TY - JOUR
AU - McMillan,, William
AB - The detection of microbial DNA in clinical samples by nucleic acid amplification methods is often limited by the low concentration of organisms present in the specimen. Adequate amounts of target DNA are produced only through time-consuming or inefficient manual concentration and lysis methods, or expensive, dedicated equipment. There is a need for new sample-processing systems that are quick, reliable, and efficient and that avoid the use of toxic or hazardous chemicals and bulky, expensive equipment. Clinical, epidemiologic, and cost concerns dictate that these systems must maintain high assay sensitivity and a short time to results. It is also desirable that these systems have the capability to function in point-of-care or critical-care environments. The two fundamental areas for sample processing are the recovery of organisms from the clinical specimen in sufficient amounts and lysis of the organisms to free the nucleic acid for subsequent amplification and detection steps. Ideally, the recovery step would also be capable of concentrating the organism from large volumes and washing away sample inhibitors. The GeneXpert system, which is being developed by Cepheid, Inc., addresses the above sample-processing issues with a disposable plastic cartridge containing filtration and ultrasonic lysis functions. This system captures the organisms on the filter membrane, washes the sample to remove inhibitors, and ultrasonically ruptures the organisms to free nucleic acid. The nucleic acid is then able to pass through the filter membrane during the subsequent elution step. Sample processing can be combined in a complete instrument system for real-time fluorescent detection of PCR-amplified target organism DNA. We developed a stand-alone filtration and lysis module to study DNA recovery and PCR sensitivity. Dilute concentrations of Bacillus Calmette-Guérin (BCG; an avirulent strain of Mycobacterium bovis) were used as a model organism. BCG is an appropriate model organism because it is a surrogate for Mycobacterium tuberculosis (Mtb) and can be processed by procedures identical to those used for clinical samples. These procedures were designed to provide a severe test for the filtration/lysis module because the sample is typically very dilute, the digestants used to liquefy sputum are PCR-inhibitory, and the organism’s cellular wall is lipid-rich, rendering it difficult to effectively lyse. Initially ultrasonic lysis of BCG was evaluated with the Cepheid tube lysis system. In this device, an ultrasonic horn is coupled by a spring-loaded holder to a special 100-μL vessel half-filled with silica beads. Power to the horn is provided by an ultrasonic power generator with controllable amplitude. Output energy is recorded vs time by a digital-analog converter interface with the appropriate software. The output energy is an indicator of the physical coupling of the ultrasonic horn and lysis vessel and can be used to monitor and standardize the ultrasonic energy applied to a sample preparation. The Cepheid one-piece valve body combines filtration, wash, and lysis functions. It is a molded plastic housing containing an immobilized submicrometer filter, lysis beads (<106 μm in diameter) within a chamber that is in direct contact with the filter surface, and chamber inlet and outlet ports. The domed exterior opposite the ports functions as the ultrasonic interface. Fluid is forced through the filter by a syringe piston, and all filtrate is collected in secondary containers. Different fluids may be used in sequence, allowing first the sample and then buffers of various compositions to successively filter-concentrate and then wash the trapped material. The valve body is clamped in place so that contact is maintained with a spring-loaded ultrasonic horn. Amplitude and duration are independently controlled, and the horn output is recorded as for the tube lysis system. BCG Montreal was cultured to midlog phase in Middlebrook 7H9 medium containing 0.5 mL/L Tween 80, 0.2 mL/L glycerol, and 100 g/L oleic-albumin dextrose complex. The sample was briefly sonicated to break up clumps, and serial 10-fold dilutions were made. A 100-μL aliquot of each dilution was plated to determine colony forming units (CFU) per unit volume. A second 100-μL aliquot of each dilution was used for the tube lysis or valve body experiments. Separate cultures and dilutions were used for the two lysis protocols. The tube lysis experiments were performed as follows. Each 100-μL aliquot of BCG was pelleted by microcentrifugation, resuspended in an equal volume of water, and added to the 100-μL lysis vessel containing silica beads. The sample was lysed for 30 s at 80% amplitude. PCR was performed on the lysed material. Sample dilutions began at 145 CFU/100 μL. All valve body filtration and lysis experiments were performed with blinded aliquots of BCG culture dilutions in phosphate-buffered saline that were prepared using the standard clinical laboratory protocol for liquefying sputum samples. The aliquots were randomly tested, and the actual concentrations were not known until the experiments were complete. A 50-μL aliquot of each BCG culture dilution was added to 3 mL of phosphate-buffered saline with concentrations beginning at 12 170 CFU/mL. This was mixed 1:1 with 20 g/L NaOH, 14.5 g/L sodium citrate, and 5 g/L N-acetyl-L-cysteine. The contents were gently swirled and incubated for 15 min at room temperature. The NaOH, citrate, and N-acetyl-L-cysteine mixture is routinely used to liquefy and decontaminate sputum samples for culture of Mtb. This digestant mixture is then diluted severalfold with buffer to minimize further activity of the reagents. This was achieved by bringing the volume to 25 mL with 0.067 mol/L phosphate buffer, pH 6.8. The entire 25-mL volume was injected into a valve body using a programmable syringe drive. The filtered samples were washed with 5 mL of water and then lysed for ∼ 20 s. After the lysis step, the material was forced through the filter by air pressure, and PCR was performed on aliquots of the filtered lysate. Typically, 100 μL of filtered lysate was recovered. All real-time fluorescence (RTF)-PCR reactions were performed using Cepheid SmartCycler® instrumentation and reaction tubes. The Mtb-specific primers and 6-carboxyfluorescein-labeled Molecular Beacon probes are described elsewhere (El-Hajj H, Marras SA, Tyagi S, Kramer FR, Alland D. A multiplex multi-colored Molecular Beacon assay for the rapid identification of Mycobacterium tuberculosis and rifampin resistance in clinical sputum samples, manuscript in preparation). Thermocycling conditions were 2 min at 95 °C, followed by 50 cycles of 10 s at 95 °C, 15 s at 58 °C, and 10 s at 72 °C. Fluorescence was measured during the 58 °C steps. RTF-PCR results demonstrated the effectiveness of ultrasonic lysis for rupturing the BCG in the tube lysis system. When we used 10-fold serial dilutions of cultured cells, 14 CFU/100-μL aliquot were detected. This demonstrated that ultrasonic lysis with silica beads was effective on BCG cells. Serial 10-fold dilutions of BCG from 1480 to 0.015 CFU/mL were processed using the one-piece valve body and then analyzed by RTF-PCR. The results for several of the samples, tested in duplicate, are shown in Fig. 1 . Sample 1 is purified Mtb DNA (single result), sample 2 is 148 CFU/mL, sample 3 is 14.8 CFU/mL, and sample 6 is 0.015 CFU/mL. Samples with <14.8 CFU/mL were not detected in this series of experiments. Control experiments showed that ultrasonic lysis was required for DNA detection and that the NaOH solution was PCR-inhibitory. Figure 1. Open in new tabDownload slide RTF results for purified Mtb DNA (Sample 1), 148 CFU/mL BCG (Sample 2), 14.8 CFU/mL BCG (Sample 3), and 0.015 CFU/mL BCG (Sample 6). Figure 1. Open in new tabDownload slide RTF results for purified Mtb DNA (Sample 1), 148 CFU/mL BCG (Sample 2), 14.8 CFU/mL BCG (Sample 3), and 0.015 CFU/mL BCG (Sample 6). These data from experiments using prototype filtration/lysis devices demonstrate the sample processing capabilities of the valve body component of the GeneXpert cartridge. The system effectively lyses BCG by use of ultrasonic energy and concentrates dilute, large volume samples by filtration. An additional advantage of the system is the ability to remove inhibitors of PCR by washing the captured sample. Removal of inhibitors will allow the use of current sputum digestant reagents, which will be important for comparison studies with culture and acid-fast bacteria staining methods of Mtb detection. The valve body is also capable of handling small or large sample volumes. These qualities are important for the range of applications planned for this system. Release of detectable amounts of DNA without lysis was not observed. This implies that the system can concentrate and wash intact organisms, efficiently concentrating the target DNA after a subsequent lysis step. On the basis of the CFU calculations, a titer not detectable by PCR was concentrated to easily detectable concentrations. Future efforts will investigate areas important for analytical sensitivity and user requirements of rapid detection. Such areas will include filter surface area, pore size and material, valve body contained volume, fluid pumping rates, buffer composition, and ultrasonic lysis conditions, and other variables. Finally, currently developed RTF multiplex PCR assays for Mtb single-nucleotide polymorphisms important in rifampin drug resistance will be adapted to the GeneXpert system. This may provide the final, critical technologic capability for the rapid, simultaneous, sensitive detection of Mtb and the presence of antibiotic resistance. Technical assistance was provided by Molly Miranda, Rick Faeth, and Kristen Lloyd. © 2001 The American Association for Clinical Chemistry This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Rapid and Sensitive Detection of Mycobacterium DNA Using Cepheid SmartCycler® and Tube Lysis System
JF - Clinical Chemistry
DO - 10.1093/clinchem/47.10.1917
DA - 2001-10-01
UR - https://www.deepdyve.com/lp/oxford-university-press/rapid-and-sensitive-detection-of-mycobacterium-dna-using-cepheid-GyWUMx0iA3
SP - 1917
VL - 47
IS - 10
DP - DeepDyve
ER -