Background and Objectives: We report a new real-time reverse transcription polymerase chain reaction (RT-PCR) method for quantification of TEL-AML1 transcripts. The method is based on hybridization probe (HybProbe) chemistry applied in LightCycler equipment. The study group comprised 44 successive cases of pediatric acute lymphoblastic leukemia (P-ALL).

Design and Methods: The quantitative estimation of TEL-AML1 transcripts was performed in 10 P-ALL TEL-AML1-positive patients. The PCR was performed in capillary tubes in 10 microL final volumes using two sets of primers: M1, which detects the long (L-form) and short (S-form) transcripts, and M2, specific for the L-form. The fluorescently labeled HybProbes (TEL 3FL and TEL 5LC) hybridize to the TEL region. TEL-AML1 expression was normalized relative to the levels of AML1 transcripts. Standard curves were prepared using serial dilutions of plasmids with TEL-AML1 or AML1 inserts.

Results: The sensitivity attained allowed the detection of TEL-AML1 transcripts at a 10-4 dilution of a cDNA sample from a patient at diagnosis. The within-assay coefficient of variation (CV) for TEL-AML1 was 7.0% and the between-assay CV was 13%. Levels of TEL-AML1 transcript and the TEL-AML1/AML1 ratio decreased by more than four log units (p <0.001) during or after the course of initial treatment. Most of the patients who achieved complete remission after 5-6 months of initial treatment were TEL-AML1 negative, although some positive samples with negligible amounts of TEL-AML1 transcripts were still detected.

Interpretation and Conclusions: This method has the sensitivity and reliability required to monitor the presence of minimal residual disease, and could be a powerful tool in monitoring the efficacy of the response to chemotherapy.

Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria are a frequent cause of food-borne gastroenteritis, hemorrhagic colitis, and hemolytic uremic syndrome. Because antimicrobial agents are generally contraindicated in patients infected with STEC, a sensitive and specific diagnostic test with rapid turnaround is essential. Current culture methods may fail to detect non-O157 STEC. We evaluated a Stx gene real-time PCR assay using hybridization probes and the LightCycler instrument with 204 prospectively collected stool specimens, which were also tested for Stx by enzyme immunoassay (EIA) (ProSpecT STEC; Remel, Lenexa, KS) and by culturing on chromogenic agar (Chromagar O157; BD BBL, Sparks, MD). In addition, 85 archived stool specimens previously tested for Stx (by EIA) and/or E. coli O157:H7 (by culture) were tested by PCR. Sample preparation for PCR included mixing the stool in sterile water and extraction of nucleic acid using the MagNA Pure LC instrument (Roche Diagnostics). The PCR assay had 100% sensitivity and specificity compared to EIA and culture for specimens collected prospectively (4 of 204 specimens were positive) and compared to culture and/or EIA for archival specimens (42 of 85 specimens were positive). Both the EIA and PCR produced positive results from a specimen containing an O103 serotype STEC in the prospective specimens, and the PCR test detected three positive specimens that contained nonviable STEC in the archived specimens. The PCR assay demonstrated 100% sensitivity and specificity compared to EIA and/or culture and more rapid turnaround than either EIA or culture.

The objective of this study was to compare the MagNA Pure 96 Pathogen Universal protocol to the MagNA Pure LC DNA Isolation Kit III that is well established in our microbiological laboratory. For this we isolated nucleic acids from ten different sample materials: body fluids (Fluids, urine, sputum), feces, swabs, as well as whole blood, EDTA-, Citrate-Plasma and serum. This spectrum is typical for a microbiology laboratory and includes easy and difficult-to-process samples, as sputum and stool are. Into these sample types we spiked classified pathogen stocks from in-house cultures. Subsequently Real-time PCR analysis of fungal, bacterial and viral targets was done using the LightCycler^® 480 Instrument.The 200 μl protocol performed effi cient and sensitive nucleic acid extraction from up to 96 samples requiring less than 60 minutes runtime. It yielded superior or similar results compared to the respective MagNA Pure LC protocol. Higher input volumes, 500 or 1000 μl, available only for the MagNA Pure 96 System, generally increased sensitivity of detection. Direct use of some routine microbiological samples without dilution by pretreatment reagents, resulted in improved Crossing Point (CP) values. For normal sample types including whole blood this made pretreatment dispensable. A pretreatment step did however improve pathogen detection in the more diffi cult-to-process samples, such as sputum, swabs and stool. Our results demonstrate that both methods allow efficient and convenient isolation of high quality nucleic acid. Compared to the Viral NA Universal protocol, the Pathogen Universal protocol is further optimized for diverse sample materials, including difficult samples and for the detection of various pathogen types. The MagNA Pure 96 Instrument thus enables high throughput sample preparation applications in microbiology laboratories.

A rapid real-time multiplex PCR assay for detecting and differentiating Bordetella pertussis and Bordetella parapertussis in nasopharyngeal swabs was developed. This assay (LC-PCR-IS) targets the insertion sequences IS481 and IS1001 of B. pertussis and B. parapertussis, respectively, and is performed using the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.). The analytical sensitivity is less than one organism per reaction. Results for Bordetella culture and/or direct fluorescent antibody testing and a second LightCycler PCR assay (target, pertussis toxin gene) were compared to results of the LC-PCR-IS assay for 111 nasopharyngeal swabs submitted for pertussis testing. Of the specimens, 12 were positive (9 B. pertussis and 3 B. parapertussis) and 68 specimens were negative by all methods. Three other specimens were positive for B. pertussis by at least two of the methods (including the LC-PCR-IS assay), and another 28 specimens were positive for B. pertussis by the LC-PCR-IS assay only. No specimens were negative by the LC-PCR-IS assay and positive by the other methods. A conventional PCR method (target, IS481) was also compared to the LC-PCR-IS assay for a different group of nasopharyngeal swab specimens (n = 96): 44 specimens were positive and 41 specimens were negative for B. pertussis with both PCR methods. Nine specimens were positive for B. pertussis by the LC-PCR-IS assay and negative by the conventional PCR assay, and two specimens were positive for B. pertussis by the conventional PCR assay and negative by the LC-PCR-IS assay. Positivity of the two assays was not significantly different (P = 0.0654). The insertion sequence IS481 is also present in Bordetella holmesii; specimens containing B. holmesii may yield false-positive results. The LC-PCR-IS assay takes approximately 45 min to complete post-nucleic acid extraction, compared to 24 h for the conventional PCR assay previously used in our laboratory. The LC-PCR-IS assay is easier to perform than the conventional PCR assay, and the closed system decreases the chance of contamination. All of these characteristics represent a significant improvement in the detection of B. pertussis and B. parapertussis in nasopharyngeal specimens.