| Área: Ecologia Microbiana ( Divisão I ) AFLP TECHNIQUE AS MOLECULAR TYPING OF STREPTOCOCCUS THERMOPHILUS STRAINS Elisa Sgarbi (UNIPR); Claudio Giorgio Bove (UNIPR); Camilla Lazzi (UNIPR); Monica Gatti (UNIPR); Juliano de Dea Lindner (UDESC); Marta Marzotto (UNIVR); Sandra Torriani (UNIVR); Erasmo Neviani (UNIPR)
ResumoStreptococcus thermophilus is a lactic acid bacteria (LAB) widely used in milk fermentation processes as a starter culture. Genotyping is a molecular tool widely used to study microbial diversity because it is able to fingerprinting specific DNA patterns that are characteristic for a single strain. In the present study we describe the optimization of an AFLP (Amplified Fragment Length Polymorphism) protocol in terms of primer combinations and experimental conditions to differentiate S. thermophilus strains at intraspecific level. Moreover the fingerprinting resolution of RAPD (Random Amplified Polymorphic DNA) and AFLP was evaluated and compared. 㰀倀 挀氀愀猀猀㴀䴀猀漀一漀爀洀愀氀 猀琀礀氀攀㴀∀䴀䄀刀䜀䤀一㨀 挀洀 挀洀 瀀琀㬀 吀䔀堀吀ⴀ䤀一䐀䔀一吀㨀 挀洀㬀 吀䔀堀吀ⴀ䄀䰀䤀䜀一㨀 樀甀猀琀椀昀礀∀㸀㰀匀倀䄀一 氀愀渀最㴀䔀一ⴀ唀匀 猀琀礀氀攀㴀∀䘀伀一吀ⴀ匀䤀娀䔀㨀 瀀琀㬀 洀猀漀ⴀ愀渀猀椀ⴀ氀愀渀最甀愀最攀㨀 䔀一ⴀ唀匀∀㸀匀攀瘀攀渀琀礀ⴀ昀漀甀爀㰀⼀匀倀䄀一㸀㰀匀倀䄀一 氀愀渀最㴀䔀一ⴀ唀匀 猀琀礀氀攀㴀∀䘀伀一吀ⴀ匀䤀娀䔀㨀 瀀琀㬀 洀猀漀ⴀ愀渀猀椀ⴀ氀愀渀最甀愀最攀㨀 䔀一ⴀ䜀䈀∀㸀 㰀⼀匀倀䄀一㸀㰀䤀㸀㰀匀倀䄀一 氀愀渀最㴀䔀一ⴀ䜀䈀 猀琀礀氀攀㴀∀䘀伀一吀ⴀ匀䤀娀䔀㨀 瀀琀㬀 洀猀漀ⴀ愀渀猀椀ⴀ氀愀渀最甀愀最攀㨀 䔀一ⴀ䜀䈀∀㸀匀⸀ 琀栀攀爀洀漀瀀栀椀氀甀猀㰀⼀匀倀䄀一㸀㰀⼀䤀㸀㰀匀倀䄀一 氀愀渀最㴀䔀一ⴀ䜀䈀 猀琀礀氀攀㴀∀䘀伀一吀ⴀ匀䤀娀䔀㨀 瀀琀㬀 洀猀漀ⴀ愀渀猀椀ⴀ氀愀渀最甀愀最攀㨀 䔀一ⴀ䜀䈀∀㸀 昀爀漀洀 搀椀昀昀攀爀攀渀琀 挀漀氀氀攀挀琀椀漀渀猀 愀渀搀 椀猀漀氀愀琀椀漀渀 猀漀甀爀挀攀猀Ⰰ ㈀ 㰀䤀 猀琀礀氀攀㴀∀洀猀漀ⴀ戀椀搀椀ⴀ昀漀渀琀ⴀ猀琀礀氀攀㨀 渀漀爀洀愀氀∀㸀䰀愀挀琀漀挀漀挀挀甀猀 最愀爀瘀椀攀愀攀㰀⼀䤀㸀Ⰰ 㰀䤀㸀䔀渀琀攀爀漀挀漀挀挀甀猀 昀愀攀挀愀氀椀猀Ⰰ 㰀⼀䤀㸀㰀匀倀䄀一 猀琀礀氀攀㴀∀洀猀漀ⴀ戀椀搀椀ⴀ昀漀渀琀ⴀ猀琀礀氀攀㨀 椀琀愀氀椀挀∀㸀 㰀䤀㸀䰀愀挀琀漀挀漀挀挀甀 氀愀挀琀椀猀㰀⼀䤀㸀 猀甀戀猀瀀⸀ 㰀䤀㸀挀爀攀洀漀爀椀猀㰀⼀䤀㸀 愀渀搀㰀⼀匀倀䄀一㸀 䰀䴀䜀 㘀㠀㤀㘀 愀猀 爀攀昀攀爀攀渀挀攀 猀琀爀愀椀渀 眀攀爀攀 甀猀攀搀 椀渀 琀栀椀猀 猀琀甀搀礀⸀ 㰀⼀匀倀䄀一㸀㰀匀倀䄀一 氀愀渀最㴀䔀一ⴀ䜀䈀 猀琀礀氀攀㴀∀䘀伀一吀ⴀ匀䤀娀䔀㨀 瀀琀㬀 洀猀漀ⴀ愀渀猀椀ⴀ氀愀渀最甀愀最攀㨀 䔀一ⴀ䜀䈀∀㸀倀爀椀洀攀爀 㰀⼀匀倀䄀一㸀㰀匀倀䄀一 氀愀渀最㴀䔀一ⴀ唀匀 猀琀礀氀攀㴀∀䘀伀一吀ⴀ匀䤀娀䔀㨀 瀀琀㬀 洀猀漀ⴀ愀渀猀椀ⴀ氀愀渀最甀愀最攀㨀 䔀一ⴀ唀匀∀㸀伀倀䤀ⴀ㈀䴀伀䐀㰀⼀匀倀䄀一㸀㰀匀倀䄀一 氀愀渀最㴀䔀一ⴀ唀匀 猀琀礀氀攀㴀∀䘀伀一吀ⴀ匀䤀娀䔀㨀 瀀琀㬀 洀猀漀ⴀ愀渀猀椀ⴀ氀愀渀最甀愀最攀㨀 䔀一ⴀ䜀䈀∀㸀 㰀⼀匀倀䄀一㸀㰀匀倀䄀一 氀愀渀最㴀䔀一ⴀ䜀䈀 猀琀礀氀攀㴀∀䘀伀一吀ⴀ匀䤀娀䔀㨀 瀀琀㬀 洀猀漀ⴀ愀渀猀椀ⴀ氀愀渀最甀愀最攀㨀 䔀一ⴀ䜀䈀∀㸀刀䄀倀䐀 眀愀猀 挀栀漀漀猀攀渀 昀漀爀 刀䄀倀䐀 挀栀愀爀愀挀琀攀爀椀稀愀琀椀漀渀㰀⼀匀倀䄀一㸀㰀匀倀䄀一 氀愀渀最㴀䔀一ⴀ唀匀 猀琀礀氀攀㴀∀䘀伀一吀ⴀ匀䤀娀䔀㨀 瀀琀㬀 洀猀漀ⴀ愀渀猀椀ⴀ氀愀渀最甀愀最攀㨀 䔀一ⴀ唀匀∀㸀⸀ 㰀⼀匀倀䄀一㸀㰀匀倀䄀一 氀愀渀最㴀䔀一ⴀ䜀䈀 猀琀礀氀攀㴀∀䘀伀一吀ⴀ匀䤀娀䔀㨀 瀀琀㬀 洀猀漀ⴀ愀渀猀椀ⴀ氀愀渀最甀愀最攀㨀 䔀一ⴀ䜀䈀∀㸀吀栀攀 䄀䘀䰀倀 瀀爀漀挀攀搀甀爀攀 眀愀猀 瀀攀爀昀漀爀洀攀搀 戀礀 洀攀愀渀猀 漀昀 琀栀攀 䄀䘀䰀倀 䴀椀挀爀漀戀椀愀氀 昀椀渀最攀爀瀀爀椀渀琀椀渀最 䬀椀琀㰀匀倀䄀一 猀琀礀氀攀㴀∀洀猀漀ⴀ猀瀀愀挀攀爀甀渀㨀 礀攀猀∀㸀☀渀戀猀瀀㬀 㰀⼀匀倀䄀一㸀⠀䄀瀀瀀氀椀攀搀 䈀椀漀猀礀猀琀攀洀ⴀ倀攀 䌀漀爀瀀漀爀愀琀椀漀渀Ⰰ 䌀愀氀椀昀漀爀渀椀愀 唀⸀匀⸀䄀⸀⤀ 眀椀琀栀 猀漀洀攀 洀漀搀椀昀椀挀愀琀椀漀渀猀⸀㰀漀㨀瀀㸀㰀⼀漀㨀瀀㸀㰀⼀匀倀䄀一㸀㰀⼀倀㸀
The RAPD analysis of the 78 strains studied showed 15 different clusters, for the majority constituted by 5-6 biotypes at most. The AFLP technique was optimized by using 200ng of template DNA and the preselective and selective primers combination EcoRIO/MseIO-EcoRIA/MseIA. The AFLP dendrogram was characterized by the presence of 46 clusters with 30 clusters formed by single strains that showed a unique pattern. The 4 strains belonging to other species were genetically differentiated from S. thermophilus strains only thanks to AFLP comparison. 㰀倀 挀氀愀猀猀㴀䴀猀漀一漀爀洀愀氀 猀琀礀氀攀㴀∀䴀䄀刀䜀䤀一㨀 挀洀 挀洀 瀀琀㬀 吀䔀堀吀ⴀ䤀一䐀䔀一吀㨀 挀洀㬀 吀䔀堀吀ⴀ䄀䰀䤀䜀一㨀 樀甀猀琀椀昀礀∀㸀㰀匀倀䄀一 氀愀渀最㴀䔀一ⴀ唀匀 猀琀礀氀攀㴀∀䘀伀一吀ⴀ匀䤀娀䔀㨀 瀀琀㬀 洀猀漀ⴀ愀渀猀椀ⴀ氀愀渀最甀愀最攀㨀 䔀一ⴀ唀匀∀㸀吀栀攀 漀瘀攀爀愀氀氀 搀愀琀愀 猀甀最最攀猀琀 琀栀愀琀 最攀渀漀琀礀瀀椀挀 挀栀愀爀愀挀琀攀爀椀稀愀琀椀漀渀 瀀攀爀昀漀爀洀攀搀 戀礀 䄀䘀䰀倀Ⰰ 瀀爀漀瘀椀搀攀 愀 戀攀琀琀攀爀 瘀椀攀眀 漀昀 洀椀挀爀漀戀椀愀氀 㰀匀倀䄀一 猀琀礀氀攀㴀∀洀猀漀ⴀ猀瀀愀挀攀爀甀渀㨀 礀攀猀∀㸀☀渀戀猀瀀㬀㰀⼀匀倀䄀一㸀搀椀瘀攀爀猀椀琀礀 漀昀㰀匀倀䄀一 猀琀礀氀攀㴀∀洀猀漀ⴀ猀瀀愀挀攀爀甀渀㨀 礀攀猀∀㸀☀渀戀猀瀀㬀 㰀⼀匀倀䄀一㸀㰀⼀匀倀䄀一㸀㰀䤀 猀琀礀氀攀㴀∀洀猀漀ⴀ戀椀搀椀ⴀ昀漀渀琀ⴀ猀琀礀氀攀㨀 渀漀爀洀愀氀∀㸀㰀匀倀䄀一 氀愀渀最㴀䔀一ⴀ䜀䈀 猀琀礀氀攀㴀∀䘀伀一吀ⴀ匀䤀娀䔀㨀 瀀琀㬀 洀猀漀ⴀ愀渀猀椀ⴀ氀愀渀最甀愀最攀㨀 䔀一ⴀ䜀䈀∀㸀匀⸀ 琀栀攀爀洀漀瀀栀椀氀甀猀㰀⼀匀倀䄀一㸀㰀⼀䤀㸀㰀匀倀䄀一 氀愀渀最㴀䔀一ⴀ䜀䈀 猀琀礀氀攀㴀∀䘀伀一吀ⴀ匀䤀娀䔀㨀 瀀琀㬀 洀猀漀ⴀ愀渀猀椀ⴀ氀愀渀最甀愀最攀㨀 䔀一ⴀ䜀䈀∀㸀 椀渀搀椀挀愀琀椀渀最 琀栀愀琀 刀䄀倀䐀 椀猀 氀攀猀猀 搀椀猀挀爀椀洀椀渀愀琀椀渀最 琀栀愀渀 䄀䘀䰀倀⸀ 㰀⼀匀倀䄀一㸀㰀匀倀䄀一 氀愀渀最㴀䔀一ⴀ䜀䈀 猀琀礀氀攀㴀∀䘀伀一吀ⴀ匀䤀娀䔀㨀 瀀琀㬀 洀猀漀ⴀ愀渀猀椀ⴀ氀愀渀最甀愀最攀㨀 䔀一ⴀ䜀䈀∀㸀䤀渀 琀栀椀猀 挀漀渀琀攀砀琀 琀栀攀 漀瀀琀椀洀椀稀愀琀椀漀渀 漀昀 愀渀 䄀䘀䰀倀 瀀爀漀琀漀挀漀氀 琀漀 搀椀昀昀攀爀攀渀琀椀愀琀攀 㰀䤀 猀琀礀氀攀㴀∀洀猀漀ⴀ戀椀搀椀ⴀ昀漀渀琀ⴀ猀琀礀氀攀㨀 渀漀爀洀愀氀∀㸀匀⸀ 琀栀攀爀洀漀瀀栀椀氀甀猀㰀⼀䤀㸀 愀琀 椀渀琀爀愀猀瀀攀挀椀昀椀挀 氀攀瘀攀氀 挀漀甀氀搀 戀攀 挀漀渀猀椀搀攀爀攀搀 㰀匀倀䄀一 猀琀礀氀攀㴀∀洀猀漀ⴀ猀瀀愀挀攀爀甀渀㨀 礀攀猀∀㸀☀渀戀猀瀀㬀㰀⼀匀倀䄀一㸀愀 渀漀瘀攀氀 愀瀀瀀氀椀挀愀琀椀漀渀 愀渀搀 猀栀漀甀氀搀 昀愀挀椀氀椀琀愀琀攀 昀甀爀琀栀攀爀 甀猀攀 漀昀 琀栀椀猀 琀攀挀栀渀椀焀甀攀 琀漀 猀琀甀搀礀 昀漀漀搀 戀愀挀琀攀爀椀愀 眀椀琀栀 琀攀挀栀渀漀氀漀最椀挀愀氀 瀀甀爀瀀漀猀攀⸀㰀⼀匀倀䄀一㸀㰀匀倀䄀一 氀愀渀最㴀䔀一ⴀ唀匀 猀琀礀氀攀㴀∀䘀伀一吀ⴀ匀䤀娀䔀㨀 瀀琀㬀 洀猀漀ⴀ愀渀猀椀ⴀ氀愀渀最甀愀最攀㨀 䔀一ⴀ唀匀∀㸀㰀漀㨀瀀㸀㰀⼀漀㨀瀀㸀㰀⼀匀倀䄀一㸀㰀⼀倀㸀㰀⼀昀漀渀琀㸀㰀⼀瀀㸀㰀戀爀㸀㰀戀㸀倀愀氀愀瘀爀愀猀ⴀ挀栀愀瘀攀㨀 㰀⼀戀㸀☀渀戀猀瀀㬀䄀䘀䰀倀Ⰰ 䰀愀挀琀椀挀 愀挀椀搀 戀愀挀琀攀爀椀愀Ⰰ 䴀漀氀攀挀甀氀愀爀 琀礀瀀椀渀最Ⰰ 刀䄀倀䐀Ⰰ 匀琀爀攀瀀琀漀挀漀挀挀甀猀 琀栀攀爀洀漀瀀栀椀氀甀猀㰀⼀琀搀㸀㰀⼀琀爀㸀㰀⼀琀愀戀氀攀㸀㰀⼀琀爀㸀㰀⼀琀搀㸀㰀⼀琀愀戀氀攀㸀㰀⼀戀漀搀礀㸀㰀⼀栀琀洀氀㸀
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