248 Abstracts I Journal of Microbiological Methods 30 (1997) 235-253 tion of several large fragments, e.g. primers reading out of repeats repMP1 (forward) and out of repMP5 (reversed) should yield fragments of 5.4, 5.6, 14.7, and 19.8 kb. The PCR products formed, or restriction patterns of these fragments should allow discrimination of M. pneumoniae strains.Initialy, 4 M. pneumo- niae strains (1 of type 1 and 3 of type 2) were used to evaluate the primer sets. Apparently due to preferential amplification of the smaller fragments (l-6 kb), the long PCR in all cases failed to produce the larger fragments (> 10 kb) expected. The repMPl- repMP5 PCR described above only yielded the 5.4 and 5.6 kb fragments, plus an unpredicted 1.5 kb fragment, but the expected 14.7 and 19.8 kb fragments were not observed. Despite this, the 4 M. pneumoniae strains could be discriminated based on the fragments formed with one of the 3 primer sets, even without restriction digestion. With a second primer set individual strains could be distinguished after restriction of the PCR fragments. This combination of primer sets and restriction endonucleases is presently being applied to a large series of M. pneumoniae isolates, in order to gain more insight into the epidemiology of this microorganism. The results of these studies will be presented. References 1. A. Cousin et al. IOM Letters 1994, 3: 494-495. 2. D. Ursi et al. J. Clin. Microbial. 1994, 2873-2875. 3. R. Himmelreich et al. Nucl. Acids Res. 1996, 24: 4420-4449. 41 Typing of Actinomycetes by DNA amplification techniques L. Gallego, F. Lopez-Otsoa, I. Pujana, J. Canduela and R. Cisterna Department of Inmunology, Microbiology and Parasitology, Uni- versity ?f the Basque Country, Apdo. 699-48080 Bilbao, Spain fntroduction: Diagnosis of infections caused by actimomycetes are sometimes hindered by several factors such as difficulties of isolation, or in the classification and identification based on conventional morphological, phisiological and biochemical prop- erties. Molecular techniques represent an alternative to the tradi- tional microbiological methods. Methods: A total of 10 isolates of Actinomycetul were analized by AP-PCR and ERIC-PCR. Chromosomal DNA was isolated by using two lytic solutions containing tween 20, triton X- 100, NP40 and lyticase and protinase K enzymes. DNA was someted to amplification using 100 pmol of the following primers AP3 (5’TCACGAATGCA3’) and ERIC2 (AAGTAAGTGACT- GGGGTGAGCG3’). The temperature cycles ramped as follows: 1 cycle of 5 min. at 94°C 5 min. at 35°C and 5 min. at 72°C 28 cycles of 1 min at 94”C, 2 min. at 35°C and 2 min. at 72”C, and a final extension step of 10 min. at 72°C. Results: Band profiles obtained with AP3 ranged from 300 to 3000 bp. and showed 5 different genotypes. Band profiles obtained with ERIC2 ranged from 200 to 2000 bp, and showed also, 5 genotypes. Differences between genotypes were due to the presence or abscence of 2-3 bands. Conclusion: PCR techniques may be a good alternative to conventional methods for typing Actinomycetales. 42 Realiability of AP-PCR and ERIC-PCR for typing Acinetobuc- ter baumannii isolates from different sources F. Lopez-Otsoa, L. Gallego, I. Pujana, J. Canduela and R. Cistema Depurtment of Inmunology, Microbiology and Parasitology, Uni- versity of the Basque Country, Apdo. 699-48080 Bilbao, Spain Introduction: Nosocomial outbreaks of infections involving strains of Acinerobrrcter buumannii are increasing. Traditional methods for identification of the strains involved have a low discriminatory power. The purpose of this study was to evaluate two PCR-based DNA fingerprinting techniques for typing Acinetobacter baumannii. Methods: A total of 34 isolates of Acinetobacter baumannii obtained from 4 differents hospitals of Spain were analized by RAPD and ERIC-PCR. Chromosomal DNA was isolated by using a modification of the CTAB methods. DNA was someted to amplification using 100 pmol of the following primers AP3- (S’TCACGAATGCA3’) and ERIC2 (AAGTAAGTGACT- GGGGTGAGCG3’). The temperature cycles ramped as follows: 1 cycle of 5 min. at 94°C 5 min. at 35°C and 5 min. at 72°C 28 cycles of 1 min at 94°C 2 min. at 35°C and 2 min. at 72°C and a final extension step of 10 min. at 72°C. Results: Band profiles obtained with AP3 ranged from 400 to 4000 bp. and showed 9 different genotypes. Band profiles obtained with ERIC2 ranged from 400 to 2500 bp, and showed 11 genotypes. The analysis of results obtained with both primers allowed us the discrimination of 12 genotypes. Conclusion: Although strains had similar profiles, differences between genotypes were due to the presence or abscence of 2-3 bands. The results obtained using the two sequence improved the level of discrimination, 43 An automated system (COBAS AMPLICORTM) for the routine diagnostic detection and quantitation of viral nucleic acids K. Gutekunst, T. Haley, K. Hasselbring, W. Iszczyszyn, K. Logan, D. Petersen, W. Schilling, S. Soviero and J.P. Spadoro Roche Molecular Systems, 1080 US Highway 202, Somerville, NJ 08876, USA Accurate quantitation of Hepatitis C Virus (HCV) RNA in serum or plasma has been shown to be a useful tool for monitoring patients on interferon therapy. This report describes an instrument system (COBAS AMPLICORTM) that performs all of the steps necessary for the PCR amplification and quantitative detection of HCV RNA that has been isolated from human serum or plasma. The general principle of the test is similar to the AMPLICOR HCV MONITORTM test that has been described previously. The amplification system amplifies all known geno- types of HCV with relatively equal efficiency. The S’UTR of HCV is amplified using a single primer pair and rTth DNA polymerase in a single-tube, single-enzyme reaction system. A quantitation standard (QS) is incorporated into each reaction during sample preparation in order to monitor both RNA recovery and amplifica- tion efficiency. The QS is used to determine the copy number of