The detection of Plasmodiophora brassicae using Loop-mediated isothermal DNA amplification

• Autorzy: Kaczmarek J. , Irzykowski W. , Burzynski A. , Jedryczka M.

Warianty tytułu

PL

Wykrywanie Plasmodiophora brassicae przy zastosowaniu amplifikacji DNA w warunkach izotermicznych z wykorzystaniem starterów zapętlających (LAMP)

• Języki publikacji: EN

Abstrakty

EN

Plasmodiophora brassicae, the cause of clubroot, is a very serious problem preventing from successful and profitable cultivation of oilseed rape in Poland. The pathogen was found in all main growing areas of oilseed rape; it also causes considerable problems in growing of vegetable brassicas. The aim of this work was to elaborate fast, cheap and reliable screening method to detect P. brassicae. To achieve this aim the Loopmediated isothermal DNA amplification (LAMP) technique has been elaborated. The set of three primer pairs was designed using LAMP software. The detection was performed with the GspSSD polymerase, isolated from bacteria Geobacillus sp., with strand displacement activity. DNA extraction from clubbed roots obtained from farmers’ fields of oilseed rape infected by P. brassicae was done using a modified CTAB method. The reaction was performed for 60 min at 62oC. The visual detection was done using CFX96 Real Time PCR Detection System (BioRad) or Gerie II Amplicatior (Optigen). The detection with LAMP proved its usefulness; it was easy, fast and accurate and independent of plant age. The detection limit was 5 spores per 1 μl of the spore suspension, so LAMP was less sensitive than quantitative PCR tests reported in the literature. However, the method is cheap and simple, so it is a good alternative, when it comes to practical use and the assessment of numerous samples.

PL

Plasmodiophora brassicae powoduje kiłę kapusty, chorobę obniżającą plonowanie i redukującą rentowność uprawy rzepaku. Patogen występuje we wszystkich głównych regionach jego uprawy, a także stanowi poważny problem dla producentów warzyw kapustowatych. Celem badań było opracowanie szybkiego, taniego i niezawodnego sposobu wykrywania P. brassicae. W tym celu opracowano metodę amplifikacji DNA w warunkach izotermicznych z wykorzystaniem starterów zapętlających (LAMP). Do detekcji wykorzystano polimerazę GspSSD, wyodrębnioną z bakterii Geobacillus sp., umożliwiającą zastępowanie nici DNA bez potrzeby zmiany profilu temperatury. Ekstrakcję DNA z wyrośli na korzeniach wykonano z zastosowaniem zmodyfikowanej metody CTAB. Reakcję prowadzono przez 60 minut w temperaturze 62oC. Wyniki zbierano przy zastosowaniu systemu CFX96 Real Time PCR Detection System (BioRad) lub Gerie II Amplificator (Optigene). Opracowana metoda była łatwa do wykonania, szybka, dokładna i niezależna od wieku badanej rośliny. LAMP był testem mniej czułym aniżeli opisane w literaturze metody ilościowego PCR; umożliwił wykrywanie 5000 zarodników w 1 mL zawiesiny. Jest to jednak metoda znacznie łatwiejsza do wykonania i zdecydowanie tańsza, co czyni ją przydatną do detekcji P. brassicae w przypadku konieczności oceny jego obecności w licznych próbach polowych.

• Wydawca: -
• Czasopismo: Acta Agrobotanica
• Rocznik: 2014
• Tom: 67
• Numer: 4
• Opis fizyczny: p.59-65,fig.,ref.

Twórcy

autor

Kaczmarek J.

• Institute of Plant Genetics, Polish Academy of Sciences, Strzeszynska34, 60-479 Poznan, Poland

autor

Irzykowski W.

• Institute of Plant Genetics, Polish Academy of Sciences, Strzeszynska34, 60-479 Poznan, Poland

autor

Burzynski A.

• Novazym Polska, Poznan Science and Technology Park, Rubiez 46, Poznan, Poland

autor

Jedryczka M.

• Institute of Plant Genetics, Polish Academy of Sciences, Strzeszynska34, 60-479 Poznan, Poland

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Identyfikatory

DOI

10.5586/aa.2014.058