Detection of pathogenic Yersinia enterocolitica in enrichment media and pork by a multiplex PCR: a study of sample preparation and PCR-inhibitory components

Lantz, Pär-Gunnar; Knutsson, Rickard; Blixt, Ylva; Al‐Soud, Waleed Abu; Borch, Elisabeth; Rådström, Peter

doi:10.1016/s0168-1605(98)00152-4

Cited by 89 publications

(66 citation statements)

References 29 publications

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“…Panels F to J show standard curves and corresponding equations when the three sets of data were used for independent analysis of DyNazyme II (F), LCTaq (G), rTth (H), Taq (I), and Tth (J). (6), which showed the same trends (data not shown). However, other factors, such as the thermal cycler model and the probe system, may affect the PCR performance.…”

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confidence: 65%

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Impact of DNA Polymerases and Their Buffer Systems on Quantitative Real-Time PCR

Wolffs¹,

Grage

Hagberg

et al. 2004

J Clin Microbiol

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An investigation of the influence of five DNA polymerase-buffer systems on real-time PCR showed that the choice of both DNA polymerase and the buffer system affected the amplification efficiency as well as the detection window. The analytical repeatability of the data for different systems changed clearly, leading us to conclude that basing quantitative measurements on single-data-set standard curves can lead to significant errors.Sequence-specific nucleic acid quantification in areas such as diagnostic PCR and molecular biology has been greatly improved by the introduction of real-time PCR technology (9). While this technology has tremendous potential for accurate and sensitive quantification, further studies addressing the quantification aspect of this technology are required before it can be widely implemented. Previous results from this laboratory (5), in which the range of detection of real-time PCR was modeled in a pure system using two different DNA polymerases, gave an indication that DNA polymerases and their buffer systems influence the performance of PCR by affecting the detection window and linear range of amplification. The aim of this work was to systematically study the effect of five DNA polymerase-buffer systems on absolute quantification using the LightCycler instrument (Roche Diagnostics, Mannheim, Germany).A primer set, Y1 and Y2, for Yersinia enterocolitica was used (6). To a commercial LightCycler kit (LCTaq) (Roche Diagnostics), a 0.4 mM concentration of each primer was added together with 4 mM MgCl 2 . Sterile Millipore water was added to a volume of 16 l and complemented with 4 l of Y. enterocolitica DNA. The concentration of DNA was fluorimetrically determined using a TD-700 fluorimeter (Turner Designs, Sunnyvale, Calif.), and the DNA was diluted to appropriate concentrations in sterile Millipore water. The four other master mixtures, contained 2.5 U of DNA polymerase and 1ϫ associated buffer, 4 mM MgCl 2 , 0.4 ml of each primer, 0.2 mM (each) deoxynucleoside triphosphate, 10,000-fold-diluted SYBR Green I, and 4 l of Y. enterocolitica DNA in a total volume of 20 l. The following DNA polymerases were used: DyNazyme II (FINNZYMES OY, Espoo, Finland), rTth (Applied Biosystems, Foster City, Calif.), and Taq (Roche Diagnostics) and Tth (Roche Diagnostics). Each amplification started with a denaturation step of 1 min at 95°C, followed by 40 cycles of 0.1 s of denaturation at 95°C, 5 s of annealing at 60°C, and elongation for 15 s at 72°C, followed by a single fluorescence measurement and finally 25 s of final elongation. Amplification was followed by melting curve analysis between 65 and 95°C and finally cooling for 1 min at 40°C. The quantification data, in terms of the crossing point value (ROM) (which is expressed as the fractional cycle number and is the intersection of the log-linear fluorescence curve with a threshold crossing line), were determined using the second derivative method of the LightCycler Software, version 3 (Roche Diagnostics).Amplification efficiency and analytical repea...

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confidence: 65%

“…A primer set, Y1 and Y2, for Yersinia enterocolitica was used (6). To a commercial LightCycler kit (LCTaq) (Roche Diagnostics), a 0.4 mM concentration of each primer was added together with 4 mM MgCl 2 .…”

mentioning

confidence: 99%

Impact of DNA Polymerases and Their Buffer Systems on Quantitative Real-Time PCR

Wolffs¹,

Grage

Hagberg

et al. 2004

J Clin Microbiol

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“…All pathogenic Y. enterocolitica and Y. pseudotuberculosis strains yielded positive PCR products from the yadA gene. However, a 203-bp PCR product was found in the pathogenic Y. enterocolitica strains but with a larger base pair than that of Y. enterocolitica in the pathogenic Y. pseudotuberculosis, which was the same as yadA primers, as described previously (18,23). The T m values of this yadA primer pair were also different between the pathogenic Y. enterocolitica (85.1°C) and Y. pseudotuberculosis (84.1°C) strains.…”

Section: Discussionmentioning

confidence: 66%

“…(34), pathogenic Yersinia spp. (18,30), Campylobacter jejuni (4,19,25,39), Vibrio cholerae (21,22), Vibrio parahaemolyticus (13,24), Vibrio vulnificus (38), Aeromonas spp. (16), Staphylococcus aureus (2), Clostridium perfringens (15), and Bacillus cereus (38).…”

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confidence: 99%

Duplex Real-Time SYBR Green PCR Assays for Detection of 17 Species of Food- or Waterborne Pathogens in Stools

2003

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A duplex real-time SYBR Green LightCycler PCR (LC-PCR) assay with DNA extraction using the QIAamp DNA Stool Mini kit was evaluated with regard to detection of 8 of 17 species of food-or waterborne pathogens in five stool specimens in 2 h or less. The protocol used the same LC-PCR with 20 pairs of specific primers. The products formed were identified based on a melting point temperature (T m ) curve analysis. The 17 species of food-or waterborne pathogens examined were enteroinvasive Escherichia coli, enteropathogenic E. coli, enterohemorrhagic E. coli, enterotoxigenic E. coli, enteroaggregative E. coli, Salmonella spp., Shigella spp., Yersinia enterocolitica, Yersinia pseudotuberculosis, Campylobacter jejuni, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Aeromonas spp., Staphylococcus aureus, Clostridium perfringens, and Bacillus cereus. No interference with the LC-PCR assay was observed when stool specimens were artificially inoculated with each bacterial species. The detection levels were approximately 10 5 food-or waterborne pathogenic bacteria per g of stool. The protocol for processing stool specimens for less than 10 4 food-or waterborne pathogenic bacteria per g of stool requires an overnight enrichment step to achieve adequate sensitivity. However, the rapid amplification and reliable detection of specific genes of greater than 10 5 food-or waterborne pathogenic bacteria per g in samples should facilitate the diagnosis and management of food-or waterborne outbreaks.The traditional cultural techniques for the direct isolation and identification of food-or waterborne pathogens from stool specimens in food poisoning outbreaks are time-consuming and laborious. Efforts have been made in diagnostic laboratories to reduce the time required for identification of food-or waterborne pathogens. Real-time PCR is currently used for the diagnosis of infectious agents, and there are few reports of the application of real-time PCR for the direct detection of Escherichia coli strain O157:H7 (11) and Plesiomonas shigelloides (20) in stool specimens. However, traditional PCR is often used for the detection of infectious agents from stool specimens and food samples, and there are a number of previous reports of PCR use for detection of food-or waterborne pathogens, including E. coli (6,14,12,27,37,38,40), Salmonella spp. (26) (38). The published PCR protocols used for detection of food-or waterborne pathogens differ and include the use of different PCR cycler machines, annealing temperatures, and buffer systems. For routine laboratory analysis, the development of streamlined PCR methods would be ideal. In addition, not all of the published PCR methods are so sensitive or specific. Therefore, careful choice of PCR primers, modification of some existing PCR primers, and development of new PCR methods for detection and identification of a wide range of food-or waterborne pathogens are all necessary for the development of a standardized PCR protocol (38). Moreover, interference and inhibitors should be considered...

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“…Real-time PCR conditions. A primer set targeting a 0.3-kb part of the 16S rRNA gene from Y. enterocolitica (16) was used to develop a real-time PCR assay using the LightCycler instrument (Roche Diagnostics, Mannheim, Germany) (28). The PCR mixture consisted of 0.75 U of Tth DNA polymerase (Roche Diagnostics); 1ϫ Tth DNA polymerase buffer (Roche Diagnostics); 4 mM MgCl 2 ; 0.4 M each primer; 0.2 mM (each) dATP, dCTP, dGTP, and dTTP; 10,000-fold-diluted SYBR Green I (Roche Diagnostics); and 4 l of sample.…”

Section: Methodsmentioning

confidence: 99%

Rapid Quantification ofYersinia enterocoliticain Pork Samples by a Novel Sample Preparation Method, Flotation, Prior to Real-Time PCR

Wolffs

Knutsson

Norling³

et al. 2004

J Clin Microbiol

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The development of real-time PCR thermal cycles in the late 1990s has opened up the possibility of accurate quantification of microorganisms in clinical, environmental, and food samples. However, a lack of suitable sample preparation methods that allow rapid quantification of the nucleic acids, remove PCR inhibitors, and prevent false-positive results due to DNA originating from dead cells has limited the use of quantitative PCR. We have used for the first time a new variant of density gradient centrifugation, called flotation, as a user-friendly sample preparation method prior to PCR. This paper describes the use of this sample preparation method, without DNA purification, for direct detection and quantification of Yersinia enterocolitica in PCR-inhibitory meat juice from pork. Since real-time PCR thermal cycles became commercially available in the late 1990s, the technology has been recognized as an outstanding tool in molecular diagnostics (4,8,19). In comparison with conventional PCR, real-time PCR facilitates automation, computerization, and quantification of nucleic acids. Also, real-time PCR, with its increased sensitivity, speed, and precision and its wide dynamic range (2, 10), has found successful applications in food microbiology. Recently, numerous rapid real-time PCR assays for qualitative detection of pathogens have been developed (3,13,14). To allow such detection, several requirements must be met during sample preparation: (i) PCR-inhibitory substances must be removed, (ii) target nucleic acids or cells must be concentrated, (iii) heterogeneous samples must be converted into a homogeneous sample (25), and (iv) detection of dead cells must be prevented (10). Culture enrichment, DNA extraction, or a combination of both is often used to meet these goals (15). However, these steps are time-consuming, and their elimination can be regarded as one of the main challenges left on the way to the most rapid PCR detection possible.Aside from the need for rapid qualitative detection, there is also a need for quantification of the bacterial load (6,22). To allow quantitative measurements, one additional requirement for sample preparation is that the selected method should not influence the amount of target, or should do so only in a controlled and predictable manner. This means that enrichment cannot be used prior to quantitative PCR (qPCR). Concerning DNA purification, which is presently almost exclusively used, there may be some disadvantages. Studies have indicated that the quality and yield of DNA available after purification can depend on the purification method, sample composition (20), and target (29), and variations in both yield and quality can affect the final quantification data (22).This study demonstrates the use of a novel sample preparation method, called flotation, based on density gradient centrifugation. The benefits of density gradient centrifugation as a sample pretreatment method are well established and include (i) the possibility of separating biological matrix particles and microorganis...

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Detection of pathogenic Yersinia enterocolitica in enrichment media and pork by a multiplex PCR: a study of sample preparation and PCR-inhibitory components

Cited by 89 publications

References 29 publications

Impact of DNA Polymerases and Their Buffer Systems on Quantitative Real-Time PCR

Impact of DNA Polymerases and Their Buffer Systems on Quantitative Real-Time PCR

Duplex Real-Time SYBR Green PCR Assays for Detection of 17 Species of Food- or Waterborne Pathogens in Stools

Rapid Quantification ofYersinia enterocoliticain Pork Samples by a Novel Sample Preparation Method, Flotation, Prior to Real-Time PCR

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