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Volume 7, Number 1—February 2001


Rapid Identification of Corynebacterium diphtheriae Clonal Group Associated with Diphtheria Epidemic, Russian Federation

Svetlana Kombarova*, Chung Kim†, Viatcheslav Melnikov*, Michael Reeves†, Olja Borisova*, Izabella Mazurova*, and Tanja Popovic*†Comments to Author 
Author affiliations: *Gabrichevsky Institute of Epidemiology and Microbiology, Moscow, Russia; †Centers for Disease Control and Prevention, Atlanta, Georgia, USA

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Random amplified polymorphic DNA (RAPD) assay and ribotyping for 79 Russian Corynebacterium diphtheriae isolatesa,b

Ribotype Biotype No. of strains Primer 3 Primer 4
G1 G 38 G1/4 G1/4
M 2 G1/4 G1/4
G4 G 25 G1/4 G1/4
M 1 G1/4 G1/4
G 1 Newc G1/4
G4v G 1 G4v G4v
M1 M 5 M1/1v M1/1v
M1v M 5 M1/1v M1/1v
New M 1 New New

aG, biotype gravis; M, biotype mitis. Cultures were kept lyophilized at room temperature or were stored in defibrinated sheep blood and held at -70°C until needed. Before use, the strains were inoculated onto blood agar plates (trypticase soy agar with 5% sheep blood; Becton Dickinson, Cockeysville, MD) and were incubated at 37°C overnight.
bDNA for ribotyping was isolated by the universal isolation procedure (5). Hybridization of restricted DNA fragments was performed using a mixture of five digoxigenin-labeled oligonucleotide probes at 37°C for 4 hours as recently described by Regnault et al. (6). Posthybridization washes were also performed at 37°C in 2X SSC (1 X SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.1% sodium dodecyl sulfate (SDS) for 2x5 minutes and in 0.1X SSC, 0.1% SDS for 2x10 minutes. Detection was performed by using the DIG Wash and Block Buffer Set (Boehringer Mannheim Biochemicals, Indianapolis, IN), sheep anti-digoxigenin antibody conjugated with alkaline phosphatase, nitroblue tetrazolium chloride (NBT), and 5-bromo-4-chloro-3-indolylphosphate (BCIP).
cNew = pattern has not been previously observed.

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