Limits...
An efficient strategy for broad-range detection of low abundance bacteria without DNA decontamination of PCR reagents.

Chang SS, Hsu HL, Cheng JC, Tseng CP - PLoS ONE (2011)

Bottom Line: To date, no satisfactory solution has been found.The spiking DNA neither interfered with template DNA amplification nor caused false positive of the reaction.When coupling with real-time and HRM analyses, it offers a new avenue for bacterial species identification with a limited source of bacterial DNA, making it suitable for use in clinical and applied microbiology laboratories.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Clinical Medical Sciences, Department of Medicine, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan, Republic of China.

ABSTRACT

Background: Bacterial DNA contamination in PCR reagents has been a long standing problem that hampers the adoption of broad-range PCR in clinical and applied microbiology, particularly in detection of low abundance bacteria. Although several DNA decontamination protocols have been reported, they all suffer from compromised PCR efficiency or detection limits. To date, no satisfactory solution has been found.

Methodology/principal findings: We herein describe a method that solves this long standing problem by employing a broad-range primer extension-PCR (PE-PCR) strategy that obviates the need for DNA decontamination. In this method, we first devise a fusion probe having a 3'-end complementary to the template bacterial sequence and a 5'-end non-bacterial tag sequence. We then hybridize the probes to template DNA, carry out primer extension and remove the excess probes using an optimized enzyme mix of Klenow DNA polymerase and exonuclease I. This strategy allows the templates to be distinguished from the PCR reagent contaminants and selectively amplified by PCR. To prove the concept, we spiked the PCR reagents with Staphylococcus aureus genomic DNA and applied PE-PCR to amplify template bacterial DNA. The spiking DNA neither interfered with template DNA amplification nor caused false positive of the reaction. Broad-range PE-PCR amplification of the 16S rRNA gene was also validated and minute quantities of template DNA (10-100 fg) were detectable without false positives. When adapting to real-time and high-resolution melting (HRM) analytical platforms, the unique melting profiles for the PE-PCR product can be used as the molecular fingerprints to further identify individual bacterial species.

Conclusions/significance: Broad-range PE-PCR is simple, efficient, and completely obviates the need to decontaminate PCR reagents. When coupling with real-time and HRM analyses, it offers a new avenue for bacterial species identification with a limited source of bacterial DNA, making it suitable for use in clinical and applied microbiology laboratories.

Show MeSH

Related in: MedlinePlus

PE-PCR specifically amplifies template bacterial DNA without co-amplification of contaminating bacterial DNA.A. The indicated amount of S. aureus genomic DNA was subjected to PE-PCR using the fusion probe M13-TstaG422 and the primer set M13 and TstaG765. B. The S. aureus genomic DNA (100 fg) was subjected to PE-PCR (upper panel) as described in panel A (lane 1), in the absence of M13 primer (lane 2), and in the artificially contaminating condition by adding 100 fg S. aureus genomic DNA into the EK mix (lane 3) or PCR mixtures (lane 4). PE-PCR was also performed in the absence of template DNA (lane 5). The presence of S. aureus genomic DNA was confirmed by species-specific PCR that amplified a chromosomal DNA fragment specific for S. aureus (lower panel). C. The indicated amount of S. aureus genomic DNA was used as the template for broad-range PE-PCR using the fusion probe M13-16S-p201F and the primer set M13 and p1370. D. The S. aureus genomic DNA (100 fg) was subject to broad-range PE-PCR (upper panel) as described in panel C (lane 1), and in the artificially contaminating condition by adding 100 fg of S. aureus genomic DNA into the EK mix (lane 2) or PCR mixtures (lane 3). Broad-range PE-PCR was also performed in the absence of template DNA (lane 4). The presence of S. aureus genomic DNA was confirmed by species-specific PCR to amplify a chromosomal DNA fragment of S. aureus (lower panel). NTC, no template control.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3102696&req=5

pone-0020303-g003: PE-PCR specifically amplifies template bacterial DNA without co-amplification of contaminating bacterial DNA.A. The indicated amount of S. aureus genomic DNA was subjected to PE-PCR using the fusion probe M13-TstaG422 and the primer set M13 and TstaG765. B. The S. aureus genomic DNA (100 fg) was subjected to PE-PCR (upper panel) as described in panel A (lane 1), in the absence of M13 primer (lane 2), and in the artificially contaminating condition by adding 100 fg S. aureus genomic DNA into the EK mix (lane 3) or PCR mixtures (lane 4). PE-PCR was also performed in the absence of template DNA (lane 5). The presence of S. aureus genomic DNA was confirmed by species-specific PCR that amplified a chromosomal DNA fragment specific for S. aureus (lower panel). C. The indicated amount of S. aureus genomic DNA was used as the template for broad-range PE-PCR using the fusion probe M13-16S-p201F and the primer set M13 and p1370. D. The S. aureus genomic DNA (100 fg) was subject to broad-range PE-PCR (upper panel) as described in panel C (lane 1), and in the artificially contaminating condition by adding 100 fg of S. aureus genomic DNA into the EK mix (lane 2) or PCR mixtures (lane 3). Broad-range PE-PCR was also performed in the absence of template DNA (lane 4). The presence of S. aureus genomic DNA was confirmed by species-specific PCR to amplify a chromosomal DNA fragment of S. aureus (lower panel). NTC, no template control.

Mentions: To provide a proof of principle, a serially diluted S. aureus genomic DNA was subject to the PE-PCR strategy. The translation elongation factor Tu (Tuf) gene of S. aureus was selected as the target for PE-PCR amplification. A fusion probe (M13-TstaG422) was designed with the M13 forward primer sequence at the 5′-end and the Tuf sequences (accession no. AF298796) at the 3′-end (Table 1). After annealing M13-TstaG422 to the template DNA, the EK mix was added into the reaction to degrade the unbound fusion probe and initiate primer extension. The primer extension product was then subject to PCR amplification using M13 and the downstream primer TstaG765 corresponding to the Tuf genomic sequences. As a result, a 391-bp single PCR product was obtained with 50 fg of bacterial DNA equivalent to 10 copies of S. aureus genome being detectable by PE-PCR. Notably, no PCR product was observed in the NTC control (Fig. 3A).


An efficient strategy for broad-range detection of low abundance bacteria without DNA decontamination of PCR reagents.

Chang SS, Hsu HL, Cheng JC, Tseng CP - PLoS ONE (2011)

PE-PCR specifically amplifies template bacterial DNA without co-amplification of contaminating bacterial DNA.A. The indicated amount of S. aureus genomic DNA was subjected to PE-PCR using the fusion probe M13-TstaG422 and the primer set M13 and TstaG765. B. The S. aureus genomic DNA (100 fg) was subjected to PE-PCR (upper panel) as described in panel A (lane 1), in the absence of M13 primer (lane 2), and in the artificially contaminating condition by adding 100 fg S. aureus genomic DNA into the EK mix (lane 3) or PCR mixtures (lane 4). PE-PCR was also performed in the absence of template DNA (lane 5). The presence of S. aureus genomic DNA was confirmed by species-specific PCR that amplified a chromosomal DNA fragment specific for S. aureus (lower panel). C. The indicated amount of S. aureus genomic DNA was used as the template for broad-range PE-PCR using the fusion probe M13-16S-p201F and the primer set M13 and p1370. D. The S. aureus genomic DNA (100 fg) was subject to broad-range PE-PCR (upper panel) as described in panel C (lane 1), and in the artificially contaminating condition by adding 100 fg of S. aureus genomic DNA into the EK mix (lane 2) or PCR mixtures (lane 3). Broad-range PE-PCR was also performed in the absence of template DNA (lane 4). The presence of S. aureus genomic DNA was confirmed by species-specific PCR to amplify a chromosomal DNA fragment of S. aureus (lower panel). NTC, no template control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102696&req=5

pone-0020303-g003: PE-PCR specifically amplifies template bacterial DNA without co-amplification of contaminating bacterial DNA.A. The indicated amount of S. aureus genomic DNA was subjected to PE-PCR using the fusion probe M13-TstaG422 and the primer set M13 and TstaG765. B. The S. aureus genomic DNA (100 fg) was subjected to PE-PCR (upper panel) as described in panel A (lane 1), in the absence of M13 primer (lane 2), and in the artificially contaminating condition by adding 100 fg S. aureus genomic DNA into the EK mix (lane 3) or PCR mixtures (lane 4). PE-PCR was also performed in the absence of template DNA (lane 5). The presence of S. aureus genomic DNA was confirmed by species-specific PCR that amplified a chromosomal DNA fragment specific for S. aureus (lower panel). C. The indicated amount of S. aureus genomic DNA was used as the template for broad-range PE-PCR using the fusion probe M13-16S-p201F and the primer set M13 and p1370. D. The S. aureus genomic DNA (100 fg) was subject to broad-range PE-PCR (upper panel) as described in panel C (lane 1), and in the artificially contaminating condition by adding 100 fg of S. aureus genomic DNA into the EK mix (lane 2) or PCR mixtures (lane 3). Broad-range PE-PCR was also performed in the absence of template DNA (lane 4). The presence of S. aureus genomic DNA was confirmed by species-specific PCR to amplify a chromosomal DNA fragment of S. aureus (lower panel). NTC, no template control.
Mentions: To provide a proof of principle, a serially diluted S. aureus genomic DNA was subject to the PE-PCR strategy. The translation elongation factor Tu (Tuf) gene of S. aureus was selected as the target for PE-PCR amplification. A fusion probe (M13-TstaG422) was designed with the M13 forward primer sequence at the 5′-end and the Tuf sequences (accession no. AF298796) at the 3′-end (Table 1). After annealing M13-TstaG422 to the template DNA, the EK mix was added into the reaction to degrade the unbound fusion probe and initiate primer extension. The primer extension product was then subject to PCR amplification using M13 and the downstream primer TstaG765 corresponding to the Tuf genomic sequences. As a result, a 391-bp single PCR product was obtained with 50 fg of bacterial DNA equivalent to 10 copies of S. aureus genome being detectable by PE-PCR. Notably, no PCR product was observed in the NTC control (Fig. 3A).

Bottom Line: To date, no satisfactory solution has been found.The spiking DNA neither interfered with template DNA amplification nor caused false positive of the reaction.When coupling with real-time and HRM analyses, it offers a new avenue for bacterial species identification with a limited source of bacterial DNA, making it suitable for use in clinical and applied microbiology laboratories.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Clinical Medical Sciences, Department of Medicine, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan, Republic of China.

ABSTRACT

Background: Bacterial DNA contamination in PCR reagents has been a long standing problem that hampers the adoption of broad-range PCR in clinical and applied microbiology, particularly in detection of low abundance bacteria. Although several DNA decontamination protocols have been reported, they all suffer from compromised PCR efficiency or detection limits. To date, no satisfactory solution has been found.

Methodology/principal findings: We herein describe a method that solves this long standing problem by employing a broad-range primer extension-PCR (PE-PCR) strategy that obviates the need for DNA decontamination. In this method, we first devise a fusion probe having a 3'-end complementary to the template bacterial sequence and a 5'-end non-bacterial tag sequence. We then hybridize the probes to template DNA, carry out primer extension and remove the excess probes using an optimized enzyme mix of Klenow DNA polymerase and exonuclease I. This strategy allows the templates to be distinguished from the PCR reagent contaminants and selectively amplified by PCR. To prove the concept, we spiked the PCR reagents with Staphylococcus aureus genomic DNA and applied PE-PCR to amplify template bacterial DNA. The spiking DNA neither interfered with template DNA amplification nor caused false positive of the reaction. Broad-range PE-PCR amplification of the 16S rRNA gene was also validated and minute quantities of template DNA (10-100 fg) were detectable without false positives. When adapting to real-time and high-resolution melting (HRM) analytical platforms, the unique melting profiles for the PE-PCR product can be used as the molecular fingerprints to further identify individual bacterial species.

Conclusions/significance: Broad-range PE-PCR is simple, efficient, and completely obviates the need to decontaminate PCR reagents. When coupling with real-time and HRM analyses, it offers a new avenue for bacterial species identification with a limited source of bacterial DNA, making it suitable for use in clinical and applied microbiology laboratories.

Show MeSH
Related in: MedlinePlus