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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

Main Article


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.

Main Article

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