Application of the 5'-nuclease PCR assay in evaluation and development of methods for quantitative detection of Campylobacter jejuni - PubMed (original) (raw)

Application of the 5'-nuclease PCR assay in evaluation and development of methods for quantitative detection of Campylobacter jejuni

H K Nogva et al. Appl Environ Microbiol. 2000 Sep.

Abstract

Campylobacter jejuni is recognized as a leading human food-borne pathogen. Traditional diagnostic testing for C. jejuni is not reliable due to special growth requirements and the possibility that this bacterium can enter a viable but nonculturable state. Nucleic acid-based tests have emerged as a useful alternative to traditional enrichment testing. In this article, we present a 5'-nuclease PCR assay for quantitative detection of C. jejuni and describe its evaluation. A probe including positions 381121 to 381206 of the published C. jejuni strain NCTC 11168 genome sequence was identified. When this probe was applied, the assay was positive for all of the isolates of C. jejuni tested (32 isolates, including the type strain) and negative for all other Campylobacter spp. (11 species tested) and several other bacteria (41 species tested). The total assay could be completed in 3 h with a detection limit of approximately 1 CFU. Quantification was linear over at least 6 log units. Quantitative detection methods are important for both research purposes and further development of C. jejuni detection methods. In this study, we used the assay to investigate to what extent the PCR signals generated by heat-killed bacteria interfere with the detection of viable C. jejuni after exposure at elevated temperatures for up to 5 days. An approach to the reduction of the PCR signal generated by dead bacteria was also investigated by employing externally added DNases to selectively inactivate free DNA and exposed DNA in heat-killed bacteria. The results indicated relatively good discrimination between exposed DNA from dead C. jejuni and protected DNA in living bacteria.

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Figures

FIG. 1

FIG. 1

Amplification products from the _C. jejuni_-specific primers (A) and a universal 16S rRNA gene PCR primer pair (39) (B) for a set of Campylobacter strains. The samples were subjected to electrophoresis in 3% (A) and 2% (B) agarose gel at 100 V for 45 min. Ten microliters of the amplification product was loaded in each lane. Lanes: CSB, C. sputorum subsp. bubulis; CL, C. lari; CFF, C. fetus subsp. fetus; CC, C. concisus; AB, A. butzleri; AS, A. skirrowii; CJJ, C. jejuni subsp. jejuni; neg, negative control; mw, molecular weight marker.

FIG. 2

FIG. 2

(A) 5′-nuclease PCR analysis of serial 10-fold dilutions of C. jejuni DNA. CT_s are plotted against the calculated copies of bacterial DNA, i.e., a 10-fold dilution of the bacterial DNA (1.04 × 106 copies/μl). The straight line, which was calculated by linear regression [y = −3.27_x (number of cells) + 40.10], shows a square regression coefficient (_R_2) of 0.988. (B) 5′-nuclease PCR analysis of serial 10-fold dilutions of C. jejuni cells. CT_s are plotted against the number of cells of C. jejuni. Template DNA was extracted from samples of cells containing serial 10-fold dilutions from approximately 4.2 × 107 CFU of C. jejuni. The straight line, which was calculated by linear regression [y = −3.66_x (number of cells) + 41.54], shows a square regression coefficient (_R_2) of 0.997.

FIG. 3

FIG. 3

Effect of heat treatment on DNA stability. Cells were incubated at 25°C (A), 55°C (B), 72°C (C), and 100°C (D) for up to 5 days, and DNA was quantified by 5′-nuclease PCR assay after 5 min, 1 h, 6 h, 24 h, and 5 days. The amounts of DNA (copy numbers) and numbers of culturable cells (CFU counts) are given relative to the values before heat treatment. The error bars show standard deviations.

FIG. 4

FIG. 4

Effect of externally added DNase on the stability of DNA in heat-treated cells. Cells were incubated for 5 min at 20°C (A), 55°C (B), 72°C (C), or 100°C (D), or for 15 min at 121°C (E) before the temperature was adjusted to 20°C and DNase was added. DNA was quantified by 5′-nuclease PCR after 5, 15, and 30 min and 1, 6, and 24 h at 20°C in both DNase-treated samples and negative controls. The stability of purified DNA treated with DNase was also investigated (F). The amounts of DNA present with (⧫) and without ([Image: see text]) DNase treatment, are reported as copy numbers relative to those present before heat treatment. The error bars show standard deviations.

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