedbe izotermne reakcije LAMP za zaznavanje bolezni rastlin na terenu

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Abstract (Slovenian)

Izotermna amplifikacija (LAMP) za testiranje rastlinskih bolezni na kraju samem je zaradi svoje praktičnosti vse bolj priznana. Čeprav je testiranje LAMP uporabniku prijazno in primerno za grobe materiale, priprava reagentov in kontrol brez kontaminacije ostaja izziv, zlasti v terenskih razmerah. Ta postopek tradicionalno zahteva dodatne korake in opremo. V projektu Q-Entry (V4-2023) smo želeli poenostaviti testiranje na terenu in zmanjšati tveganje kontaminacije z izvedbo celovite študije stabilnosti vnaprej pripravljenih oligonukleotidov in pozitivnih kontrolnih materialov za test LAMP, za zaznavanje bakterij Ralstonia spp. Ta študija temelji na našem predhodno razvitem standardiziranem protokolu (Lenarčič in sod., 2014; Pirc in sod., 2021). Vnaprej smo pripravili mešanico šestih oligonukleotidnih začetnikov in kontrolno DNK znanega izolata R. solanacearum ter jih razporedili v testne trakove. V obdobju dveh let smo te materiale testirali vsake tri mesece, pri čemer smo predhodno pripravljenim mešanicam dodali 2x Isothermal Master Mix (OptiGene) in reakcije izvajali pri konstantni temperaturi 60°C z instrumentom GenieII (OptiGene). Rezultate smo ovrednotili s primerjavo kvalitativnih rezultatov in časa do pozitivne reakcije v različnih časovnih intervalih z začetnimi ugotovitvami. Naši rezultati potrjujejo stabilnost predhodno pripravljenih reagentov in kontrolnega materiala za najmanj dve leti, če so shranjeni pri temperaturi ≤ -15 °C. Ta stabilnost omogoča njihovo predhodno pripravo, s čimer se učinkovito ločijo naloge, za katere sta koristna nadzorovano okolje in specializirana oprema (za delo z majhnimi volumni), od dejanskega izvajanja testov na terenu. Zato ta pristop poenostavi postopke, saj zmanjša število potrebnih korakov na kraju samem, s tem pa poveča praktičnost diagnostike rastlinskih bolezni na terenu.

Abstract (English)

The adoption of isothermal Loop-Mediated Isothermal Amplification (LAMP) for on-site testing of plant diseases is increasingly recognized for its practicality. Although LAMP testing is user-friendly and accommodates crude sample materials, preparing reagents and controls without contamination remains a challenge, especially in field conditions. This process traditionally requires additional steps and equipment, posing operational complexities. In the Q-Entry project (V4-2023), we aimed to simplify field testing and reduce contamination risks by conducting a comprehensive stability study of premixed oligonucleotides and positive control materials for the LAMP test targeting Ralstonia spp. This study builds upon our standardized protocol previously developed (Lenarčič et al., 2014; Pirc et al., 2021). We prepared a blend of six LAMP oligonucleotide primers and control DNA from a known R. solanacearum isolate in advance, arranging them into testing strips. Over a two-year period, we tested these materials every three months, adding 2x Isothermal Master Mix (OptiGene) to the premixed primers, and conducting reactions at a constant temperature of 60°C using a GenieII instrument (OptiGene). We evaluated the results by comparing the qualitative outcomes and the time to positivity at various intervals with the initial findings. Our results affirm the stability of both premixed reagents and control material for a minimum of two years when stored at temperatures ≤ -15 °C. This stability enables their advance preparation, effectively segregating tasks that benefit from a controlled environment and specialized equipment (for handling small volumes) from the actual test execution in the field. Consequently, this approach simplifies field operations by reducing the number of steps required on-site, thereby enhancing the efficiency and reliability of plant disease diagnostics.