Genetic Testing

Rapid tests discussed earlier for detection and characterization of microorganisms were based on phenotypic expressions of genotypic characteristics of microorganisms. The phenotypic expressions are subject to growth conditions such as temperature, pH, nutrient availability, oxidation-reduction potentials, environmental and chemical stresses, toxins, and water activities. Phenotypic expression, even including immunological tests, depends on cells' ability to produce the target antigens to be detected by the available antibodies or vice versa. The conventional "gold standards'' of diagnostic microbiology rely on phenotypic expression or traits that are inherently subject to variation.

Genotypic characteristics of a cell are far more stable than its phenotype. The natural mutation rate of a bacterial culture is about 1 in 100 million cells. Thus, there has been a push in recent years to make genetic test results the confirmative and definitive identification step in diagnostic microbiology. The debate is still continuing, and the final decision has not been reached by governmental and regulatory bodies for microbiological testing. Genetic-based diagnostic and identification systems are discussed in this section.

Hybridization of the deoxyribonucleic acid (DNA) sequence of an unknown bacterium by a known DNA probe is the first stage of genetic testing. The Genetrak system (Framingham, MA) provides a sensitive and convenient method to detect pathogens such as salmonella, listeria, campylo-bacter, and E. coli O157 in foods. Initially, the system utilized radioactive isotopes bound to DNA probes to detect complementary DNA of unknown cultures. The drawbacks of the first generation of this type of probes are (1) most food laboratories are not eager to work with radioactive materials in routine analysis and (2) there are limited copies of DNA in a cell. The second generation of probes uses enzymatic reactions to detect the presence of the pathogens and uses RNA as the target molecule. In a cell, there is only one complete copy of DNA; however, there may be 1,000 to 10,000 copies of ribosomal RNA. Thus, the new generation of probes is designed to detect target RNA using color reactions. After enrichment of cells (e.g., salmonella) in a food sample for about 18 hours, the cells (target cells as well as other microbes) are lysed by a detergent to release cellular materials (DNA, RNA, and other molecules) into the enrichment solution. Two RNA probes (designed to react with one piece of target salmonella RNA) are added into the solution. The capture probe with a long tail of a nucleotide (e.g., polyadenine tail or AAAAA) is designed to capture the RNA onto a dipstick with a long tail of thymine (TTTTT). The reporter probe, with an enzyme attached, will react with the other end of the RNA fragment. If salmonella RNA molecules are present, the capture probes will attach to one end of the RNA, and the reporter probes will attach to the other end. A dipstick coated with many copies of a chain of complementary nucleotide (e.g., thymine, TTTTT) will be placed into the solution. Since adenine (A) will hybridize with thymine (T), the chain (TTTTT) on the dipstick will react with the AAAAA and thus capture the target RNA complex onto the stick. After washing away debris and other molecules in the liquid, a chromagen is added. If the target RNA is captured, then the enzyme present in the second probe will react with the chromagen and will produce a color reaction indicating the presence of the pathogen in the food. In this case, the food is positive for salmonella. The system developed by Genetrak has been evaluated and tested for many years and has AOAC International approval of the procedure for many food types. More recently, Genetrak has adapted a microtiter format for more efficient and automated operation of the system.

PCR is now an accepted method to detect pathogens by amplification of the target DNA and detecting the target PCR products. Basically, a DNA molecule (double helix) of a target pathogen (e.g., salmonella) is first denatured at about 95°C to form single strands, then the temperature is lowered to about 55°C for two primers (small oligonucleotides specific for salmonella) to anneal to specific regions of the single stranded DNA. The temperature is increased to about 70°C for a special heat-stable polymerase, the TAQ enzyme from Thermus aquaticus, to add complementary bases (A, T, G, or C) to the single-stranded DNA and complete the extension to form a new double strand of DNA. This is called a thermal cycle. After this cycle, the tube will be heated to 95°C again for the next cycle. After one thermal cycle, one copy of DNA will become two copies. After about 21 cycles and 31 cycles, one million and one billion copies of the DNA will be formed, respectively. This entire process can be accomplished in less than an hour in an automatic thermal cycler. Theoretically, if a food contains one copy of salmonella DNA, the PCR method can detect the presence of this pathogen in a very short time. After PCR reactions, one still needs to detect the presence of the PCR products to indicate the presence of the pathogen. Four commercial kits for PCR reactions and detection of PCR products are briefly discussed in the following.

The BAX system (Qualicon, Inc., Wilmington, DE) for screening food-borne pathogens combines DNA amplification and automated homogeneous detection to determine the presence or absence of a specific target. All primers, polymerase, and deoxynucleotides necessary for PCR as well as a positive control and an intercalating dye are incorporated into a single tablet. The system works directly from an overnight enrichment of the target organisms. No DNA extraction is required. Assays are available for salmonella, E. coli O157:H7, listeria genus, and Listeria monocytogenes. The system uses an array of 96 blue LEDs as the excitation source and a photomultiplier tube to detect the emitted fluorescent signal. This integrated system improves the ease-of-use of the assay. In addition to simplifying the detection process, the new method converts the system to a homogeneous PCR test. The homogenous detection process monitors the decrease in fluorescence of a double-stranded DNA (dsDNA) intercalating dye in solution with dsDNA as a function of temperature. Following amplification, melting curves are generated by slowly ramping the temperature of the sample to a denaturing level (95°C). As the dsDNA denatures, the dye becomes unbound from the DNA duplex, and the fluorescent signal decreases. This change in fluorescence can be plotted against temperature to yield a melting curve waveform. This assay thus eliminates the need for gel-based detection and yields data amenable to storage and retrieval in an electronic database. In addition, this method reduces the hands-on time of the assay and reduces the subjectivity of the reported results. Further, melting curve analysis makes possible the ability to detect multiple PCR products in a single tube. The inclusivity and exclusivity of the BAX system assays reach almost 100% meaning that false positive and false negative rates are almost zero. The automated BAX system can now be used with assays for the detection of Cryptosporidium parvum and Campylobacter jejuni/coli and for the quantitative and qualitative detection of genetically modified organisms in soy and corn. The new BAX system is far more convenient than the old system in which a gel electrophoresis step was required to detect PCR products after thermal cycling.

The following two methods also have been developed to bypass the elec-trophoresis step to detect PCR products. These methods are called ''real-time PCR'' because they involve a solution in which a fluorescent signal increases if the target sequence is present in the solution. They rely on the use of fluorescent molecules and can directly measure the amplification products while amplification is in progress. The more target DNA in the solution, the sooner the number of PCR products will reach the detection threshold and can be detected since fewer thermal cycles are needed, compared to a solution with a smaller number of target DNA molecules. With the use of different fluorescent dyes in the same solution, several target DNA molecules can be studied simultaneously. This is called a multiplex PCR system.

The TaqMan system of Applied Biosystems (Foster City, CA) also amplifies DNA by a PCR protocol. However, during the amplification step a special molecule is annealed to the single-stranded DNA to report the linear amplification. The molecule has the appropriate sequence for the target DNA. It also has two attached particles. One is a fluorescent particle, and another one is a quencher particle. When the two particles are close to each other no fluorescence occurs. However, when the TAQ polymerase is adding bases to the linear single strand of DNA, it will break this molecule away from the strand (like the PacMan in computer games). As this occurs, the two particles will separate from each other, and fluorescence will occur. By measuring fluorescence in the tube, a successful PCR reaction can be determined. Note that the reaction and reporting of a successful PCR protocol occur in the same tube. The author's research team developed a TaqMan procedure to detect rapidly Yersinia enterocolitica in foods [18].

A new system called Molecular Beacon Technology (Stratagene, La Jolla, CA) was developed and can be used for food microbiology in the future [19]. In this technology, all reactions are again in the same tube. A Molecular Beacon is a tailor-made hairpin-shaped hybridization probe. The probe is used to attach to target PCR products. On one end of the probe there is attached a fluorophore, and on the other end a quencher. In the absence of the target PCR products the beacon is in a hairpin shape, and there is no fluorescence. However, during PCR reactions and the generation of target PCR products, the beacons will attach to the PCR products and cause the hairpin molecule to unfold. As the quencher moves away from the fluorophore, fluorescence will occur, and this can be measured. The measurement can be done as the PCR reaction is progressing, thus allowing "real-time" detection of target PCR products, and thus the presence of the target pathogen in the sample. This system has the same efficiency as the TaqMan system, but the difference is that the beacons detect the PCR products themselves, while in the TaqMan system they only report the occurrence of a linear PCR reaction and not the presence of the PCR product directly. By using molecular beacons containing different fluorophores, one can detect different PCR products in the same reaction tubes, and thus it is possible to perform "multiplex" tests of several target pathogens or molecules. The use of this technology is very new and not well known in food microbiology areas.

One of the major problems of PCR systems is contamination of PCR products from one test to another. Thus, if any PCR products from a positive sample (e.g., salmonella PCR products in a previous run) enter the reaction system of the next analysis, they may cause a false positive result. The Probelia system, developed by Institut Pasteur (Paris, France), attempts to eliminate PCR product contamination by substituting the base uracil for the base thymine in the entire PCR protocol. Thus, in the reaction tube there are adenine, uracil, guanine, and cytosine, and no thymine. During the PCR reaction, the resultant Probelia PCR products will be AUGC pairing and not the natural ATGC pairings. The PCR products are read by hybridization of known sequences in a microtiter plate. The report of the hybridization is by color reaction similar to an ELISA test in the microtiter system.

After one experiment is completed, a new sample is added into another tube for the next experiment. In the tube there is an enzyme, uracil-D-glycosylase (UDG), which will hydrolyze any DNA molecules that contain a uracil. Therefore, if there are contaminants from a previous run, they will be destroyed before the beginning of the new run. Before a new PCR reaction, the tube with all reagents is heated to 56° C for 15 minutes for UDG to hydrolyze any contaminants. During the DNA denaturization step, the UDG will be inactivated and will not act on the new PCR products containing uracil. Currently, Probelia can detect salmonella and Listeria monocytogenes from foods. Other kits under development include E. coli O157:H7, campylobacter, and Clostridium botulinum.

Theoretically, PCR systems can detect one copy of target pathogen DNA from a food sample (e.g., salmonella DNA). In practice, about 200 cells are needed to be detected by current PCR methods. Thus, even in a PCR protocol, bacteria in the food must be enriched for a period of time, e.g., overnight or at least 8 hours' incubation of food in a suitable enrichment liquid, so that there are enough cells for the PCR process to be reliable.

Besides the technical manipulations of the systems which can be complicated for many food product microbiology laboratories, two major problems need to be addressed: inhibitors of PCR reactions and the question of live and dead cells. In food, there are many enzymes, proteins, and other compounds that can interfere with the PCR reaction and result in false negatives.

These inhibitors must be removed or diluted. Since the PCR reaction amplifies target DNA molecules, even DNA from dead cells can be amplified, and thus food with dead salmonella can be declared as salmonella positive by PCR results. In this situation, food properly cooked but containing DNA of dead cells may be unnecessarily destroyed because of a positive PCR test. PCR can be a powerful tool for food microbiology once all the problems are solved, and analysts are convinced of its applicability in routine analysis of foods.

The aforementioned genetic methods are for detection of target pathogens in foods and other samples. They do not provide identification of the cultures to the species and subspecies level which is critical in epidemiological investigations of outbreaks or routine monitoring of occurrence of microorganisms in the environment. The following discussions will center around developments in the genetic characterization of bacterial cultures.

The RiboPrinter microbial characterization system (DuPont Qualicon, Wilmington, DE) characterizes and identifies organisms to genus, species, and subspecies levels automatically. To obtain a RiboPrint of an organism, the following steps are followed:

1. A pure colony of bacteria suspected to be the target organism (e.g., salmonella) is picked from an agar plate by a sterile plastic stick.

2. Cells from the stick are suspended in a buffer solution by mechanical agitation.

3. An aliquot of the cell suspension is loaded into the sample carrier to be placed into the instrument. Each sample carrier has space for eight individual colony picks.

4. The instrument will automatically prepare the DNA for analysis by restriction enzyme and lysis buffer to break the cell envelope, release and cut DNA molecules. The DNA fragments will go through an electrophoresis gel to separate DNA fragments into discrete bands. Lastly, the DNA probes, conjugate, and substrate will react with the separated DNA fragments, and light emission from the hybridized fragments is then photographed. The data are stored and compared with known patterns of the particular organism. The entire process takes eight hours for eight samples. However, at two-hour intervals, another eight samples can be loaded for analysis.

Different bacteria will exhibit different patterns (e.g., salmonella versus E. coli), and even the same species can exhibit different patterns (e.g., Listeria monocytogenes has 49 distinct patterns). Examples of numbers of RiboPrint patterns for some important food pathogens are: salmonella, 145; listeria, 89; Escherichia coli, 134; staphylococcus, 406; and vibrio, 63. Additionally, the database includes 300 lactobacillus, 43 lactococcus, 11 leuconostoc, and 34 pediococcus patterns. The current identification database provides 3267 RiboPrint patterns representing 98 genera and 695 species.

One of the values of this information is that in the case of a foodborne outbreak, scientists not only can identify the etiological agent (e.g., Listeria monocytogenes) but can pinpoint the source of the responsible subspecies. For example, in the investigation of an outbreak of Listeria monocytogenes, cultures were isolated from a slicer of the product and also from the drains of the plant. The question was: which source was responsible for the outbreak? By matching RiboPrint patterns of the two sources of L. monocytogenes against the foodborne outbreak culture, it was found that the isolate from the slicer matched the outbreak culture, thus determining the true source of the problem. The RiboPrinter system is a very powerful tool for electronic datasharing worldwide.

These links can monitor the occurrence of foodborne pathogens and other important organisms as long as different laboratories utilize the same system for obtaining the RiboPrint patterns.

Another important system concerns the pulsed-field gel electrophoresis patterns of pathogens. In this system, pure cultures of pathogens are isolated and digested with restriction enzymes, and the DNA fragments are subjected to a system known as pulsed-field gel electrophoresis which effectively separates DNA fragments on the gel (DNA fingerprinting). For example, in a foodborne outbreak of E. coli O157:H7, biochemically identical E. coli O157:H7 cultures can exhibit different patterns. By comparing the gel patterns from different sources, one can trace the origin of the infection or search for the spread of the disease and thereby control the problem.

In order to compare data from various laboratories, the Pulse Net System was established under the National Molecular Subtyping Network for Food-borne Disease Surveillance at the Centers for Disease Control and Prevention (CDC). An extensive training program has been established so that all the collaborating laboratories use the same protocol and are electronically linked to share DNA fingerprinting patterns of major pathogens. As soon as a suspect culture is noted as a possible source of an outbreak, all the collaborating laboratories are alerted to search for the occurrence of the same pattern to determine the scope of the problem and share information in real time.

There are many other genetic-based methods, but they are not directly related to food microbiology and are beyond the scope of this review. It is safe to say that many genetic-based methods are slowly but surely finding their way into food microbiology laboratories, and they will provide valuable information for quality assurance, quality control, and food safety programs in the future.

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