Differentiation ofLactobacillus Strains byRibotyping

AG-3'), theunderlined sequencebeing aSaclrestriction site, andSalI-T7-PC5 (5'-GGTCGACCGTTAATACGACTCAC TATAGGGATACCITGTTACGACT'T-3'), theunderlined sequencebeing a Sallrestriction siteandthesequencein boldface typebeing theT7promoter. TheDNA was amplified byusing anAutogene programmable cycling waterbath(Grant Instruments Ltd., Cambridge, United Kingdom), about 100ng oftotal DNA ina100-pl reaction mixture containing 10,ul of 1Oxpolymerase chain reaction buffer (500 mM KCI,100mM Tris, 15mM MgCl2, 40,ug ofbovine serumalbumin), 40pmol ofeachprimer, 200,uM(final concentration) eachdeoxynucleoside triphosphate, and1.5U ofTaqpolymerase (AmershamInternational, Amersham, United Kingdom). Amplification conditions consisted ofonecycle of5minat95°C; 30 cycles of1minat93°C, 1minat53°C, and3minat72°C; plus one additional cycle witha final 10-min chainelongation. Theamplification product was cleaned withGeneclean (Bio 101, LaJolla, Calif.). A probecontaining both16Sand23Sribosomal cDNA was synthesized byreversetranscription ofE.coli MRE600 16S+23SrRNA(Boehringer GmbH,Mannheim, Germany) withavianmyeloblastosis virus reversetranscriptase and randomoligonucleotide primers. Thetranscription reaction mixture consisted of1,ugofrRNA,2,ul ofrandomprimers [pd(N)6, 10mg/ml] (Pharmacia, Uppsala, Sweden), 2U of avianmyeloblastosis virus reversetranscriptase (Bethesda Research Laboratories Inc., Gaithersburg, Md.),6,ul of5x reversetranscriptase buffer, 1,ul ofRNaseinhibitor (2,500 U/ml;Bethesda Research Laboratories), 1plof100mM dithiothreitol (Sigma), 2plofeachdeoxynucleoside triphosphateat10mM (dATP,dGTP,anddTTP),and1plof [a-32P]dCTP (Amersham) ina total volumeof30plI. The mixturewas incubated for1hat37°C, andthereaction was stopped byaddition of1plof0.5M EDTA (pH8.0). The transcription products were confirmed byelectrophoresis in a3%(wtlvol) polyacrylamide gelfor2hat25V.ThecDNA bands were visualized byautoradiography withX-rayfilm (Cronex; Dupont[Australia Ltd.], Sydney, Australia) at -70°Cfor6h.