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Development of an automated sample preparation method for HCV
240
Abstracts I Journal
of Microbiological Methods 30 (1997) 235-253
Goessens, S. Deelen, N. Lemmens-den Toom, J.H. van RijsoortVos and H.A. Verbrugh Departments of Medical Microbiology and Infectious Diseases and Dermatology, Erasmus University Medical Center Rotterdam, Rotterdam, the Netherlands A prospective evaluation was done to determine the performance of the LCR (Lc,, Abbott Labs), PCR (COBAS AMPLICOR, Roche Diagnostic Systems) and TMA (AMPLIFIED CT, Genprobe Inc) for the detection of C.trachomatis (Ct) in urine. From 1,000 patients, first voided urine (FVU), urethral specimen and cervical specimen (if applicable) were obtained. Sensitivity and specificity were calculated using a new golden standard. A sample was considered to be true positive if two or more techniques were positive. Discrepant results were retested using an in-house PCR. The prevalence in males (n=544) and females (n=456) was 12.1% and 12.5%, respectively. The sensitivity and specificity of cell culture was 56 and 99%, respectively. The sensitivity of LC,, COBAS AMPLICOR and TMA was 840/c, 93% and 85%. respectively. A decreased sensitivity was observed when using female urines. The specificity exceeded 99% for all amplification techniques. Inhibition of the PCR reaction was monitored using the internal control that was incorporated in the COBAS AMPLICOR. 18 (4%) female patients and 8 (2%) male patients had results indicating inhibition of the PCR when using FVU. We conclude that urine may serve as an adequate alternative patient specimen for the detection of Chlamydia trachomatis when using amplification assays. The sensitivity of the amplification assays for the detection of Chlamydia trachomatis in urine strongly depends on the sex of the patient. The COBAS AMPLICOR seems to be least affected by this phenomenon. 17 Development of an automated sample preparation method for HCV J.R. Hildebrandt”, T. Picone”, E. Reitz”, Z. Li”, M. Fairchild”, K.C. Young”, .I. Akers”, N. DiDomenicob, R. Herronh and W. Rey’ “Roche Molecular Systems, 1145 Atlantic Avenue, Alameda, California 94501, USA ‘Roche Molecular Systems, I080 U.S. Highway 202, Somerville, New Jersey 08876, USA ‘Tegimenta AG, Roche Diagnostics Instrument Center, CH-6343 Rotkreuz, Switzerland Automation of PCR amplification and detection has already been successfully implemented on the COBAS AMPLICORTM. The next phase in this process is the automation of specimen preparation. An instrument has been developed to accomplish fully automated specimen processing. Using Hepatitis C Virus (HCV) as a model, a method was developed based on hybridization of viral target with oligonucleotide probe followed by magnetic particle capture of the hybridized probe-RNA complex. The processed specimen was shown to be compatible with the HCVTM and the AMPLICOR HCVTM, COBAS AMPLICOR AMPLICOR HCV MONITORTM Tests. Serum and plasma specimens processed using manual or automated methods were analyzed with the AMPLICOR HCV MONITOR Test and gave equivalent results with both sample processing methods over the
full IO’-1Oh dynamic range of the test. A significant increase in the stability of the processed RNA specimen was achieved with the automated method. In addition, the sensitivity of the qualitative AMPLICOR HCV Test has been improved approximately 50-fold to 30 copies/ml. Further improvements in the instrument will allow for improved throughput with less user intervention than manual methods. Complete automation of the PCR process, including sample preparation, amplification, and detection will insure further incorporation of PCR in the routine diagnostic laboratory. 18 Diagnosis and typing of plant viruses by immunocapture RT-PCR coupled to a fluorogenic exonuclease assay G. Nolasco”, Z. Sequeira”, M.T. Santosh, C. Sequeira”, O.A. Sequeirah, V.J. Febres’, B. Cevik’, R.F. Lee’ and C.L. Niblett’ ‘Univetxidade do Algarve, 8000 Faro, Portugal hEsta@o Agronomica National, 2780 Oeiras, Portugal ‘Univ. Florida, Gainesville, FL 32611, USA Genomic amplification by PCR methodology has proven to be a very sensitive tool for the detection of plant viruses. Despite that, it has not yet been adopted in large scale screenings. It is understood that PCR based methods require special skills and are time consuming compared with the widely used ELISA assay. We present a format of PCR based assay that completely overcomes the above stated drawbacks: nucleic acids extraction is completely avoided by an immunocapture step (ICIRT-PCR) performed by antibodies immobilised on the inner surface of PCR tubes (Nolasco et al., 1993) and the analysis of PCR products is performed by an homogeneous fluorogenic 5’ exonuclease assay (Holland et al., 1991), thus avoiding any post-PCR manipulation. This assay was developed taking as model two plant viruses: Grapevine Leafroll 3 Closterovims (GLR3V) and Citrus Tristeza Closterovirus (CTV). In both cases the assay proved to be more sensitive than ELISA, enabling the diagnosis at an earlier stage of crop development and allowing to analyse larger pools of composite samples. In addition, this assay has allowed the typing of CTV strains. This was achieved by including in the same reaction two strain discriminating fluorogenic probes labelled with different fluorochromes. These probes undergo a differential rate of cleavage according to the composition of the mixture of strains present in the plant. This ability is very important in certain citrus growing areas as it enables to selectively erradicate the most damaging isolates.Parts of this work were supported by the NATO SFS grant PO-Plant Virus. 19 chain reaction for the detection of A nested polymerase Borreliu burgdorferi sensu late based on a multiple sequence analysis of the hbb gene Claudio Valsangiacomo and Jean-Claude Piffaretti Istituto Cantonale Batteriosierologico, Via Ospedale 6, 6900 Lugano, Switzerland A highly sensitive nested Polymerase Chain Reaction (PCR) method was designed for the detection of a wide spectrum of strains from Borrelia burgdoqeri sensu late, the etiologic agent of
Abstracts I Journal
of Microbiological Methods 30 (1997) 235-253
Goessens, S. Deelen, N. Lemmens-den Toom, J.H. van RijsoortVos and H.A. Verbrugh Departments of Medical Microbiology and Infectious Diseases and Dermatology, Erasmus University Medical Center Rotterdam, Rotterdam, the Netherlands A prospective evaluation was done to determine the performance of the LCR (Lc,, Abbott Labs), PCR (COBAS AMPLICOR, Roche Diagnostic Systems) and TMA (AMPLIFIED CT, Genprobe Inc) for the detection of C.trachomatis (Ct) in urine. From 1,000 patients, first voided urine (FVU), urethral specimen and cervical specimen (if applicable) were obtained. Sensitivity and specificity were calculated using a new golden standard. A sample was considered to be true positive if two or more techniques were positive. Discrepant results were retested using an in-house PCR. The prevalence in males (n=544) and females (n=456) was 12.1% and 12.5%, respectively. The sensitivity and specificity of cell culture was 56 and 99%, respectively. The sensitivity of LC,, COBAS AMPLICOR and TMA was 840/c, 93% and 85%. respectively. A decreased sensitivity was observed when using female urines. The specificity exceeded 99% for all amplification techniques. Inhibition of the PCR reaction was monitored using the internal control that was incorporated in the COBAS AMPLICOR. 18 (4%) female patients and 8 (2%) male patients had results indicating inhibition of the PCR when using FVU. We conclude that urine may serve as an adequate alternative patient specimen for the detection of Chlamydia trachomatis when using amplification assays. The sensitivity of the amplification assays for the detection of Chlamydia trachomatis in urine strongly depends on the sex of the patient. The COBAS AMPLICOR seems to be least affected by this phenomenon. 17 Development of an automated sample preparation method for HCV J.R. Hildebrandt”, T. Picone”, E. Reitz”, Z. Li”, M. Fairchild”, K.C. Young”, .I. Akers”, N. DiDomenicob, R. Herronh and W. Rey’ “Roche Molecular Systems, 1145 Atlantic Avenue, Alameda, California 94501, USA ‘Roche Molecular Systems, I080 U.S. Highway 202, Somerville, New Jersey 08876, USA ‘Tegimenta AG, Roche Diagnostics Instrument Center, CH-6343 Rotkreuz, Switzerland Automation of PCR amplification and detection has already been successfully implemented on the COBAS AMPLICORTM. The next phase in this process is the automation of specimen preparation. An instrument has been developed to accomplish fully automated specimen processing. Using Hepatitis C Virus (HCV) as a model, a method was developed based on hybridization of viral target with oligonucleotide probe followed by magnetic particle capture of the hybridized probe-RNA complex. The processed specimen was shown to be compatible with the HCVTM and the AMPLICOR HCVTM, COBAS AMPLICOR AMPLICOR HCV MONITORTM Tests. Serum and plasma specimens processed using manual or automated methods were analyzed with the AMPLICOR HCV MONITOR Test and gave equivalent results with both sample processing methods over the
full IO’-1Oh dynamic range of the test. A significant increase in the stability of the processed RNA specimen was achieved with the automated method. In addition, the sensitivity of the qualitative AMPLICOR HCV Test has been improved approximately 50-fold to 30 copies/ml. Further improvements in the instrument will allow for improved throughput with less user intervention than manual methods. Complete automation of the PCR process, including sample preparation, amplification, and detection will insure further incorporation of PCR in the routine diagnostic laboratory. 18 Diagnosis and typing of plant viruses by immunocapture RT-PCR coupled to a fluorogenic exonuclease assay G. Nolasco”, Z. Sequeira”, M.T. Santosh, C. Sequeira”, O.A. Sequeirah, V.J. Febres’, B. Cevik’, R.F. Lee’ and C.L. Niblett’ ‘Univetxidade do Algarve, 8000 Faro, Portugal hEsta@o Agronomica National, 2780 Oeiras, Portugal ‘Univ. Florida, Gainesville, FL 32611, USA Genomic amplification by PCR methodology has proven to be a very sensitive tool for the detection of plant viruses. Despite that, it has not yet been adopted in large scale screenings. It is understood that PCR based methods require special skills and are time consuming compared with the widely used ELISA assay. We present a format of PCR based assay that completely overcomes the above stated drawbacks: nucleic acids extraction is completely avoided by an immunocapture step (ICIRT-PCR) performed by antibodies immobilised on the inner surface of PCR tubes (Nolasco et al., 1993) and the analysis of PCR products is performed by an homogeneous fluorogenic 5’ exonuclease assay (Holland et al., 1991), thus avoiding any post-PCR manipulation. This assay was developed taking as model two plant viruses: Grapevine Leafroll 3 Closterovims (GLR3V) and Citrus Tristeza Closterovirus (CTV). In both cases the assay proved to be more sensitive than ELISA, enabling the diagnosis at an earlier stage of crop development and allowing to analyse larger pools of composite samples. In addition, this assay has allowed the typing of CTV strains. This was achieved by including in the same reaction two strain discriminating fluorogenic probes labelled with different fluorochromes. These probes undergo a differential rate of cleavage according to the composition of the mixture of strains present in the plant. This ability is very important in certain citrus growing areas as it enables to selectively erradicate the most damaging isolates.Parts of this work were supported by the NATO SFS grant PO-Plant Virus. 19 chain reaction for the detection of A nested polymerase Borreliu burgdorferi sensu late based on a multiple sequence analysis of the hbb gene Claudio Valsangiacomo and Jean-Claude Piffaretti Istituto Cantonale Batteriosierologico, Via Ospedale 6, 6900 Lugano, Switzerland A highly sensitive nested Polymerase Chain Reaction (PCR) method was designed for the detection of a wide spectrum of strains from Borrelia burgdoqeri sensu late, the etiologic agent of