Amplification Based Methods

Sensitivity of detection can be greatly improved through the use of the different in vitro amplification methods [polymerase chain reaction (PCR), ligase chain reaction or self-sustained sequence replication, nucleic acid sequence-based amplification (NASBA)]. With the exception of PCR and NASBA, the above-mentioned in vitro amplification methods have had only limited practical relevance in food monitoring (e.g., Stubbs et al. 1994).

2.2.1 Polymerase Chain Reaction

Since its discovery, a considerable number of PCR-based assays have been developed, but they have been applied most often to clinical and environmental samples and more rarely for the detection of food-borne microorganisms. The PCR technique allows rapid and selective identification and/or detection of microorganisms in different matrices by amplifying specific gene fragments. The reaction cycle consists of three steps: (a) denaturation of the double-stranded DNA (dsDNA), (b) annealing of short DNA fragments (primers) to single DNA strands, and (c) extension of the primers with a thermostable DNA polymerase. Following the completion of one cycle, the sample is denatured for the next annealing and extension steps during which not only the original target region is amplified, but also the amplification product of the first cycle, leading to an exponential increase of the number of copies of the target DNA. The detection of amplification products is possible through gel electrophoresis, ethidium bromide staining and visual examination of the gel using ultraviolet light. To increase the sensitivity and to confirm the identity of the amplification product, Southern blotting and hybridization with a specific probe can also be carried out (Hendolin et al. 2000; Loffler et al. 2000; Sandhu et al. 1995). Attempts have been made to increase the specificity of PCR reactions using other methods, including post-PCR hybridization (Sandhu et al. 1995), PCR-ELISA (enzyme linked immunosorbent assay) or ELOSA (enzyme linked oligosorbent assay) reactions (Grimm and Geisen 1998; Schnerr et al. 2001), RFLP (restriction fragment length polymorphism) analysis of the PCR products (Yamagishi et al. 1999), denaturing gradient gel electrophoresis (DGGE, Cocolin et al. 2001), fluorescent capillary electrophoresis (Turenne et al. 1999), or nested PCR, where one set of primers is used to amplify DNA fragments from target DNA, and a second set of primers complementary to an internal sequence of the product of the first PCR reaction is used to score and confirm the results (Ibeas 1997). In case of closely related species, single nucleotide differences can be visualized by using single strand conformation polymorphism (SSCP) analysis (Kumeda and Asao 1996), heteroduplex mobility assay (Olicio et al. 1999), heteroduplex panel analysis (Kumeda and Asao 2001), or by sequence analysis (Cappa and Cocconcelli 2001).

2.2.2 Nucleic Acid Sequence-Based Amplification

An alternative to PCR analysis, NASBA has also been applied to detect fungi (Loffler et al. 2001; Widjojoatmodjo et al. 1999). The NASBA is an isothermal nucleic acid amplification technology that specifically amplifies RNA sequences using T7 RNA polymerase (Compton 1991). Major advantages of NASBA over PCR is that it is performed isothermally at 41 °C, no separate reverse transcription step is required for RNA amplification, and since RNA is less stable than DNA-provides a better estimate of living cells in the sample analyzed.

2.2.3 Quantification of Results

Besides qualitative detection of spoilage microbes, it is also often desirable to know their abundance in foods and feeds. Quantification of the PCR results can be carried out by different approaches including limiting dilution of the DNA samples, densitometric measurement of PCR products, by HPLC with a UV detector (Katz 1996), by quantitative competitive PCR (QC-PCR), or by real-time PCR (Cross 1995). Two of them, QC-PCR and real-time PCR are the most promising tools for quantifying fungi in foods and feeds.

The QC-PCR using internal DNA standard provide the means for determining relative amounts of target DNA. The principle of QC-PCR is the coamplification of standard DNA together with target DNA. The competitor DNA is of known sequence (typically identical with the target DNA with added deletions or insertions) and has the same primer binding sites as the target DNA (Baek and Kenerley 1998; Haugland et al. 1999).

During conventional PCR, an endpoint analysis is carried out by examining the fluorescence of ethidium bromide stained amplification products separated by gel electro-phoresis. With real-time PCR, the continuous analysis of amplification-associated fluorescence during the whole PCR

reaction gives a graphic display of the time course of amplification of the PCR product of interest. Both direct and indirect methods are used for generating the fluorescence monitored during the PCR cycle (Walker 2001 and references therein; Figure 1). Indirect assays employ a system that yields fluorescence generated during the process of primer extension during amplification. This is employed in the Taqman system. Taqman probes utilize the intrinsic 5' nuclease activity of Taq DNA polymerase to digest a probe that has annealed to the specific gene of interest. The probe consists of quencher and reporter fluorochromes separated by a specific DNA sequence. Taq DNA polymerase-associated 5' nuclease digestion of the probe results in degradation of the probe and separation of the fluorochromes, so loss of quenching results in amplification-associated production of fluorescence. Direct methods refer to those systems in which fluorescence is a direct result of some binding of a fluorescence molecule to the amplified product or a direct incorporation of a fluorescence interference probe into the amplified product. A simple direct method involves the use of a fluorescent dye such as SYBR Green. This dye possesses selective affinity to dsDNA. Binding of the dye to dsDNA enhances fluorescence at 530 nm proportionally with dsDNA concentration. Specific hybridization probes can also be used that fluoresce only when bound to the gene of interest. All these probes are based on fluorescent resonance energy transfer (FRET; Walker 2001). Molecular beacons are essentially hairpin probes that employ fluorescence interference (Figure 1). A variation on the hairpin probe concept has been employed to develop unimolecular probe systems such as the Sunset or Scorpion primers, which are incorporated into the amplified sequence during the PCR reaction. A variety of PNA-based probes have also been developed for real-time PCR applications (Stender et al. 2002).

The LightCycler hybridization probe system consists of a labeled donor and acceptor probe. Fluorescence from the acceptor will only be generated when both probes are annealed to the product (Figure 1). The level of fluorescence is proportional to the amount of DNA generated during the PCR reaction (Loffler et al. 2000). The PCR reaction can be carried out in small volumes in a glass capillary that speeds up the heating process. The LightCycler technology combines rapid in vitro amplification in glass capillaries with real-time determination and quantification of DNA, enabling a 35 cycle PCR with 32 samples to be completed in 45 min.

2.2.4 Limits of PCR

One of the main drawbacks of PCR-based detection methods is that besides living cells, dead cells with relatively intact DNA are also detected, thus leading to false positive results. This limitation can be circumvented by including a propagation step prior to PCR analysis, or by using RNA as template in reverse transcription coupled with PCR, or NASBA reactions (Vaitilingom et al. 1998; Widjojoatmodjo et al. 1999). Although the detection of dead cells is a

Wiley Barnard

Figure 1 Fluorescence systems used in real-time PCR systems. S, SYBR Green; R, reporter fluorochrome; Q, quencher fluorochrome. Separation of the quencher from proximity to the reporter enables the fluorescence of the reporter to be measured [from Walker (2001); reprinted by permission of John Wiley & Sons, Inc.].

Figure 1 Fluorescence systems used in real-time PCR systems. S, SYBR Green; R, reporter fluorochrome; Q, quencher fluorochrome. Separation of the quencher from proximity to the reporter enables the fluorescence of the reporter to be measured [from Walker (2001); reprinted by permission of John Wiley & Sons, Inc.].

disadvantage for spoilage bacteria and yeasts, Geisen (1998) suggested that it is advantageous in the case of mycotoxin-producing fungi. Mycotoxins are stable molecules, and detection of the producing fungi in a sample could be used as prediction for the presence of mycotoxins.

Much of the difficulty in implementing PCR for the analysis of food samples lies in the problems encountered during the preparation of template DNAs from food matrices. The DNA extraction method used must achieve two aims: efficiently purify fungal DNA with minimal loss, and remove compounds inhibiting the PCR reaction. Foods are complex matrices and may contain several compounds that interfere with the PCR reaction leading to false negative results. These compounds include nucleases, chelating agents, or inhibitors of the polymerase itself. Cheese was found to be an extremely problematic matrix (Scheu et al. 1998). Several attempts have been made to develop a DNA extraction protocol to remove inhibitors from the samples (Dickinson et al. 1995; Lantz et al. 1999; Rossen et al. 1991; Rossen et al. 1992). The use of additional reagents during DNA extraction was suggested by different authors (e.g., DNA binding agents such as hexadecyl-trimethylammonium bromide, or polyvinyl pyrro-lidon for elimination of polyphenols). Additionally, DNA samples can be further purified by dialysis, gel filtration, or by other chromatographic methods. Besides careful extraction, other methods have also been suggested, including extensive dilution of the contaminating substances, or application of internal standard DNA in the PCR reaction (Geisen 1998). Separation of the microbes from the food matrix can be carried out by subculturing, or by using magnetic beads coated with specific binding proteins (lectins, antibodies; Patel et al. 1993).

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