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Chapter 8 : Preanalytical Sample Preparation and Analyte Extraction

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Abstract:

Sample preparation methods aim at separation of the target cells or virus particles from the surrounding food matrix. Down-stream methods, such as cell disruption, analyte purification, and detection via, for example, real-time PCR, have to be taken into consideration individually when designing or choosing a suitable preparation method. Sample treatment is defined as the preanalytical step in the method protocol, which is also necessary for reduction of the sample volume while maintaining the initial target number, as much as possible. This chapter first describes optimal performance characteristics of sample preparation. Next, the chapter talks about practical solutions to physical separation methods, biochemical and biological separation methods. Then, it discusses chemical and enzymatic digestion of the food sample matrix for preseparation. Analyte extraction is the subsequent step following sample preparation. It is necessary to obtain access to the molecules that are the target of molecular detection assays such as real-time PCR. In order to gain access to the analyte molecules, nucleic acids, or proteins, the integrity of the target cells has to be destroyed in most cases. Since analyte extraction as a part of the detection chain in food pathogen detection is similar to the application in basic research, a brief summary is given.

Citation: Rossmanith P, Hedman J, Rådström P, Hoorfar J, Wagner M. 2011. Preanalytical Sample Preparation and Analyte Extraction, p 121-136. In Hoorfar J (ed), Rapid Detection, Characterization, and Enumeration of Foodborne Pathogens. ASM Press, Washington, DC. doi: 10.1128/9781555817121.ch8
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Image of FIGURE 1
FIGURE 1

Transmission electron microscopic pictures of serovar Typhimurium and after using “First generation” chemical treatment in sample preparation for solubilization of food matrices. (A and F) Exposure to 4 M guanidine-isothiocyanate and 2 M NaCl according to D’Urso et al. ( ). (B and E) Treatment with 0.6% NaCl and 6% sodium citrate according to Stevens and Jaykus ( ). (C and D) 2% Sodium citrate treatment according to Ulve et al. ( ).

Citation: Rossmanith P, Hedman J, Rådström P, Hoorfar J, Wagner M. 2011. Preanalytical Sample Preparation and Analyte Extraction, p 121-136. In Hoorfar J (ed), Rapid Detection, Characterization, and Enumeration of Foodborne Pathogens. ASM Press, Washington, DC. doi: 10.1128/9781555817121.ch8
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Image of FIGURE 2
FIGURE 2

Transmission electron microscopic pictures of serovar Typhimurium after exposure to chemical treatments used in sample preparation for solubilization of food matrices (matrix lysis). (A) Control group, an overnight culture of untreated serovar Typhimurium cells. (B) A penicillin G-treated culture for comparison of cell wall damage. (C) Treatment with 8 M urea and 1% SDS. The visible secondary structure was generated from the remnants of several lysed cells. (D) Exposure to 1 M MgCl. The cellular integrity is intact; DNA seems to reversibly precipitate within the cells. (E) Treatment with 7.5% [Emim]SCN. The cells show appearance comparable to that of cells treated with 1 M MgCl with more effect of the chemical towards the cell wall; also, the precipitation seems more advanced. (F) Treatment with 8 M urea and 1% Lutensol. The cellular integrity is mostly intact, but the shape of the cells is changed and the cell wall is affected.

Citation: Rossmanith P, Hedman J, Rådström P, Hoorfar J, Wagner M. 2011. Preanalytical Sample Preparation and Analyte Extraction, p 121-136. In Hoorfar J (ed), Rapid Detection, Characterization, and Enumeration of Foodborne Pathogens. ASM Press, Washington, DC. doi: 10.1128/9781555817121.ch8
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Image of FIGURE 3
FIGURE 3

Transmission electron microscopic pictures of after exposure to chemical treatments used in sample preparation for solubilization of food matrices (matrix lysis). (A) Control group, an overnight culture of cells including storage for 4 weeks at 4°C to demonstrate the natural degradation of the cells. (B) A penicillin G-treated culture for comparison of cell wall damage. (C) Treatment with 8 M urea and 1% SDS. The cellular appearance reflects the harsh chemical stress that affected the cells. (D) Treatment with 8 M urea and 1% Lutensol. The cells show significant influence of the reagent, with nevertheless intact cell walls. (E) Exposure to 1 M MgCl. The cells remain seemingly unaffected by the treatment. (F) Treatment with 7.5% [Emim]SCN. The cellular appearance also reflects the high rate of recovery obtained by the sample preparation method using this reagent.

Citation: Rossmanith P, Hedman J, Rådström P, Hoorfar J, Wagner M. 2011. Preanalytical Sample Preparation and Analyte Extraction, p 121-136. In Hoorfar J (ed), Rapid Detection, Characterization, and Enumeration of Foodborne Pathogens. ASM Press, Washington, DC. doi: 10.1128/9781555817121.ch8
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Image of FIGURE 4
FIGURE 4

Schematic chemical structure of 1-ethyl-3-methylimidazolium thiocyanate.

Citation: Rossmanith P, Hedman J, Rådström P, Hoorfar J, Wagner M. 2011. Preanalytical Sample Preparation and Analyte Extraction, p 121-136. In Hoorfar J (ed), Rapid Detection, Characterization, and Enumeration of Foodborne Pathogens. ASM Press, Washington, DC. doi: 10.1128/9781555817121.ch8
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Image of FIGURE 5
FIGURE 5

Schematic overview of preanalytical sample preparation methods that are usually used to disrupt the cellular structure of target bacteria in food samples.

Citation: Rossmanith P, Hedman J, Rådström P, Hoorfar J, Wagner M. 2011. Preanalytical Sample Preparation and Analyte Extraction, p 121-136. In Hoorfar J (ed), Rapid Detection, Characterization, and Enumeration of Foodborne Pathogens. ASM Press, Washington, DC. doi: 10.1128/9781555817121.ch8
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