TY - JOUR
AU - Northrup, M, Allen
AB - Molecular testing for the diagnosis of bacterial or viral infections in raw clinical specimens requires complex, multistep procedures to release and isolate nucleic acid before PCR amplification (1)(2). Laboratory bench-top sample preparation procedures are very labor- and equipment-intensive, which reflects on total assay cost and increased susceptibility to sample and reagent carryover (3)(4). The need for automation has led to the development and introduction of robotics-based laboratory instruments with discrete operations that simulate the basic functions of a laboratory technician. These systems are typically developed for high-throughput applications and usually require intermittent operator involvement. In addition, because they directly emulate the operator’s manual functions for processing boluses of sample and reagents, the issue of sample or reagent carryover and carryin remains unresolved, and the complexity of the robotic mechanisms themselves contributes to high capital costs and poor reliability. Several factors currently contribute to and facilitate the development of the next generation of automated and integrated diagnostics instruments. One factor includes the recent advances in the microfluidics field of miniaturized integrated platforms and supporting technologies, which potentially enable the seamless execution of a sequence of protocols (5). Miniaturized processor-controlled microfluidic platforms contain chambers and valves, and encapsulate entities such as filter membranes or solid-phase particles. They are able to execute important processes such as cell capture, lysis, and fluid mixing, which currently require discrete laboratory instruments (6). Another factor is the development of a miniature, rapid PCR thermocycler device with real-time optical detection capable of multiplex analysis (7)(8)(9). Its modularity and adaptability allows for easy integration into an instrument platform downstream of a sample preparation module. It is possible to envision a one-step operation covering sample preparation and analysis, from raw sample to results reporting. Finally, the utilization of inexpensive materials, such as molded plastics, allows for the design of devices capable of simultaneously handling large sample volumes, up to many milliliters (required for clinical sensitivity), and microfluidic volumes, down to a few microliters (desirable for expensive analytical reagents) (8)(10). This report describes progress in the development of a fully integrated and automated instrument for which all sample preparation and PCR functions are integrated in a single platform. The platform is a plastic cartridge (Fig. 1A )mounted in an instrument (Fig. 1B ). The instrument contains a miniature, single-site thermally controlled fluorometer consisting of an assembly of two heater plates with embedded heaters and thermal sensors, a cooling fan, an optical subsystem with LEDs and detectors, and an analog printed circuit board. Heating and cooling rates of 7–8 °C and 3–4 °C, respectively, have been achieved for a 100-μL reaction volume (data not shown). The cartridge itself comprises three flat plastic acrylic parts sandwiched together with laser-cut thin [0.762-mm (0.030-inch)] silicone gasket layers that provide fluid sealing and surfaces for the valves and an ultrasonic horn. It contains chambers for the sample, wash buffer, lysis buffer, neutralization buffer, PCR mastermix, and waste. In addition, there is a chamber for holding filters for cell capture and lysis and an integrated 100-μL PCR reaction tube. Details of the filter stack assembly are shown in Fig. 1C . It consists of layers of 1.27-cm (0.5-inch) filters and glass beads: a 5 μm filter (LoProDyne; Pall Corp), a 1.2 μm filter (Versapor, Pall Corp), glass beads (≤106 μm; Sigma), and a 0.2 μm filter (Durapore; Millipore). The tip of an ultrasonic horn (Selfridge and Associates) is interfaced with the cartridge by pressing against the 0.762-mm (0.030-inch) silicone gasket membrane that forms one wall of the filter stack chamber. All of the cartridge chambers are interconnected by D-shaped microfluidic lines [diameter, 0.813 mm (0.032 inches)]. Fluid flow is driven pneumatically through one or more of six ports on top of the cartridge by a minipump in the instrument, and the flow rate is controlled by digital regulators (Omega). The direction of the fluid flow is controlled by nine membrane valves individually actuated by external solenoid valves pinching against the silicone membrane. A Visual Basic software program is used to control the system, and the interface is shown schematically in Fig. 1D . The area within the dashed red square in Fig. 1D represents the physical part of the cartridge, and the area outside of the square is the instrument. The specimen containing the bacterial cells is pumped from the sample chamber through the filter stack chamber and into waste. After cell capture and washing, the fluid is pressurized, and cells are lysed by sonication and DNA is released. Chamber pressurization is required to ensure adequate coupling between the horn and the silicone gasket membrane. In a study to optimize the fluidic protocol, type E Chlamydia trachomatis (elementary and reticulate bodies; Intracel Bartels) and gonococcal cells from an overnight culture were diluted into 5.5 mL of pooled normal urine to create a mock clinical specimen. The final dilution of the Chlamydia was 1:250 000, and the concentration of gonococcal cells was 20 000 colony forming units/mL of urine. To load the cartridge, 5.0 mL of the urine specimen was added to the “sample” chamber. In addition, 2.0 mL of wash solution (10 mmol/L Tris, 1 mmol/L EDTA, 0.2 g/L sodium azide, pH 8.3), 1.5 mL of lysis solution (Roche Amplicor Chlamydia Resuspension Diluent), 0.5 mL of neutralizer solution (Roche Amplicor Chlamydia Urine Diluent), and 100 μL of PCR mastermix was also added to other respective chambers. The cartridge was then placed in the instrument, and test protocols were automatically executed. To evaluate recovery, a PCR mastermix was developed for the multiplex detection of the Chlamydia and gonococcal DNA using TaqMan® probes. The multimix was composed of 200 nmol/L FAM/TAMRA probe and 200 nmol/L TET/TAMRA probe specific for gonococcal and Chlamydia DNA, respectively; 500 nmol/L primers; 5 U of Platinum® Taq (Life Technologies) per reaction; 200 μmol/L dNTPs; 50 mmol/L KCl; 8 mmol/L MgCl2; 0.5 g/L bovine serum albumin; and 1 mL/L Tween 20 in 10 mmol/L Tris, pH 8.3. The primer and probe sequences for Neisseria gonorrhoeae were designed using Primer ExpressTM software to amplify a portion (256 bp) of a 2-kb region of chromosomal DNA shown to be specific for N. gonorrhoeae with no cross-reactivity to N. meningitidis (11): Forward primer: 5′-TATTACGTTCAGGCGAAGCTGTATAACTTTG-3′ Reverse primer: 5′-TTAATTCCAACATACGGCGTGTTTTACCGCT-3′ Probe: 5′-FAM-TGGCGTACCTCAATTTCGTGAACGTGTGCT-TAMRA-3′ In addition, primer and probe sequences selected by Primer Express software were used to detect a fragment (311 bp) of the endogenous plasmid associated with C. trachomatis. The sequences were designed with melting temperatures suitable for multiplexing with the N. gonorrhea-specific sequences. Forward primer: 5′-TTGAGCGTATAAAGGGAAGGCTTGACAGTG-3′ Reverse primer: 5′-GTTGAGTAACCGCAAGATTTATCGCCATGT-3′ Probe: 5′-TET-TATATTCTCACAGTCAGAAATTGGAGTGCTGGCTCGTATA-TAMRA-3′ After the fluidic protocol was complete, samples were taken from the PCR tube, from the mastermix chamber, or the neutralization chamber, and amplification with real-time detection was performed in a Cepheid Smart Cycler® thermocycler. Cycling conditions were as follows: hold at 95 °C for 60 s; 45 cycles of 5 s at 95 °C and 30 s at 65 °C. Time to completion was typically <30 min. Panels E and F in Fig. 1 show the PCR results for neutralized lysate and a complete reaction mixture obtained from the mastermix chamber, respectively. To separately evaluate the efficiency of the ultrasonics-based lysis procedure, Chlamydia cells were also lysed in a PCR reaction tube, containing glass beads, pressed against an externally mounted ultrasonic horn. Recovery was measured using an FDA-approved kit (Roche Amplicor®Chlamydia Test kit). The efficiency of lysis was comparable to that obtained with the reference kit (Fig. 1G ). We have shown significant progress in the development of a cartridge-based instrument for automated sample preparation and subsequent DNA detection of bacteria. Once the reagents and samples are placed in the cartridge, the entire process of sample preparation from 5 mL of urine sample was completed within 2 min. Rapid lysis of bacteria and release of DNA was achieved by sonication in the presence of glass beads, using an ultrasonic horn separated from the liquid sample by a flexible membrane. To date, we have demonstrated feasibility for the individual sample preparation steps, a fluidic management scheme, a complete fluidic protocol, and a rapid PCR-based amplification. We currently are developing a fully integrated system with improved sample handling, more optimal fluidic protocol, and a more sensitive, four-color fluorometer module. Figure 1. Open in new tabDownload slide Description (A–D) and performance (E–G) of the prototype GeneXpert DNA diagnostic platform. (A), plastic sample preparation cartridge with integrated PCR tube; (B), instrument; (C), exploded view of cartridge filter stack assembly; (D), software fluidic interface. Area within the red dashed square in D represents the physical part of the cartridge; the area outside of the square is the instrument. (E), multiplex PCR results for a sample obtained from the neutralization chamber. (F), PCR results for a sample obtained from the mastermix chamber. (G), lysis efficiency of the ultrasonic-based method vs the reference method. Figure 1. Open in new tabDownload slide Description (A–D) and performance (E–G) of the prototype GeneXpert DNA diagnostic platform. (A), plastic sample preparation cartridge with integrated PCR tube; (B), instrument; (C), exploded view of cartridge filter stack assembly; (D), software fluidic interface. Area within the red dashed square in D represents the physical part of the cartridge; the area outside of the square is the instrument. (E), multiplex PCR results for a sample obtained from the neutralization chamber. (F), PCR results for a sample obtained from the mastermix chamber. (G), lysis efficiency of the ultrasonic-based method vs the reference method. References 1 Saiki RK, Scharf S, Faloona F, Mullis KB, Horn HA, Arnheim N. Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 1985 ; 230 : 1350 -1354. Crossref Search ADS PubMed 2 Vet JAM, Majithia AR, Marras SAE, Tyagi S, Dube S, Poiesz BJ, Kramer FR. Multiplex detection of four pathogenic retroviruses using molecular beacons. Proc Natl Acad Sci U S A 1999 ; 96 : 6394 -6399. Crossref Search ADS PubMed 3 Vahey MT, Wong MT, Michael NL. A standard PCR protocol: rapid isolation of DNA and PCR assay for globin. In: Dieffebach CW, Dveksler GS, eds. PCR primer: a laboratory manual. New York: Cold Spring Harbor Laboratory Press, 1995:17–22.. 4 McCormick RM. A solid-phase extraction procedure for DNA purification. Anal Biochem 1989 ; 181 : 66 -74. Crossref Search ADS PubMed 5 Wilding P, Kricka LJ, Cheng J, Hvichia G, Shoffner MA, Fortina P. Integrated cell isolation and polymerase chain reaction analysis using silicon microfilter chambers. Anal Biochem 1998 ; 257 : 95 -100. Crossref Search ADS PubMed 6 Waters LC, Jacobson SC, Kroutchinia H, Khandurina J, Foote RS, Ramsey JM. Microchip device for cell lysis, multiplex PCR amplification, and electrophoretic sizing. Anal Chem 1998 ; 70 : 158 -162. Crossref Search ADS PubMed 7 Wittwer CT, Fillmore GC, Garling DJ. Minimizing the time required for DNA amplification by efficient heat transfer to small samples. Anal Biochem 1990 ; 186 : 328 -331. Crossref Search ADS PubMed 8 Northrup MA, Christel LA, McMillan WA, Petersen K, Pourahmadi F, Western L, Young S. A new generation of PCR instruments and nucleic acid concentration systems. In: Innis MA, Gelfand DH, Sninsky JJ, eds. PCR application: protocols for functional genomics. San Diego: Academic Press, 1996:105–26.. 9 Ibrahim MS, Lofts RS, Jahrling PB, Henchal EA, Weedn VW, Northrup MA, Belgrader P. Real-time microchip PCR for detecting single-base differences in viral and human DNA. Anal Chem 1998 ; 70 : 2013 -2017. Crossref Search ADS PubMed 10 Ogura M, Agata Y, Watanabe K, McCormick RM, Hamaguchi Y, Aso Y, Mitsuhashi M. RNA chip: quality assessment of RNA by microchannel linear gel electrophoresis in injection-molded plastic chips. Clin Chem 1998 ; 44 : 2249 -2255. PubMed 11 Miyada CG, Born TL. A DNA sequence for the discrimination of Neisseria gonorrhoeae from other Neisseria species. Mol Cell Probes 1991 ; 5 : 327 -335. Crossref Search ADS PubMed © 2000 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 - Toward a Rapid, Integrated, and Fully Automated DNA Diagnostic Assay for Chlamydia trachomatis and Neisseria gonorrhoeae
JF - Clinical Chemistry
DO - 10.1093/clinchem/46.9.1511
DA - 2000-09-01
UR - https://www.deepdyve.com/lp/oxford-university-press/toward-a-rapid-integrated-and-fully-automated-dna-diagnostic-assay-for-IASr1VQt08
SP - 1511
VL - 46
IS - 9
DP - DeepDyve
ER -