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Brief Communication |
Correspondence: 1Corresponding Author: Jason Sawyer, Technology Transfer Unit, Biotechnology Department, Veterinary Laboratories Agency – Weybridge, New Haw, Addlestone, Surrey, KT15 3NB United Kingdom, e-mail: j.sawyer{at}vla.defra.gsi.gov.uk
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Abstract |
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Key Words: Molecular diagnostics • PCR • PCR master mixes • quality control • routine PCR • RT-PCR
Real-time polymerase chain reaction (PCR) is seen as an important step forward in making PCR a routine testing tool because of the closed-tube format and lack of gel electrophoresis step.1 In an effort to make routine diagnostic PCR more user-friendly, the possibility of freezing complete real-time PCR and real-time reverse-transcription PCR (RT-PCR) reagents as single-use assays, which required only thawing and the addition of template nucleic before thermal cycling, was explored.
For real-time PCR, 2 hybridization probe4 assays and a 5' nuclease TaqMan assay2 were examined. For real-time RT-PCR, a 1-step 5' nuclease TaqMan assay was examined.
The first hybridization probe PCR assay detected mammalian mitochondrial DNA (mtDNA). A PCR master mix was prepared using the LC-FastStart DNA master Hybridization Probes Kita and aliquoted (in 18-µL volumes) into 0.5-mL microfuge tubes. Reactions were used immediately or frozen at –70°C. Concentrations in the final working PCR mixture were 4 mM MgCl2, 1 x reaction buffer, 0.5 µM forward and reverse primers, 0.2 µM probeb labelled at the 5' end with LightCyclera Red 640 and at the 3' end with phosphate, and 0.2 µM probeb labelled at the 3' end with fluorescein. Bovine DNA,c to be used as template, was diluted to approximately 10 ng/µL, aliquoted into single-use units, and frozen at –70°C. After storage times of up to 8 months, assays to be used were removed from the freezer, allowed to thaw, and 2 µL of bovine DNA was added as template. After adding template DNA to the PCR mixture, samples were transferred to LightCycler capillariesa and cycled on the LightCycler instrumenta as follows: 95°C for 10 minutes; 45 cycles of 95°C for 10 seconds, 55°C for 10 seconds, and 72°C for 20 seconds (all at a temperature ramping rate of 20°C/seconds). Threshold cycle (CT) values were determined by the LightCycler softwarea (Version 5.32) using the second derivative method with arithmetic baseline adjustment.
The second hybridization probe assay was designed to detect Leptospira DNA. A PCR master mix was prepared and aliquoted as described above. Reactions were used immediately or frozen at –70°C. Concentrations in the final working PCR mixture were 3 mM MgCl2, 1 x reaction buffer, 0.5 µM forward and reverse primersd 0.2 µM probeb labelled at the 5' end with LightCycler Red 640 and at the 3' end with phosphate, and 0.2 µM probeb labelled at the 3' end with fluorescein. The DNA template used was a boiled L. interrogans bratislava culture diluted 10-fold in water (approximate concentration 50 ng/µL). This template was aliquoted into single-use units and frozen at –70°C. After storage times of up to 7 months, assays to be used were removed from the freezer, allowed to thaw, and 2 µL of Leptospire DNA was added as template. Thermal cycling and analysis were as described for the mammalian mtDNA assay above.
The 5' nuclease TaqMan assay also detected mammalian mtDNA. Master mixes were prepared using TaqMan Universal PCR Master Mixe or QuantiTect Probe PCR Mixf and dispensed into 0.5 mL microfuge tubes in 23-µL aliquots. Assays were used immediately or frozen at –70°C. Concentrations in the final working PCR mixture were as follows: 2.5 mM MgCl2, 1 x reaction buffer, 0.3 µM forward and reverse primers,d 0.1 µM TaqMan probee labelled at the 5' end with FAM (6-carboxyfluorescein) and at the 3' end with TAMRA (6-carboxytetramethylrhodamine). After storage times of up to 6 months, assays to be used were removed from the freezer, allowed to thaw, and 2 µL of bovine DNA was added as template. Reaction mixes were transferred to 0.2 mL PCR tubes and cycled on an MX3000p instrumentg as follows: 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, and 54°C for 1 minute. The CT values were determined via the MX3000p software (version 1.2) using the amplification-based threshold determination and adaptive baseline analysis options.
The performance of the mtDNA 5' nuclease TaqMan assay after freezing was also examined over a range of template concentration. A PCR master mix was prepared as described above but aliquoted directly into 0.2-mL PCR tubes. Assays were used immediately or frozen at –70°C. Bovine DNA (approximately 50 ng/µL) was aliquoted into single-use units and frozen at –70°C. After storage times of up to 6 months, assays to be used were removed from the freezer, allowed to thaw, and used to amplify a 5-fold dilution series of the stored bovine DNA. Cycling and analysis were performed as described above.
The RT-PCR assay was a 1-step 5' nuclease TaqMan assay, which detected bovine viral diarrhea virus (BVDV). Master mixes were prepared using QuantiTect Probe RT-PCR Mixf and dispensed into 0.2 mL PCR tubes in 20-µL aliquots. Assays were used immediately or frozen at –70°C. Concentrations in the final working PCR mixture were as follows: 4 mM MgCl2, 1 x reaction buffer, 0.4 µM forward and reverse primers,h 0.2 µM TaqMan probeh labelled at the 5' end with FAM and at the 3' end with TAMRA. Template RNA was extracted from BVDV-infected bovine cell-culture fluid using the QIAamp viral RNA mini kit.f The RNA (approximately 2 ng/µL) was aliquoted into single-use units and frozen at –70°C. After storage times of up to 1 month, assays to be used were removed from the freezer, allowed to thaw, and used to amplify a 5-fold dilution series of the stored RNA. Next, 5 µL of template RNA was added to each reaction. Reactions were cycled on an MX3000p instrumentg as follows: 50°C for 30 minutes; 95°C for 15 minutes; and 40 cycles of 95° C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds. The CT values were determined by the MX3000p software (version 1.2) using the amplification-based threshold determination and adaptive baseline-analysis options.
The CT values recorded for both the hybridization probe and the TaqMan probe PCR assays (Fig. 1a–c) showed that freezing complete real-time PCR reaction mixes was possible without deterioration in assay performance, even after extended periods of storage at –70°C. Any deterioration in assay performance would have been expected to result in an increase in CT value. Use of real-time PCR and measurement of CT is an ideal system with which to determine the performance of PCR assays, and it is more accurate than the end-point measurement typically used in conventional PCR.
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Results with the TaqMan assays showed that the 2 different commercial master mixes tested exhibited similar stability when frozen (Fig. 1c). Interestingly, however, the 2 master mixes consistently produced different CT values, indicating different assay sensitivity. This mirrors other experimental findings (Jason Sawyer, 2000–2005, unpublished observations) within this laboratory where the relative performance of commercial master mixes is assay specific and probably reflects the particular buffer and reactions conditions required for optimal performance of different PCR assays. This emphasizes the need for caution when changing commercial master mixes.
After the encouraging results obtained with real-time PCR reagents, the possibility of freezing complete real-time RT-PCR reagents (excluding template) was examined. Amplification of a dilution series of BVDV RNA demonstrated that real-time RT-PCR reactions can also be frozen in a similar manner to real-time PCR reactions as no deterioration in assay performance was seen after storage of master mix for up to 1 month (Fig. 1e). Although storage was not extended longer than 1 month, it seems likely that the storage period could be extended further. The finding that real-time RT-PCR assays can be frozen, without deterioration in performance, means this method may potentially be adopted for a wider range of organisms of veterinary significance, including the RNA viruses.
The approach of preparing and freezing complete master mixes has several advantages. It is convenient and user-friendly, and assays are easy to use; the required number of tubes are simply removed from storage before adding template and cycling. Assay consistency should be improved by reducing the scope for pipetting or experimental error. Additionally, the assays may be used more easily by nonspecialist staff. This approach would be particularly useful for assays that are low throughput and/or for which there is an unpredictable demand. Another application would be a disease-outbreak situation where large batches of reagents could be produced centrally and distributed to laboratories for testing.
Interestingly, given the accepted wisdom that it is prudent to avoid excessive freeze-thawing, it had been expected that the avoiding repeated freeze-thawing of reagents inherent in this approach would be an advantage in terms of reagent stability. However, both PCR and RT-PCR master mixes were remarkably resilient to the effects of freeze-thawing. No deterioration in assay performance was seen when master mixes were subjected to up to 6 freeze-thaw cycles before PCR and RT-PCR cycling (data not shown).
Some of the most widely used types of real-time PCR and RT-PCR assays, using common fluorescent labels and reagents, were tested in this study. The fact that the performance of these assays was not affected by freezing master mixes suggests that this approach should be applicable to other real-time PCR assays. However, it will be necessary to validate individual PCR assays, kits, and chemistries before adopting this approach. The study reported here builds on previous work3,4 by examining real-time PCR reagents based on the use of Taq polymerase and using CT to measure assay performance; it extends the storage times examined and shows that real-time RT-PCR reactions can also be frozen without deterioration of assay performance, at least for a storage period of 4 weeks.
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Acknowledgments |
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a. Roche Applied Science, Lewes, UK.
b. TibMolBiol, Berlin, Germany.
e. Applied Biosystems, Warrington, UK.
g. Stratagene, Amsterdam, The Netherlands./p>
h. Sigma Genosys, Cambridge UK.
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