Identification of Foodborne Bacterial Pathogens by Gene Probes
Fig 1
Fig 2
The identification of bacteria by DNA probe hybridization methods is based on the presence or absence of particular genes. This is in contrast to most biochemical and immunological tests that are based on the detection of gene products such as antigens or chemical end products of a metabolic pathway.
The physical basis for gene probe tests stems from the structure of DNA molecules themselves. Usually, DNA is composed of two strands of nucleotide polymers wound around each other to form a double helix.
These long nucleotide chains are held together by hydrogen bonds between specific pairs of nucleotides. Adenine (A) in one strand binds to thymine (T) in the complementary strand. Similarly, guanine (G) in one strand forms a hydrogen bond with cytosine (C) in the opposite strand. or a discussion of the structure of DNA and nucleic acid hybridization, see Watson et al. (107). An overview (49) of DNA hybridization technology gives a more detailed explanation of hybridization theory, sample preparation, labeling, and formats.
The hydrogen bonds holding the strands together can usually be broken by raising the pH above 12 or the temperature above 95°C. Single-stranded molecules result and the DNA is considered denatured. When the pH or temperature is lowered, the hydrogen bonds are reestablished between the AT and GC pairs, reforming double-stranded DNA. The source of the DNA strands is inconsequential as long as the strands are complementary. If the strands of the double helix are from different sources, the molecules are called hybrids and the process is termed hybridization.
A gene probe is composed of nucleic acid molecules, most often double-stranded DNA. It consists of either an entire gene or a fragment of a gene with a known function. Alternatively, short pieces of single-stranded DNA can be synthesized, based on the nucleotide sequence of the known gene. These are commonly referred to as oligonucleotides. Both natural and synthetic oligonucleotides are used to detect complementary DNA or RNA targets in samples.
Double-stranded DNA probes must be denatured before the hybridization reaction; oligonucleotide and RNA probes, which are single-stranded, do not need to be denatured.
Target nucleic acids are denatured by high temperature or high pH, and then the labeled gene probe is added. If the target nucleic acid in the sample contains the same nucleotide sequence as that of the gene probe, the probe will form hydrogen bonds with the target.
Thus the labeled probe becomes specifically associated with the target (Fig. 1). The unreacted, labeled probe is removed by washing the solid support, and the presence of probe-target complexes is signalled by the bound label.
In addition to DNA, probes and/or their targets can be made of RNA. A number of commercially available gene probe kits use synthetic DNA probes specific for ribosomal RNA targets. DNA:RNA and RNA:RNA hybrids are somewhat more thermally stable than DNA:DNA duplexes, but RNA molecules are quite labile at alkaline pH.
Fig. 1. A) Oligonucleotide of known sequence (derived from gene of known function) is end-labeled with radioactivity, using AT32P and bacteriophage T4 polynucleotide kinase. B) DNA probe is allowed to incubate with DNA extracted from a sample. If target DNA (immobilized on a solid support) contains sequences complementary to those of the probe, the probe (and its radioactive label) will bind with the sample DNA. For purposes of illustration, this schematic shows the two strands of DNA parallel to each other. In reality, they are wrapped around one another to form a double helix.
Colony Hybridization
DNA hybridization tests may be performed in many ways. One format, the colony hybridization assay (29,59), will be described here. Generally, an aliquot of a homogenized food is spread-plated on an appropriate agar.
After incubation, the colonial pattern is transferred to a solid support (usually a membrane or paper filter) by pressing the support onto the agar surface. Next, the cells are lysed in situ by a combination of high pH and temperature (0.5 M NaOH and/or steam or microwave irradiation), which also denatures and affixes the DNA to the support. The solid support with the attached target DNA is incubated with a 32P- or enzyme-labeled probe. The labeled probe DNA that fails to reform the double helix is removed by washing the probe-target complexes on the support at an appropriate temperature and salt concentration.
Great care must be taken to ensure that the washing temperature is correct; this parameter is usually determined empirically.
If the temperature of the washing solution is too high, all the hydrogen bonds between the probe and target may be broken, producing a false-negative result. If the washing temperature is too low, strands of DNA will not match up accurately, and noncomplementary strands may be formed, leading to a false-positive outcome.
If the temperature allows only accurately rejoined strands to remain together, the conditions are termed "high stringency." If the temperature is too low, so that mismatched strands exist, the stringency is low. For a review of hybridization using solid supports, see Meinkoth and Wahl (62).
The radioactive probe DNA that is bound to the target on the support is often detected by autoradiography. An X-ray film is placed over the support. Radioactive decays expose the film, so that when it is developed, black spots appear where cells are harboring the same gene as the probe (Fig. 2). If an enzyme-labeled probe is used, a chromogenic substrate is added. Where the probe-associated enzyme is present, a colored spot will develop. Each spot represents a bacterial colony that has arisen from a single cell.
The number of cells harboring the target gene in the original sample can be calculated by multiplying the number of spots by the dilution factor.
The first step in developing a gene probe assay is to decide what information is needed. If a particular taxonomic group is to be identified, the probe must be directed toward a gene or region of a gene that is conserved throughout a particular species or genus. On the other hand, one may want to know if a microorganism carrying a particular gene is present.
Table 1 lists probes that have been used or are of potential use for detecting bacterial pathogens in foods. In the section, "Probes and Their Targets," the development of each probe is described briefly along with what is known about the probe target and its significance. The first probes designed to detect all members of a taxonomic group were constructed by screening randomly cloned DNA fragments. As data on the evolution of ribosomal RNA nucleotide sequences accumulate, probes are being directed toward these targets. Conserved regions can be used to identify large taxons, whereas the variable regions may be unique for a particular genus or species. Furthermore, as a cell contains more than 1000 copies of ribosomal RNA, test sensitivity is increased, because fewer cells are required to produce a positive signal.
Refer to the second diagram above
DNA Hybridization
The identification of bacteria by DNA probe hybridization methods is based on the presence or absence of particular genes. This is in contrast to most biochemical and immunological tests that are based on the detection of gene products such as antigens or chemical end products of a metabolic pathway.
The physical basis for gene probe tests stems from the structure of DNA molecules themselves. Usually, DNA is composed of two strands of nucleotide polymers wound around each other to form a double helix.
These long nucleotide chains are held together by hydrogen bonds between specific pairs of nucleotides. Adenine (A) in one strand binds to thymine (T) in the complementary strand. Similarly, guanine (G) in one strand forms a hydrogen bond with cytosine (C) in the opposite strand. or a discussion of the structure of DNA and nucleic acid hybridization, see Watson et al. (107). An overview (49) of DNA hybridization technology gives a more detailed explanation of hybridization theory, sample preparation, labeling, and formats.
The hydrogen bonds holding the strands together can usually be broken by raising the pH above 12 or the temperature above 95°C. Single-stranded molecules result and the DNA is considered denatured. When the pH or temperature is lowered, the hydrogen bonds are reestablished between the AT and GC pairs, reforming double-stranded DNA. The source of the DNA strands is inconsequential as long as the strands are complementary. If the strands of the double helix are from different sources, the molecules are called hybrids and the process is termed hybridization.
A gene probe is composed of nucleic acid molecules, most often double-stranded DNA. It consists of either an entire gene or a fragment of a gene with a known function. Alternatively, short pieces of single-stranded DNA can be synthesized, based on the nucleotide sequence of the known gene. These are commonly referred to as oligonucleotides. Both natural and synthetic oligonucleotides are used to detect complementary DNA or RNA targets in samples.
Double-stranded DNA probes must be denatured before the hybridization reaction; oligonucleotide and RNA probes, which are single-stranded, do not need to be denatured.
Target nucleic acids are denatured by high temperature or high pH, and then the labeled gene probe is added. If the target nucleic acid in the sample contains the same nucleotide sequence as that of the gene probe, the probe will form hydrogen bonds with the target.
Thus the labeled probe becomes specifically associated with the target (Fig. 1). The unreacted, labeled probe is removed by washing the solid support, and the presence of probe-target complexes is signalled by the bound label.
In addition to DNA, probes and/or their targets can be made of RNA. A number of commercially available gene probe kits use synthetic DNA probes specific for ribosomal RNA targets. DNA:RNA and RNA:RNA hybrids are somewhat more thermally stable than DNA:DNA duplexes, but RNA molecules are quite labile at alkaline pH.
Fig. 1. A) Oligonucleotide of known sequence (derived from gene of known function) is end-labeled with radioactivity, using AT32P and bacteriophage T4 polynucleotide kinase. B) DNA probe is allowed to incubate with DNA extracted from a sample. If target DNA (immobilized on a solid support) contains sequences complementary to those of the probe, the probe (and its radioactive label) will bind with the sample DNA. For purposes of illustration, this schematic shows the two strands of DNA parallel to each other. In reality, they are wrapped around one another to form a double helix.
Colony Hybridization
DNA hybridization tests may be performed in many ways. One format, the colony hybridization assay (29,59), will be described here. Generally, an aliquot of a homogenized food is spread-plated on an appropriate agar.
After incubation, the colonial pattern is transferred to a solid support (usually a membrane or paper filter) by pressing the support onto the agar surface. Next, the cells are lysed in situ by a combination of high pH and temperature (0.5 M NaOH and/or steam or microwave irradiation), which also denatures and affixes the DNA to the support. The solid support with the attached target DNA is incubated with a 32P- or enzyme-labeled probe. The labeled probe DNA that fails to reform the double helix is removed by washing the probe-target complexes on the support at an appropriate temperature and salt concentration.
Great care must be taken to ensure that the washing temperature is correct; this parameter is usually determined empirically.
If the temperature of the washing solution is too high, all the hydrogen bonds between the probe and target may be broken, producing a false-negative result. If the washing temperature is too low, strands of DNA will not match up accurately, and noncomplementary strands may be formed, leading to a false-positive outcome.
If the temperature allows only accurately rejoined strands to remain together, the conditions are termed "high stringency." If the temperature is too low, so that mismatched strands exist, the stringency is low. For a review of hybridization using solid supports, see Meinkoth and Wahl (62).
The radioactive probe DNA that is bound to the target on the support is often detected by autoradiography. An X-ray film is placed over the support. Radioactive decays expose the film, so that when it is developed, black spots appear where cells are harboring the same gene as the probe (Fig. 2). If an enzyme-labeled probe is used, a chromogenic substrate is added. Where the probe-associated enzyme is present, a colored spot will develop. Each spot represents a bacterial colony that has arisen from a single cell.
The number of cells harboring the target gene in the original sample can be calculated by multiplying the number of spots by the dilution factor.
Target Selection
The first step in developing a gene probe assay is to decide what information is needed. If a particular taxonomic group is to be identified, the probe must be directed toward a gene or region of a gene that is conserved throughout a particular species or genus. On the other hand, one may want to know if a microorganism carrying a particular gene is present.
Probes to specific determinants of virulence are useful in assessing a risk to public health posed by bacterial contamination.
Table 1 lists probes that have been used or are of potential use for detecting bacterial pathogens in foods. In the section, "Probes and Their Targets," the development of each probe is described briefly along with what is known about the probe target and its significance. The first probes designed to detect all members of a taxonomic group were constructed by screening randomly cloned DNA fragments. As data on the evolution of ribosomal RNA nucleotide sequences accumulate, probes are being directed toward these targets. Conserved regions can be used to identify large taxons, whereas the variable regions may be unique for a particular genus or species. Furthermore, as a cell contains more than 1000 copies of ribosomal RNA, test sensitivity is increased, because fewer cells are required to produce a positive signal.
Refer to the second diagram above
Fig. 2. Aliquot of homogenized sample is spread-plated on appropriate medium (cross-hatched area) and incubated until colonies are formed.
Colonies are transferred by gentle contact to solid support such as a filter (hatched area). Colony cells are lysed in situ by high pH and/or steam or microwave irradiation, which immobilizes single-stranded target DNA. Filters are then incubated with a labeled gene probe. (In this figure, a radioactive label was used.)
Unbound probe is removed by washing the filter at a temperature that allows well-matched double strands to remain joined; poorly matched strands are separated. If DNA from a colony contains the same genetic information as the probe, that area of the filter will become radioactive. Radioactivity is observed as a dark spot on an X-ray film.
Count the spots to calculate the number of cells containing specific gene present in the original sample.
Probe Specificity
The relatively short length of synthetic oligonucleotide probes means that they are specific for particular regions of DNA. There is only about 1 chance in 15,000 that a sequence length of 18 bases would appear more than once in the E. coli genome. With a 22-base probe, the chance drops to about 1 in 4 million. To avoid mismatches that reduce specificity, filter washings are conducted at high stringency so that a single base-pair difference between target and probe could not result in hybridization and produce a negative result.
Such changes occur as the result of rare mutations. The use of two nonoverlapping probes would significantly reduce the probability of false negatives.
Construction of Probes
Recombinant DNA techniques have made gene probes possible.
Probe tests require preparations of relatively pure, specific segments of DNA.
The first probes were obtained by inserting these regions into plasmids and transforming the plasmids into the appropriate host cells to increase the amount of probe DNA.
Plasmids were purified, and in some cases the inserted fragments were isolated. These cloned, natural DNA probes served quite well, although a considerable amount of effort was required for their production and purification. Through the development of DNA sequencing and automated oligonucleotide synthesis, short (18-30 bases) DNA probes were produced in the laboratory by chemical means. The ready availability of probes considerably expanded their use and application.
Probe Labeling
For probes consisting of cloned DNA fragments, the nick translation method (89) for labeling DNA with radioactivity is very popular. Cloned DNA can also be labeled by a random priming technique (18). Several kits to perform these reactions are commercially available; however, these techniques are unsatisfactory for labeling short oligonucleotides. Oligonucleotide probes are usually labeled on the 5' end with 32P, using bacteriophage T4 polynucleotide kinase and gamma-AT 32P (88). Although radioactive gene probes seem to have the greatest sensitivity in colony hybridization procedures, they are a potential biohazard, and disposal of radioactive waste can be expensive.
Many schemes are being examined for the nonradioactive labeling of gene probes. Some of these techniques have been incorporated into commercial tests designed to signal the presence or absence of a particular gene. For example, alkaline phosphatase has been conjugated to synthetic oligonucleotides without affecting the kinetics or specificity of the hybridization reaction (40).
The Polymerase Chain Reaction
At present it is not practical to use gene probes to detect bacteria directly in foods.
Current methods require about 105-106 copies of the target sequence to yield a clear, positive result. To make this number of copies, cells are allowed to replicate in liquid media or to form colonies on agar plates. The growth period is usually overnight, adding 16-24 hours to the length of the test.
It is now possible to amplify specific DNA segments enzymatically to a million-fold in 1-3 hours. This process is called the polymerase chain reaction (PCR) (92). The reaction has been automated by using a thermostable enzyme and a programmable heating block (93). Because of the rapid amplification of target DNA, 1-day probe tests may be developed in the near future. A review of PCR has been published (16). PCR has been used to detect enteroinvasive E. coli and Shigella spp. (54), V. vulnificus (37) Hepatitis A virus (see Chapter 26), and V. cholerae (see Chapter 28) in foods.
Description of Probes and Their Development
The design and construction of gene probes requires careful scientific experimentation and a series of complex decisions.
A first step is to determine if the gene probe is to be targeted to a particular pathogenic strain or to an entire taxonomic group. A target must be chosen so that all of the microorganisms to be detected contain such a gene.
For probes designed to detect all members of a genus or species, ribosomal RNA has been a popular target because it contains both conserved and variable regions.
If a pathogenic strain is sought, a probe is usually targeted to a virulence factor gene responsible for causing disease. A considerable amount of research is needed to identify the genes involved and the role they play in pathogenesis.
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