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`JOURNAL OF CLINICAL MICROBIOLOGY,
`Dec. 2001, p. 4518–4519
`0095-1137/01/$04.00⫹0 DOI: 10.1128/JCM.39.12.4518–4519.2001
`Copyright © 2001, American Society for Microbiology. All Rights Reserved.
`
`Vol. 39, No. 12
`
`Automation of Fluorescence-Based PCR for Confirmation of
`Meningococcal Disease
`M. A. DIGGLE, G. F. S. EDWARDS, AND S. C. CLARKE*
`Scottish Meningococcus and Pneumococcus Reference Laboratory, Glasgow, United Kingdom
`
`Received 26 June 2001/Accepted 13 September 2001
`
`A fluoresence-based PCR method was developed, fully automated, and used to confirm infection with
`Neisseria meningitidis by detection of the meningococcus-specific ctrA gene. The method provided a highly
`sensitive, high-throughput assay that was reproducible and less labor-intensive than manual methods.
`
`There are a number of methods available for the detection
`or amplification of specific DNA sequences (2, 7, 8, 9). Some
`of these have disadvantages, including low sensitivity, lack of
`specificity, high cost, or laborious methodology (10). During
`the past decade, PCR has provided an invaluable tool and has
`been partly responsible for the explosion in molecular biology
`(4). The method has found its place in many areas that utilize
`molecular techniques in research and nonresearch environ-
`ments, including microbiology, animal and human genetics,
`and clinical diagnostics. Traditionally, PCR is performed in a
`commercial thermocycler and the products are visualized with
`a gel-based system (3). However, various technologies are now
`available to further exploit the PCR method. New chemistries,
`such as Taqman and Molecular Beacons, have been developed
`commercially to provide real-time PCR methods that are more
`sensitive than the equivalent gel-based system because they are
`fluorescence based (2, 8). These chemistries have also allowed
`the further expansion of the applications of PCR into areas
`such as single-nucleotide polymorphism analysis (5), while
`standard PCR has been developed into providing amplicons
`for microarray analysis (6). Automation has also recently be-
`come more affordable and is therefore accessible to more
`laboratories. It is now used heavily in the pharmaceutical in-
`dustry and more recently has found use in academic research
`and clinical diagnostics.
`Presented here is a novel application of one type of PCR
`chemistry with the capacity for high throughput and full auto-
`mation. We have termed this method dual-labeled end-point
`fluorescence PCR (DEF-PCR), and it is based on a previously
`described chemistry (2) whereby oligonucleotide primers are
`dual labeled with a reporter dye, carboxyfluorescein, covalently
`linked to the 5⬘ end and the quencher dye, carboxy-tetrameth-
`ylrhodamine, linked to the 3⬘ end. A probe hybridizes to a
`specific DNA sequence upon PCR product formation but is
`subsequently digested by 5⬘ exonuclease activity of Taq DNA
`polymerase during primer extension, thus releasing reporter
`dye and increasing fluorescence emissions. The procedure is
`fully automated on a liquid handling robot, and the formation
`
`* Corresponding author. Mailing address: Scottish Meningococcus
`and Pneumococcus Reference Laboratory, North Glasgow University
`Hospitals NHS Trust, Department of Microbiology, House on the Hill,
`Stobhill Hospital, Balornock Rd., Glasgow G21 3UW, United King-
`dom. Phone: 44 141 201 3836. Fax: 44 141 201 3836. E-mail: stuart
`.clarke@northglasgow.scot.nhs.uk.
`
`of PCR products is analyzed via the alteration and subsequent
`increase in fluorescence emissions using an integrated 96-well-
`format fluorimeter.
`Nonculture diagnosis of certain infectious diseases is becom-
`ing increasingly important as antibiotics are given prior to
`hospital admission. One such example is meningococcal infec-
`tion whereby a rapid confirmation of disease is required for
`both patient treatment and case contact prophylaxis. We there-
`fore used this method to demonstrate and confirm disease in
`patients clinically suspected to have meningococcal infection.
`To do this we used ctrA primers for the detection of Neisseria
`meningitidis DNA, and the PCR conditions were those used
`previously (1). This gene target has previously been shown to
`be sensitive and specific for this purpose and does not amplify
`DNA from other neisseriae or other species which may cause
`septicaemia or meningitis (1). Due to its fluorescence-based
`chemistry, the method is highly sensitive and here we have also
`fully automated the method.
`DNA was extracted using the Nucleospin Blood protocol
`(ABgene, Surrey, United Kingdom) for the isolation of
`genomic DNA from whole blood, serum, and plasma. The
`reproducibility of the method was tested on 96 individual sam-
`ples comprising 48 N. meningitidis DNA controls of various
`serogroups (Table 1) and 48 negative controls (sterile distilled
`water). After DNA extraction, samples were transferred into
`1.8-ml non-cross-contamination tubes and placed in the sam-
`ple rack of the Roboseq 4204 SE robotic liquid handling sys-
`tem, possessing an integrated thermocycler and fluorescence
`reader (MWG Biotech, Milton Keynes, United Kingdom). All
`components of the system were in a 96-well microtiter plate
`format, allowing standardization and high-throughput method-
`ology. Programming of the liquid handling robot was per-
`formed according to the manufacturer’s instructions. All PCR
`reagents were maintained at 4°C on the robotic platform. Each
`reaction was performed in a final volume of 50 ␮l consisting of
`48 ␮l of 1.1⫻ Reddymix PCR Master Mix (ABgene) contain-
`ing 1.25 U of Taq DNA polymerase; 75 mM Tris-HCl (pH 8.8
`at 25°C); 20 mM (NH4)2; 1.5 mM MgCl2; 0.01% (vol/vol)
`Tween 20; a 0.2 mM concentration (each) of dATP, dCTP,
`dGTP, and DTTP; 1 ␮l of each primer (1 pmol) (MWG Bio-
`tech); 1 ␮l of dual-labeled probe (0.5 pmol) (MWG Biotech);
`and 2 ␮l of extracted DNA. Each reaction was set up auto-
`matically by the robot within a refrigerated 96-well microtiter
`plate using disposable tips. Cross-contamination was avoided
`
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`VOL. 39, 2001
`
`NOTES
`
`4519
`
`TABLE 1. Fluorescence emission cutoff values for the detection of
`the ctrA gene from Neisseria meningitidis
`
`Control sample
`
`No. of
`samples
`
`N. meningitidis serogroup B
`N. meningitidis serogroup C
`N. meningitidis serogroup Y
`N. meningitidis serogroup W135
`N. meningitidis serogroup X
`N. meningitidis serogroup NG
`
`Total
`
`Negative control
`
`8
`27
`2
`4
`5
`2
`
`48
`
`48
`
`Detection of ctrA by
`DEF-PCR
`
`No. (%)
`positive
`
`8 (100)
`24 (89)
`2 (100)
`4 (100)
`4 (80)
`2 (100)
`
`44 (92)
`
`No. (%)
`negative
`
`0 (0)
`3 (11)
`0 (0)
`0 (0)
`1 (20)
`0 (0)
`
`4 (8)
`
`0 (0)
`
`100 (100)
`
`by the use of these tips, which were discarded automatically
`into a waste container. After PCR setup, optically clear dis-
`posable strips (ABgene) were manually placed over the wells
`to seal the contents. The microtitre plate was then automati-
`cally placed into the integrated thermocycler for the period of
`thermocycling. After amplification, the microtiter plate was
`automatically removed from the thermocycler into the inte-
`grated Bio-Tek FL600 fluorescence plate reader. Selected
`wavelengths of 485 to 420 nm and 530 to 525 nm for excitation
`and emissions respectively were used to detect the fluorescence
`emissions caused by carboxyfluorescein. A total of 100 end-
`point readings were taken from each well, and the average was
`calculated by using the KC4 software (MWG Biotech). The
`KC4 software was programmed to calculate a cutoff value
`based on the subtraction of the average of the three controls
`from the positive control.
`A threshold value was determined as 0.5 standard deviation
`above the mean of the background fluorescence emission for
`all wells after endpoint calculations. This standard deviation
`and subsequent cutoff value was calculated using the 48 posi-
`tive and 48 negative controls. Of the positive controls, 44 were
`positive by the DEF-PCR assay, providing a sensitivity of 92%.
`Of the negative controls, all 48 (100%) were negative. The
`reproducibility and sensitivity of the method was therefore
`demonstrated, with all positive control samples being detected,
`with a sensitivity of 92% and with all negative control samples
`being negative (Table 1).
`The method is relatively cheap compared with similar meth-
`ods, as (i) the chemistry is widely available without the need for
`specialist equipment and, as such, can be performed manually
`using conventional setup techniques and (ii) results can be
`
`read with a manually operated fluorescent plate reader pos-
`sessing the appropriate filter set. Although we developed the
`method using N. meningitidis control DNA, the method is now
`under ongoing evaluation in the laboratory for the nonculture
`confirmation of meningococcal and pneumococcal infection.
`The assays are now set up to include one positive control, three
`negative controls, and up to 92 test samples in a 96-well mi-
`crotiter plate format.
`Despite the fact that the method can be performed manu-
`ally, we have described full automation here, although addi-
`tional automation can be achieved with robotic systems by the
`inclusion of an integrated vacuum manifold for DNA extrac-
`tion from body fluids. Although we used the 96-well microtiter
`plate format, many robotic systems can be designed with the
`384-well format to achieve even higher throughput. In conclu-
`sion, DEF-PCR has been demonstrated as an effective tool for
`the high throughput and sensitive detection of meningococcal
`DNA for the nonculture confirmation of meningococcal infec-
`tion. However, the method could be applied to other bacterial
`and viral infections but also has the potential for application in
`many areas including microbiological research, clinical diag-
`nostics, and the pharmaceutical industry.
`
`We thank MWG-Biotech Ltd. for the loan of the RoboSeq 4202 SE
`and the integrated Bio-Tek FL600 microtiter fluorescence reader and
`ABgene for providing support (consumables) for the PCR.
`
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