Successful approach for detection of low numbers of enterotoxigenic Escherichia coli in minced meat by using the polymerase chain reaction

Wernars, K.; Delfgou, E.; Soentoro, P.S.S.; Notermans, S.

doi:10.1128/aem.57.7.1914-1919.1991

Cited by 50 publications

(14 citation statements)

References 18 publications

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“…In these cases, it is essential to detect only infective E.a. Wernars et al (1991) and Schaad et al (1995) proposed to select for the detection of viable and infective bacterial cells in biological sample extracts by a short, growth-enrichment step, to be included before the PCR assay (BIO-PCR [Schaad et al, 1995]). But it remains difficult to evaluate the viability of inactive bacterial cells inside plant tissue.…”

Section: Discussionmentioning

confidence: 99%

**Identification and sensitive endophytic detection of the fire blight pathogen Erwinia amylovora with 23S ribosomal DNA sequences and the polymerase chain reaction**

1996

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Two short sequences, situated in the bacterial 23S rDNA gene, were used as primers for the PCR detection of Erwinia amylovora bacteria. All 34 E. amylovora strains tested, coming from different geographical and host plant origins and of different virulence, produced a 565‐bp PCR fragment. The E. amylovora bacteria could be discriminated from all other phytobacteria with which no PCR product was observed. Only Escherichia coli bacteria were cross‐recognized by the production of a weaker PCR band of similar size to E. amylovora. In a fast PCR protocol, where two temperatures were cycled, E. amylovora in pure culture could be detected on gel at concentrations as low as 3 × 102 cfu mL–1. This corresponds to a detection limit of 1.5 bacteria per PCR. However, reliable PCR detection in woody host plant tissue was only obtained with PVP/PVPP‐treated sample extracts. Using E. amylovora‐spiked plant extracts and extracts of fruit tree shoots artificially infected with E. amylovora, the PCR detection sensitivity was determined to be 6.6 × 102 cfu mL–1 of extract. Starting from the plant samples, the PCR detection results were visualized on gels within 5 h.

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Section: Discussionmentioning

confidence: 99%

**Identification and sensitive endophytic detection of the fire blight pathogen Erwinia amylovora with 23S ribosomal DNA sequences and the polymerase chain reaction**

1996

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“…ϩ, specific 387-bp fragment found; Ϫ, specific 387-bp fragment not found. (1,10,13,16,17,24,34,45,47,49,50). It is concluded that components of food, enrichment media, or a high concentration of DNA may lead to inhibition of the PCR.…”

Section: Discussionmentioning

confidence: 99%

Sequence of a cloned pR72H fragment and its use for detection of Vibrio parahaemolyticus in shellfish with the PCR

Lee

Pan

Chen

1995

Appl Environ Microbiol

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The nucleotide sequence of pR72H cloned from Vibrio parahaemolyticus 93 was determined. We examined all V. parahaemolyticus gene sequences published in the GenBank-EMBL databases for homology and found that no other DNA sequence of V. parahaemolyticus was highly homologous to the sequence reported in this study. A pair of primers, VP33-VP32, derived from a pR72H fragment were selected to detect V. parahaemolyticus. The sensitivity of PCR detection for a pure culture of V. parahaemolyticus was 10 cells from crude bacterial lysates. Furthermore, a detection level of 2.6 fg, equivalent to 1 cell, was obtained by using purified chromosomal DNA as the template. The expected PCR products were obtained from all V. parahaemolyticus strains tested (n ‫؍‬ 124), while no PCR amplicons were found in other vibrios or related genera (n ‫؍‬ 50). High levels (10 6 to 10 10 CFU/ml) of Escherichia coli cells did not affect the PCR assay sensitivity. The presence of 10 8 V. parahaemolyticus cells or 10 9 E. coli cells in the PCR mixtures completely inhibited the PCR. When oyster samples were inoculated with V. parahaemolyticus 93 and cultured in tryptic soy broth containing 3% NaCl for 3 h at 35؇C, an initial sample inoculum level of 9.3 CFU/g was detected in a PCR assay with crude bacterial lysates. The PCR assay with enrichment culturing in salt polymyxin broth was compared with the conventional method for naturally contaminated shellfish and fish samples. We conclude that this PCR assay with enrichment culturing is a good alternative method for the detection of V. parahaemolyticus.

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“…Since its first introduction in 1985 (13), PCR has already become a widespread technique in numerous research laboratories. In addition to pure research (8,10,11) and medical (7,9,12) applications, PCR technology has recently been applied to water (1, 2, 6), sediment (16), and food samples (19). In the present study, we have coupled a rapid direct DNA extraction method (18) with the PCR to detect a low cell density or a low gene copy number of bacterial cells in soil and sediments.…”

mentioning

confidence: 99%

Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction

Tsai

Olson

1992

Appl Environ Microbiol

322

126

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Polymerase chain reaction was used to amplify the low copy number of two 16S ribosomal gene fragments from soil and sediment extracts. Total DNA for polymerase chain reaction was extracted from 1 g of seeded or unseeded samples by a rapid freeze-and-thaw method. Amplified DNA fragments can be detected in DNA fractions isolated from seeded soil containing less than 3 Escherichia coli cells and from seeded sediments containing less than 10 cells. This research demonstrated that coupling polymerase chain reaction to direct DNA extraction improves sensitivity by 1 and 2 orders of magnitude for sediments and soils, respectively. This technique could become a powerful tool for genetic ecology studies.

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Successful approach for detection of low numbers of enterotoxigenic Escherichia coli in minced meat by using the polymerase chain reaction

Cited by 50 publications

References 18 publications

**Identification and sensitive endophytic detection of the fire blight pathogen Erwinia amylovora with 23S ribosomal DNA sequences and the polymerase chain reaction**

**Identification and sensitive endophytic detection of the fire blight pathogen Erwinia amylovora with 23S ribosomal DNA sequences and the polymerase chain reaction**

Sequence of a cloned pR72H fragment and its use for detection of Vibrio parahaemolyticus in shellfish with the PCR

Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction

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