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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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Comparison of broad-range real-time PE-PCR and broad-range real-time PCR with DNase I pretreatment of PCR reagents.A–C. The indicated amounts of S. aureus genomic DNA were subject to broad-range real-time PE-PCR using the fusion probe M13-16S-p201F and the primer set M13 and p1370 in the presence of LCGreen I plus HRM dye (panel A and B). Alternatively, the PCR reaction mixtures with or without pretreatment of DNase I (1 U and 2.5 U) were used for broad-range real-time PCR to amplify the indicated amounts of S. aureus genomic DNA (panel C). The PCR product was subject to HRM analysis using HR-1 instrument. The amplification (panel A and left panel of C) and derivative plots (panel B and right panel of C) were shown. NTC, no template control.
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pone-0020303-g004: Comparison of broad-range real-time PE-PCR and broad-range real-time PCR with DNase I pretreatment of PCR reagents.A–C. The indicated amounts of S. aureus genomic DNA were subject to broad-range real-time PE-PCR using the fusion probe M13-16S-p201F and the primer set M13 and p1370 in the presence of LCGreen I plus HRM dye (panel A and B). Alternatively, the PCR reaction mixtures with or without pretreatment of DNase I (1 U and 2.5 U) were used for broad-range real-time PCR to amplify the indicated amounts of S. aureus genomic DNA (panel C). The PCR product was subject to HRM analysis using HR-1 instrument. The amplification (panel A and left panel of C) and derivative plots (panel B and right panel of C) were shown. NTC, no template control.

Mentions: To further enhance the power of this method, we modified the protocol to incorporate real-time PCR and HRM analysis into the broad-range PE-PCR platform. A 10-fold serially diluted S. aureus genomic DNA (10 pg–10 fg) was subject to broad-range real-time PE-PCR in the presence of HRM dye LCGreen I plus. As revealed by the amplification plots, 10 fg of template DNA resulted in amplicon-specific amplification and no PCR product in the NTC reaction (Fig. 4A and 4B). The unique nucleotide contents in the PCR amplicon of S. aureus produced a distinctive derivative plot while no melting peak was observed for the NTC reaction. The probability of obtaining a positive PCR signal for 100, 50, 25, and 10 fg of template DNA was 100%, 90%, 50%, and 30%, respectively (n = 10).


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)

Comparison of broad-range real-time PE-PCR and broad-range real-time PCR with DNase I pretreatment of PCR reagents.A–C. The indicated amounts of S. aureus genomic DNA were subject to broad-range real-time PE-PCR using the fusion probe M13-16S-p201F and the primer set M13 and p1370 in the presence of LCGreen I plus HRM dye (panel A and B). Alternatively, the PCR reaction mixtures with or without pretreatment of DNase I (1 U and 2.5 U) were used for broad-range real-time PCR to amplify the indicated amounts of S. aureus genomic DNA (panel C). The PCR product was subject to HRM analysis using HR-1 instrument. The amplification (panel A and left panel of C) and derivative plots (panel B and right panel of C) were shown. NTC, no template control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020303-g004: Comparison of broad-range real-time PE-PCR and broad-range real-time PCR with DNase I pretreatment of PCR reagents.A–C. The indicated amounts of S. aureus genomic DNA were subject to broad-range real-time PE-PCR using the fusion probe M13-16S-p201F and the primer set M13 and p1370 in the presence of LCGreen I plus HRM dye (panel A and B). Alternatively, the PCR reaction mixtures with or without pretreatment of DNase I (1 U and 2.5 U) were used for broad-range real-time PCR to amplify the indicated amounts of S. aureus genomic DNA (panel C). The PCR product was subject to HRM analysis using HR-1 instrument. The amplification (panel A and left panel of C) and derivative plots (panel B and right panel of C) were shown. NTC, no template control.
Mentions: To further enhance the power of this method, we modified the protocol to incorporate real-time PCR and HRM analysis into the broad-range PE-PCR platform. A 10-fold serially diluted S. aureus genomic DNA (10 pg–10 fg) was subject to broad-range real-time PE-PCR in the presence of HRM dye LCGreen I plus. As revealed by the amplification plots, 10 fg of template DNA resulted in amplicon-specific amplification and no PCR product in the NTC reaction (Fig. 4A and 4B). The unique nucleotide contents in the PCR amplicon of S. aureus produced a distinctive derivative plot while no melting peak was observed for the NTC reaction. The probability of obtaining a positive PCR signal for 100, 50, 25, and 10 fg of template DNA was 100%, 90%, 50%, and 30%, respectively (n = 10).

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