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

Abstract

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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