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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.

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Related in: MedlinePlus

The principle of PE-PCR for bacterial DNA amplification and detection.A fusion probe is designed with the sequences at the 3′-end corresponding to the bacterial genomic sequences and a non-bacterial tag sequence at the 5′-end. The reaction is initiated by annealing the fusion probe to the template bacterial DNA after heat-denaturing at 95°C for 5 min (Step 1 and 2). An enzyme mix (EK mix) of exo I and Klenow DNA polymerase is then added into the reaction mixture and incubated at 37°C for 2 h (Step 3a and 3b). Following heat-inactivation of EK mix at 80°C for 20 min (Step 3c), a forward primer (non-bac-F) corresponding to the non-bacterial sequence of the fusion probe and a reverse primer (bac-R) targeting bacterial genomic sequence downstream of the fusion probe are used for PCR amplification of the primer extension product (Step 4). In this setting, only template bacterial DNA but not the endogenous contaminated bacterial DNA is amplified (Step 5).
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pone-0020303-g002: The principle of PE-PCR for bacterial DNA amplification and detection.A fusion probe is designed with the sequences at the 3′-end corresponding to the bacterial genomic sequences and a non-bacterial tag sequence at the 5′-end. The reaction is initiated by annealing the fusion probe to the template bacterial DNA after heat-denaturing at 95°C for 5 min (Step 1 and 2). An enzyme mix (EK mix) of exo I and Klenow DNA polymerase is then added into the reaction mixture and incubated at 37°C for 2 h (Step 3a and 3b). Following heat-inactivation of EK mix at 80°C for 20 min (Step 3c), a forward primer (non-bac-F) corresponding to the non-bacterial sequence of the fusion probe and a reverse primer (bac-R) targeting bacterial genomic sequence downstream of the fusion probe are used for PCR amplification of the primer extension product (Step 4). In this setting, only template bacterial DNA but not the endogenous contaminated bacterial DNA is amplified (Step 5).

Mentions: The PE-PCR strategy is an approach that combines primer extension and PCR to circumvent the problem of endogenous DNA contamination. As illustrated in Fig. 2, the key component of this strategy is a fusion probe that comprises a bacterial sequence on its 3′-end and a non-bacterial tag sequences on its 5′-end. Using this fusion probe as an extension primer, the PE-PCR reaction was initiated by annealing an excess amount of the fusion probes to the template bacterial DNA after heat-denaturing (Step 1 and 2). An enzyme mix (EK mix) of exonuclease I (exo I) and Klenow DNA polymerase was then added into the reaction mixture. As a result, the unbound/free fusion probes were degraded by exo I and the primer extension reaction was initiated by Klenow DNA polymerase (Step 3a and 3b). Following heat-inactivation of EK mix, a forward primer (non-bac-F) corresponding to the non-bacterial sequence of the fusion probe and a reverse primer (bac-R) targeting bacterial genomic sequence downstream of the fusion probe were used for PCR amplification of the primer-extended product (Step 4). By tagging the template bacterial sequences with a non-bacterial tag sequence prior to PCR amplification (Step 4), the templates are distinguished from the contaminants contained in the PCR reagents. In theory, our PE-PCR strategy should amplify only the tagged template bacterial genomic DNA, thereby, rendering the contaminants a non-issue (Step 5).


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)

The principle of PE-PCR for bacterial DNA amplification and detection.A fusion probe is designed with the sequences at the 3′-end corresponding to the bacterial genomic sequences and a non-bacterial tag sequence at the 5′-end. The reaction is initiated by annealing the fusion probe to the template bacterial DNA after heat-denaturing at 95°C for 5 min (Step 1 and 2). An enzyme mix (EK mix) of exo I and Klenow DNA polymerase is then added into the reaction mixture and incubated at 37°C for 2 h (Step 3a and 3b). Following heat-inactivation of EK mix at 80°C for 20 min (Step 3c), a forward primer (non-bac-F) corresponding to the non-bacterial sequence of the fusion probe and a reverse primer (bac-R) targeting bacterial genomic sequence downstream of the fusion probe are used for PCR amplification of the primer extension product (Step 4). In this setting, only template bacterial DNA but not the endogenous contaminated bacterial DNA is amplified (Step 5).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020303-g002: The principle of PE-PCR for bacterial DNA amplification and detection.A fusion probe is designed with the sequences at the 3′-end corresponding to the bacterial genomic sequences and a non-bacterial tag sequence at the 5′-end. The reaction is initiated by annealing the fusion probe to the template bacterial DNA after heat-denaturing at 95°C for 5 min (Step 1 and 2). An enzyme mix (EK mix) of exo I and Klenow DNA polymerase is then added into the reaction mixture and incubated at 37°C for 2 h (Step 3a and 3b). Following heat-inactivation of EK mix at 80°C for 20 min (Step 3c), a forward primer (non-bac-F) corresponding to the non-bacterial sequence of the fusion probe and a reverse primer (bac-R) targeting bacterial genomic sequence downstream of the fusion probe are used for PCR amplification of the primer extension product (Step 4). In this setting, only template bacterial DNA but not the endogenous contaminated bacterial DNA is amplified (Step 5).
Mentions: The PE-PCR strategy is an approach that combines primer extension and PCR to circumvent the problem of endogenous DNA contamination. As illustrated in Fig. 2, the key component of this strategy is a fusion probe that comprises a bacterial sequence on its 3′-end and a non-bacterial tag sequences on its 5′-end. Using this fusion probe as an extension primer, the PE-PCR reaction was initiated by annealing an excess amount of the fusion probes to the template bacterial DNA after heat-denaturing (Step 1 and 2). An enzyme mix (EK mix) of exonuclease I (exo I) and Klenow DNA polymerase was then added into the reaction mixture. As a result, the unbound/free fusion probes were degraded by exo I and the primer extension reaction was initiated by Klenow DNA polymerase (Step 3a and 3b). Following heat-inactivation of EK mix, a forward primer (non-bac-F) corresponding to the non-bacterial sequence of the fusion probe and a reverse primer (bac-R) targeting bacterial genomic sequence downstream of the fusion probe were used for PCR amplification of the primer-extended product (Step 4). By tagging the template bacterial sequences with a non-bacterial tag sequence prior to PCR amplification (Step 4), the templates are distinguished from the contaminants contained in the PCR reagents. In theory, our PE-PCR strategy should amplify only the tagged template bacterial genomic DNA, thereby, rendering the contaminants a non-issue (Step 5).

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