Combination of competitive quantitative PCR and constant-denaturant capillary electrophoresis for high-resolution detection and enumeration of microbial cells - PubMed (original) (raw)

Combination of competitive quantitative PCR and constant-denaturant capillary electrophoresis for high-resolution detection and enumeration of microbial cells

E L Lim et al. Appl Environ Microbiol. 2001 Sep.

Abstract

A novel quantitative PCR (QPCR) approach, which combines competitive PCR with constant-denaturant capillary electrophoresis (CDCE), was adapted for enumerating microbial cells in environmental samples using the marine nanoflagellate Cafeteria roenbergensis as a model organism. Competitive PCR has been used successfully for quantification of DNA in environmental samples. However, this technique is labor intensive, and its accuracy is dependent on an internal competitor, which must possess the same amplification efficiency as the target yet can be easily discriminated from the target DNA. The use of CDCE circumvented these problems, as its high resolution permitted the use of an internal competitor which differed from the target DNA fragment by a single base and thus ensured that both sequences could be amplified with equal efficiency. The sensitivity of CDCE also enabled specific and precise detection of sequences over a broad range of concentrations. The combined competitive QPCR and CDCE approach accurately enumerated C. roenbergensis cells in eutrophic, coastal seawater at abundances ranging from approximately 10 to 10(4) cells x ml(-1). The QPCR cell estimates were confirmed by fluorescent in situ hybridization counts, but estimates of samples with <50 cells x ml(-1) by QPCR were less variable. This novel approach extends the usefulness of competitive QPCR by demonstrating its ability to reliably enumerate microorganisms at a range of environmentally relevant cell concentrations in complex aquatic samples.

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Figures

FIG. 1

FIG. 1

Electropherograms of C. roenbergensis DNA fragments amplified from pure culture (A) and Boston Harbor seawater (B) analyzed by CDCE. PCR products from both amplifications were mixed in equal amounts and electrophoresed (C). The primer peaks correspond to the FITC-labeled 187F primer that remained after PCR. The smaller primer peaks are fractions of labeled primers which disintegrated into shorter pieces during PCR.

FIG. 2

FIG. 2

Melting profile of the 130-bp target and internal competitor DNA fragments. A substitution was introduced at position 231 of the internal competitor fragment (GC to AT, vertical arrow). Bp 41 on the map corresponds to base position 168 of the C. roenbergensis 18S rRNA gene. The Tm of the low-melting-temperature domain of target sequence is 0.5°C higher than that of the internal competitor. Std., standard.

FIG. 3

FIG. 3

Electropherograms of target and internal competitor DNAs separated by CDCE at 71.8°C. The DNA fragments were coamplified from pure target and internal competitor DNA templates (A) and from pure template added to seawater cell lysate (B) at the same concentration as in panel A.

FIG. 4

FIG. 4

Comparison of amplification efficiencies in Eel Pond seawater of C. roenbergensis and internal competitor DNA as a function of PCR cycle number (two-stage amplification). PCR products were quantified by integrating the target and internal competitor DNA peaks and multiplying the ratios of the target to total peak area and primer to total peak area by the concentration (copies per microliter) of primer used in the PCR. The error bars represent the standard deviation of the mean of quadruple measurements. Linear regressions were performed on data points from cycles 35 to 45.

FIG. 5

FIG. 5

QPCR standard curve for C. roenbergensis. The ratio of target peak to internal competitor peak areas was plotted against cell number in 20-μl PCR mixes. All the data points are the mean values of four measurements, and the error bars represent the standard deviation of the mean. The regression (_R_2 = 0.99) was performed on the standard-curve data points only. Values from the spiked samples which fall on the regression line indicate a 1:1 agreement between the expected and QPCR-estimated concentrations of C. roenbergensis in the spiked samples.

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