Vibrio spp. detektatzeko CARD-FISH protokolo baten egokitzea

Abstract

[EUS] Giza jarduerek eragindako klima-aldaketa dela eta, ozeanoetako tenperatura igo egin da azken mendean, eta ondorioz, aldatu egin da mikroorganismo-populazioen aniztasuna eta banaketa. Esaterako, Vibrio generoko bakterioak ugaritzen ari dira, mundu-mailako bibriosiaren intzidentziaemendioa kausatuz. Bakterio Gram negatibo hauentzat diseinaturiko hazkuntza-medioak ez dira oso eraginkorrak izaten detekzioari dagokionean; horregatik, mikroorganismoen kultiboan oinarritzen ez diren zenbaketa- eta detekzio-teknikak garatu dira, besteak beste, in situ fluoreszentzia hibridazioa (FISH) eta honen hobekuntza den Catalyzed Reporter Deposition (CARD)-FISH. Teknika hauetan, mikroorganismoak fluoreszentziaz markatzen dira, itu-mikroorganismoen 16S rRNA-rekiko osagarria den zunda espezifiko baten bidez. Kasu honetan, GV 841 zunda erabili da, espezifikotasuna eta estaldura beste zunda batzuekin konparatu ostean. Zunda espezifikoa bilatzeaz gain, Pernthaler eta lankideen (2004) CARD-FISH protokoloa egokitu egin da Vibrio spp. detektatzeko; horretarako, lisozimaren kontzentrazioa igo da bakterioen horma iragazkorrago bihurtzeko, eta laginen berehalako mikroskopio-behaketa egin da. Emaitzek adierazi dute CARD-FISH, GV 841 zundarekin konbinatuta, baliozko metodoa dela V. harveyi-en detekziorako. Hala ere, metodoa aplikatu aurretik kontutan hartu behar da, alde batetik, metodoak zenbait muga dituela, eta bestetik, espezie ezberdinekin eta lagin naturalekin esperimentu-kontrolak egin behar direla.

[EN] Due to the climate change caused by human activity, ocean temperature has increased during the last century. Thus, the diversity and distribution of the microbe populations have changed. For example, bacteria of genus Vibrio are growing on number, thereby increasing the incidence of vibriosis worldwide. In terms of identification, the culture media designed for these gram-negative bacteria do not result very useful. Because of this, some culture-independent identification and detection techniques have been developed, such as the fluorescence in situ hybridization (FISH) and Catalyzed Reporter Deposition (CARD)-FISH. In these techniques, microorganisms are fluorescently stained by specific probes, complementary to the 16S rRNA of the target cells. In this case, the GV 841 probe has been used, after comparing its specificity and coverage with other probes. Also, increasing the lysozyme concentration used to enhance the permeability of the bacteria cell wall and carrying out immediate microscopic observation, Pernthaler et al. (2004) CARD-FISH protocol has been adjusted for the detection of Vibrio spp. The results have shown that the combination of the CARD-FISH technique with the GV 841 probe is a valid method for the detection of V. harveyi. This method, however, has some limitations to take into account before applying it and also, it is necessary to make experimental controls using different Vibrio species and environmental samples.