Elimination of Bacterial DNA from Taq DNA Polymerases by

0 downloads 0 Views 711KB Size Report
The incidence of false positives due to the presence of bacterial DNA in Taq DNA polymerase is an obstacle to the use of PCR in the diagnosis of infection.
JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1999, p. 3402–3404 0095-1137/99/$04.00⫹0 Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Vol. 37, No. 10

Elimination of Bacterial DNA from Taq DNA Polymerases by Restriction Endonuclease Digestion NORA M. CARROLL,† PETER ADAMSON,

AND

NARCISS OKHRAVI*

Department of Clinical Ophthalmology, The Institute of Ophthalmology, London EC1V 9EL, United Kingdom

The incidence of false positives due to the presence of bacterial DNA in Taq DNA polymerase is an obstacle to the use of PCR in the diagnosis of infection. We describe a method that uses a restriction enzyme to destroy the ability of contaminating sequences to act as templates for a nested PCR which uses primers based on the 16S rRNA genes. The method was used prior to a PCR that amplified 10 fg of bacterial DNA. This method can be readily adapted to suit other sensitive PCRs required for clinical applications. amplification was used as supplied. Details of the two primer pairs used in this study appear in Table 1. PCR mixtures contained 60 ␮M each deoxynucleoside triphosphate (Pharmacia), 0.3 ␮M each primer, 3.0 mM Mg2⫹, and 1 U of various Taq DNA polymerases in 25 ␮l. First-round amplifications were conducted with 2.5 pmol each of primers 16SF and 16SR, with an initial denaturation at 95°C (3 min) and cycling as follows: 95°C for 10 s, 54.2°C for 10 s, and 72°C for 15 s for 30 cycles (Genius Thermal Cycler; TECHNE). The second-round amplification was carried out as was the first, except that the Mg2⫹ concentration was 2.5 mM and 5 pmol of each primer was used. One microliter of the product from the first-round amplification was amplified with primers NF and NR as follows: denaturation at 95°C (3 min) and cycling at 95°C for 7 s, 60°C for 7 s, and 72°C for 10 s (30 cycles). Reagent controls from the first round were always subjected to a second round of amplification to control for contamination. Amplicons were resolved on a 1% agarose–Tris-acetateEDTA gel, visualized by using ethidium bromide under UV illumination, and recorded by using the UVP gel documentation system (UVP Ltd.). Samples for Southern analysis were purified by using Qiaquik PCR purification kits (Qiagen) and slot blotted onto Hybond N⫹ (Amersham). Probes for Southern hybridizations were 3⬘ labelled and detected by using the ECL 3⬘ labelling kit (Amersham). Contaminating sequences in Taq DNA polymerase are readily amplified by using universal bacterial primers based on ribosomal genes. All assays to check for DNA in Taq DNA polymerase were carried out by using fresh reagents to rule out the possibility of exogenous contamination. The level of contamination was insufficient to give a detectable product after one round of PCR but was easily detected in all preparations after two rounds of amplification (Fig. 1). Amplitaq LD

Due to its ability to exponentially amplify regions of DNA, the PCR has the potential to be used as a diagnostic tool. It has been used in the detection and identification of a wide range of bacterial species using oligonucleotide primers based on the conserved regions of rRNA sequences. The detection of organisms which are difficult to cultivate has been improved by the use of this technique (1, 5, 8), as it relies on the presence of DNA and not the viability of the organism. The disadvantage of primers with broad specificity is the concomitant amplification of contaminating DNA, which gives rise to false-positive results. The elimination of false positives is an essential prerequisite to the development of PCR protocols for use in a clinical setting. This objective has been partly achieved by rendering PCR amplicons unsuitable for reamplification and also by the elimination of contaminating template DNA in PCR reagents. The presence of bacterial DNA in preparations of Taq DNA polymerase is well established (3, 4). Several methods have been used to eradicate this contamination (6, 7, 9), but none were found to be 100% effective. Diagnosis by culture is especially difficult with clinical samples when a limited volume is available. A set of nested primers was developed for the detection of bacteria which were capable of detecting 10 fg of bacterial DNA (unpublished observation). However, these primers were found to be of limited use, as every reagent control which was subjected to two rounds of amplification gave rise to a product of the size expected for a bacterial template, although none was added. In this paper, a method is outlined for the removal of contaminating DNA sequences from Taq DNA polymerase preparations which can be easily applied to other PCR assays. Pretreatment of Taq DNA polymerase to remove contaminating bacterial DNA. Prior to PCR amplification, the water, buffer, MgCl2 and Taq DNA polymerase components were mixed and incubated for 30 min at 37°C with 1.0 U of Sau3AI (Boehringer Mannheim) per U of Taq DNA polymerase. The restriction enzyme was inactivated by incubation at 95°C for 2 min, following which the deoxynucleoside triphosphates, primers, and template DNA were added and PCR amplification commenced. Taq DNA polymerase for the second round of

TABLE 1. Names, sequences, and positions of oligonucleotide primers used in this study Primer

* Corresponding author. Mailing address: Department of Clinical Ophthalmology, The Institute of Ophthalmology, Bath St., London EC1V 9EL, United Kingdom. Phone: 0171-608-6872. Fax: 0171-6086931. E-mail: [email protected]. † Present address: Department of Medical Biochemistry, University of Stellenbosch, Tygerberg 7505, South Africa.

16SF 16SR NF NR

3402

Sequence

5⬘ 5⬘ 5⬘ 5⬘

TTGGAGAGTTTGATCCTGGCTC 3⬘ ACGTCATCCCCACCTTCCTC 3⬘ GGCGGCAKGCCTAAYACATGCAAGT 3⬘ GACGACAGCCATGCASCACCTGT 3⬘

Position on E. coli rRNA gene sequence

4–25 1174–1194 42–66 1044–1067

Downloaded from http://jcm.asm.org/ on April 14, 2015 by University of Manchester Library

Received 2 April 1999/Returned for modification 4 June 1999/Accepted 6 July 1999

VOL. 37, 1999

routinely demonstrated the least PCR product production following nested amplification. Prior to first-round PCR amplification, Amplitaq LD was treated with Sau3AI as already described. It can be seen from Fig. 2 that use of very high levels of Sau3A1 (4 U/U of Taq DNA polymerase) as a pretreatment resulted in reduced product formation from 20 ng of template DNA. Lower levels of Sau3AI achieved the aim of avoiding false positives while maintaining an acceptable level of sensitivity after two rounds of PCR amplification. Treatment of the Amplitaq LD prior to the second round of amplification was not performed, as the subsequent sensitivity of detection was compromised (data not shown). Southern hybridization of the negative controls in Fig. 2B showed that the untreated negative control was the only sample that gave a positive signal (data not shown). As can be seen in Fig. 3, inclusion of 1 U of Sau3AI/U of Amplitaq LD allowed the amplification of 10 fg of bacterial DNA, illustrating that this method does not compromise the sensitivity or specificity of this PCR. Taq DNA polymerase is contaminated with bacterial DNA (3, 7). Amplitaq LD from Perkin-Elmer, an ultrapure prepa-

FIG. 2. Effect on product amplification of treating Taq DNA polymerase with decreasing amounts of Sau3A1. Positive reactions contained 20 ng of Escherichia coli NTCC 11151 DNA. Negative reaction mixtures contained no added template. (A) First round of amplification with primers 16F and 16R. Lanes 1, 3, 5, 7, 9, 11, and 13 were positive reaction mixtures treated with 4, 2, 1, 0.5, 0.25, 0.13, and 0 U of Sau3AI, respectively. Lanes 2, 4, 6, 8, 10, 12, and 14 were negative reaction mixtures treated with 4, 2, 1, 0.5, 0.25, 0.13, and 0 U of Sau3AI, respectively. Lane 15 contained the molecular size marker (GIBCO, Paisley, Scotland). (B) Second round of amplification with primers NF and NR. Lanes 1 to 7 contained 1 ␮l of the negative reaction mixtures from round 1 that had been treated with 4, 2, 1, 0.5, 0.25, 0.13, and 0 U of Sau3AI, respectively. Lane 8 contained the positive control for the amplification, and lane 9 contained the reagent control. Lane 10 contained the molecular size marker (GIBCO).

3403

FIG. 3. Sensitivity of nested PCR amplification of E. coli NTCC 11151 DNA using primer pairs 16F plus 16R and NF plus NR. The template was E. coli NTCC 11151 DNA purified as described in the text. Lanes 1 to 8 contained the products of the amplification of 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, and 10 fg of DNA or no DNA using primers 16F and 16SR and Amplitaq LD that had been pretreated with Sau3AI. Lane 9 contained the 1-kb size marker (Promega). Lanes 10 to 17 contain the products of the amplification of 1 ␮l of each of the first-round samples with primers NF and NR. Lane 18 contains a reagent control for the second round of amplification.

ration, is guaranteed to contain less than 10 copies of bacterial 16S rRNA gene sequences per 2.5-␮l aliquot, which was sufficient to produce a false-positive result under our conditions of amplification. To eliminate this source of contamination, a pretreatment protocol was developed which successfully rendered this DNA unamplifiable while maintaining the sensitivity of the reaction. Additional criteria for choosing Sau3A1 were its ease of inactivation by heat and its wide availability. Several investigators have reported methods for reducing or eliminating the amplification of contaminating DNA sequences in Taq DNA polymerase. These included pretreatment of Taq DNA polymerase with DNase I or purification on a CsCl2 density gradient (1–6). DeFilippes (2) reported that false positivity was reduced by the use of restriction endonucleases but did not address the question of contamination of the Taq DNA polymerase itself. The large amounts of enzyme used and the long incubation time made his procedure unwieldy, expensive, and impractical for routine use. The method described in this paper can be easily incorporated into current PCR protocols, as it utilizes an enzyme that is active in the PCR buffer, reducing the amount of sample handling and opportunities for sample contamination. In addition, the short reaction time merely extends the total time required for the PCR by 30 min. In a clinical context, this protocol will allow confident reporting of positive results and its extreme sensitivity will also allow the use of negative PCR results. This is significant, as clinical diagnoses are often complicated by immune-mediated processes which can appear indistinguishable from infection. However, as this technique is used to remove contaminating sequences only from Taq DNA polymerase, it is no substitute for general cleanliness and the usual precautions in the PCR laboratory, i.e., separation of pre- and post-PCR areas. N.C. was supported by Oclyx Ltd. P.A. was supported by Fight for Sight. N.O. was supported by Wellcome Vision Research Fellowship 045203 and locally organized research funds from Moorfields Eye Hospital (221 and 271). We thank Susan Lightman, Department of Clinical Ophthalmology, The Institute of Ophthalmology, for the opportunity to undertake this study. REFERENCES 1. Boddinghaus, B., T. Rogall, T. Flohr, H. Blocker, and E. C. Bottger. 1990 Detection and identification of mycobacteria by amplification of rRNAs. J. Clin. Microbiol. 28:1751–1759. 2. DeFilippes, F. M. 1991. Decontaminating the polymerase chain reaction. BioTechniques 10:26–30. 3. Hughes, M. S., L.-A. Beck, and R. A. Skuce. 1994. Identification and elimi-

Downloaded from http://jcm.asm.org/ on April 14, 2015 by University of Manchester Library

FIG. 1. Nested PCRs using primer pairs 16F plus 16R and NR plus NF amplify a product in the absence of an added template. Lanes 1 to 5 were amplified by using Amplitaq LD (Perkin-Elmer, Cheshire, United Kingdom), lanes 6 to 10 were amplified with Amplitaq (Perkin-Elmer), and lanes 11 to 15 were amplified with Taq DNA polymerase (Stratagene, Amsterdam, The Netherlands). Lanes 1, 6, and 11 were positive controls for the outer PCRs; lanes 2, 7, and 12 were reagent controls for the outer reaction; lanes 3, 8, and 13 were positive controls for the nested reaction; lanes 4, 9, and 14 contained 1 ␮l of the first-round reagent control amplified with the nested primers; and lanes 5, 10, and 15 were reagent controls for the nested PCR. Lane 16 contained the molecular size marker (Promega, Wis.).

NOTES

3404

NOTES

nation of DNA sequences in Taq polymerase. J. Clin. Microbiol. 32:2007– 2008. 4. Maiwald, M., H. J. Ditton, H. G. Sonntag, and M. von-Knebel-Doeberitz. 1994. Characterisation of contaminating DNA in Taq polymerase which occurs during amplification with a primer set for Legionella 5S ribosomal RNA. Mol. Cell. Probes 8:11–14. 5. Mariani, B. D., M. J. Levine, R. E. Booth, Jr., and R. S. Tuan. 1995. Development of a novel, rapid processing protocol for polymerase chain reaction-based detection of bacterial infections in synovial fluids. Mol. Biotechnol. 4:227–237. 6. Meier, A., D. H. Persing, M. Finken, and E. C. Bottger. 1993. Elimination of contaminating DNA within polymerase chain reaction reagents: implications

J. CLIN. MICROBIOL.

7. 8. 9. 10.

for a general approach to detection of uncultured pathogens. J. Clin. Microbiol. 31:646–652. Rand, H. R., and H. Houck. 1990. Taq polymerase contains bacterial DNA of unknown origin. Mol. Cell. Probes 4:445–450. Relman, D. A., T. M. Schmidt, R. P. MacDermott, and S. Falkow. 1992. Identification of the uncultured bacillus of Whipple’s disease. N. Engl. J. Med. 327:293–301. Sarkar, G., and S. S. Sommer. 1993. Removal of DNA contamination in polymerase chain reaction reagents by ultraviolet irradiation. Methods Enzymol. 218:381–388. Schmidt, T. M., B. Pace, and N. R. Pace. 1991. Detection of DNA contamination in Taq polymerase. BioTechniques 11:176–177.

Downloaded from http://jcm.asm.org/ on April 14, 2015 by University of Manchester Library