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. For 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.

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.

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.

Probes and Their Targets

Campylobacter jejuni: Ribosomal RNA

A probe that is specific for C. jejuni ribosomal RNA genes has been developed (86,87) and is available commercially. A pool of randomly selected and tested chromosomal fragments is also specific for C. jejuni, but the target has not been reported (83).

Escherichia coli: Heat-labile enterotoxin genes

The heat-labile enterotoxins (LT) of E. coli are a closely related group of proteins; they are distinguished from heat-stable enterotoxins (ST) by being immunogenic and are inactivated by heating at 60°C for 10 Min (31). The toxins stimulate adenylate cyclase (30) and can be detected by tissue culture assays of Chinese hamster ovary cells (30) or mouse Y-l adrenal cells (13). Using these tests, So et al. (102) localized and cloned the structural gene for LT; Dallas et al. (8) recloned a smaller fragment into plasmid pEWD299. Although there are several different genes for LT, as evidenced by their nucleotide sequences (56,73,103,110,111), they all share a significant amount of genetic similarity. The region of the LT genes chosen as a gene probe target is identical in each of these genes, so that all strains with the genetic potential to produce LTs should be detected.

The LT probe, eltA11, is a 20 base synthetic oligonucleotide that encodes amino acids 45-51 of the A subunit of the E. coli LT (111).

E. coli: Heat-stable enterotoxin genes

The heat-stable enterotoxin (ST) of E. coli is distinguished from LT (above) by heat stability and lack of immunogenicity. It can be detected by the suckling mouse bioassay (12) and acts by stimulating guanylate cyclase (22). There are at least two different types: ST I (also known as STa and STP) and ST II (also known as STb and STH). The latter toxin is not active in the infant mouse assay. These genes have been cloned and the nucleotide sequences of the region encoding STa and STb have been determined (74,82,101).

The STP probe is a 22 base synthetic oligonucleotide for the toxin type strain first isolated from pigs. It targets the region of the gene that encodes amino acids 4-12 of the toxin protein.

The STH probe is targeted to the ST elaborated by a strain of E. coli isolated from a human. The probe is also 22 bases long and targets the region of the STH  that encodes amino acids 19-26 of the toxin.

Both of these probes have been tested for their specificity, and data are available on their ability to detect a few ST-producing cells against a high level of ST-negative microorganisms (35). The reliability of the colony hybridization technique with oligonucleotide probes was tested by collaborative study, using pure cultures of strains harboring the STH or STP genes (36).

Enteroinvasive Escherichia coli (EIEC) and Shigella: Invasive gene

Some strains of E. coli invade colonic epithelial cells, multiply intracellularly, and spread intercellularly, causing a dysenteric enteritis similar to that caused by Shigella (15). However, an important difference is that the infectious dose for Shigella may be as low as 1-10 organisms, whereas 10⁸ EIEC cells are necessary to cause disease. A number of genetic determinants that encode virulence factors of EIEC and Shigella spp. are located on a large [220 kilobase (kb) pair] invasion plasmid (96). Loss of this virulence plasmid renders the bacterium avirulent (97). A 17 kb EcoRI fragment was used as a hybridization probe to detect invasive Shigella species and EIEC (4).

Small and Falkow (100) demonstrated that a 2.5 kb pair HindIII fragment of the large plasmid is required for invasion of human epithelial cells. Plasmid DNA involved in the invasion of HeLa cells by S. flexneri has also been cloned (61). These regions of the plasmid have been sequenced and are genetically similar (54). A probe from this region of the plasmid is specific for tissue culture cell-invasive EIEC and Shigella. Such probes also identify strains that are invasive in the guinea pig eye assay (98). Of 41 probe-positive isolates tested by the guinea pig method, 2 were negative, indicating that a few strains may be invasive in tissue culture assays but not in tests that require a greater number of pathogenic determinants (108). A synthetic probe of 18 bases has been constructed. Its target is within a gene that encodes for a virulence factor.

Enterohemorrhagic E. coli (EHEC): Shiga-like toxin (SLT) genes

Human illnesses ranging from simple diarrhea to hemorrhagic colitis and hemorrhagic uremic syndrome have been associated with strains of E. coli that produce moderate to high levels of Shiga-like toxins (SLTs). Strains of E. coli serotype O157:H7 are the most significant pathogens associated with hemorrhagic colitis; strains of serotype O26:H11 are also classified as EHEC. More than 50 other serotypes of E. coli that produce SLTs have been identified, but the correlation of these serotypes with disease is uncertain. Two related but distinct cytotoxins, SLT I and SLT II, have been characterized. Individual strains produce one or both cytotoxins. For example, E. coli O157:H7 produces SLT I, SLT II, or both, whereas O26:H11 produces only SLT I. The DNA sequences of SLT I and SLT II structural genes have been published, and analysis shows that SLT I has 99% homology with the Shiga toxin gene of S. dysenteriae type 1, but SLT II has only 60% homology (41). Two synthetic oligodeoxyribonucleotide probes were prepared from sequence data from the A-subunit regions of the SLT I and SLT II genes (nucleotides 473-490 and 472-490, respectively). HC agar (M62) (see ref. 105) was the selective medium chosen to screen isolates and foods for E. coli strains that carry the SLT gene because the growth of E. coli O157:H7 is less inhibited on HC agar than on other selective media. HC agar contains NaCl and a lower concentration of bile salts No. 3. Its plating efficiency of a strain of E. coli O157:H7 at 37 and 43°C for 17 h was similar to that of plate count agar. Plating efficiencies of other E. coli serotypes that carry the SLT gene have not been determined. The modified enrichment procedure of Doyle and Schoeni (14) is included in the method for detection of low level contamination of foods. For additional information about enterohemorrhagic strains of E. coli, see the review by Karmali (46).

Enterohemorrhagic E. coli (EHEC): O157:H7 serotype-specific probe

The fluorogenic MUG assay for E. coli is based on the activity of the -glucuronidase (GUD) enzyme, which is encoded by the uidA gene in E. coli. Although isolates of serotype O157:H7 are negative with the MUG assay, genetic studies have shown that this EHEC serogroup also contains uidA gene sequences for the GUD enzyme (21). Sequencing analysis has determined that the uidA gene of O157:H7 serotype contains several base mutations; therefore, it is not identical to the uidA gene of MUG assay (+) E. coli. Although the base mutations in the uidA allele of O157:H7 do not appear to be responsible for the absence of the MUG phenotype, one of the base changes was found to be conserved among the O157:H7 serogroup. An oligonucleotide probe, PF-27, directed to this base alteration was developed and determined to be specific only for EHEC isolates of serotype O157:H7. Other SLT-producing EHEC and other pathogenic E coli or enteric bacteria failed to hybridize with PF-27 (20).

Listeria monocytogenes: Invasion-associated protein (iap) and hemolysin (hly) genes

Of the seven Listeria species that have been isolated from a variety of foods, including dairy, vegetable, meat, and poultry products, only L. monocytogenes has been implicated in human disease. Genetic and physiological studies have incriminated an extracellular hemolysin as one of the virulence factors in L. monocytogenes (5,26,47). This hemolysin (also called listeriolysin O or alpha-listeriolysin) has been cloned and sequenced (11,55,64). Several oligonucleotides (including AD13) were constructed by using the sequence of the listeriolysin O gene (64) and can specifically identify L. monocytogenes in foods by colony hybridization (11,70).

A 5.3 kb DNA fragment encoding a 60 kilodalton (Kdal) protein (msp) associated with hemolytic activity has been cloned (24). Kuhn and Goebel (53) reported the cloning and sequencing of a gene (iap) whose product (a 60 Kdal protein) may be involved in the uptake of L. monocytogenes by nonprofessional phagocytes. Sequence analysis revealed that the msp and iap share extensive homology, which indicates that msp and iap may be the same gene (51). An internal region of this gene was sequenced (Datta, unpublished results) and a synthetic probe, AD07, was used to identify and enumerate L. monocytogenes in a number of foods (9,10,34). Thus, either AD07 (for the iap gene) or AD13 (for the hly gene) can be used to detect and enumerate L. monocytogenes in foods. To avoid false-negative results because of "silent" mutations in the gene (nucleotide changes that affect DNA probe binding but do not change the gene function), both probes should be used in combination (designated AD713).

Salmonella species:

Originally, several restriction endonuclease fragments selected randomly from the Salmonella chromosome were used as probes to identify members of the genus (23). Although these molecules served as specific probes, the role played by the target genes was never reported. More recently, probes were developed for regions of the bacterial ribosomal gene that are unique for salmonellae. These probes were used to develop a commercial kit that also uses a nonisotopic labeling and detection system (7).

Staphylococcus aureus: entB probe

Six groups (A, B, C₁, C₂, D, and E) of related enterotoxins associated with pathogenicity are elaborated by some strains of S. aureus and can cause symptoms of staphylococcal food poisoning if ingested (1). The genes for enterotoxins A, B, C₁, and E (entA, entB, entC₁, and entE) have been cloned and sequenced (2,3,6,43,85).

Three synthetic oligonucleotide probes were synthesized according to the sequence of the entB gene and used to test 210 strains of S. aureus (78). One probe was specific for entB; the others hybridized with strains producing enterotoxin C. The former probe was used to detect EntB-producing S. aureus in artificially contaminated crabmeat (Trucksess and Williams, manuscript in preparation). Although the nucleotide sequences of enterotoxin genes for groups A, C₁, and E are known, synthetic probes have not been reported.

Vibrio cholerae: Cholera toxin

The classical cholera enterotoxin (CT) is a major virulence factor in pathogenic strains of V. cholerae. The mechanism of action and immunological reactivity is quite similar to the LT of E. coli. Genes encoding this multisubunit protein were cloned and sequenced (57,58,63). Non-O1 V. cholerae enterotoxin genes are apparently similar to classical CT (33). Two sequences from the A subunit structural gene for production of the classical enterotoxin are used as probes to detect the CT gene: ctxA11 (bases 702-721) and ctxA12 (bases 718-735).

Vibrio parahaemolyticus: Thermostable direct hemolysin

An important foodborne pathogen often associated with seafood, V. parahaemolyticus can produce a thermostable direct hemolysin (TDH) (95), also referred to as the Kanagawa phenomenon-associated hemolysin (69). This phenotype is commonly associated with strains isolated from humans with gastroenteritis but rarely found in environmental isolates (44). It is not yet known if this hemolysin is a virulence factor, but epidemiological evidence suggests that it is. The gene for the hemolysin has been cloned and sequenced (45,75,106). The specificity of both the cloned probes (76) and a synthetic oligonucleotide probe, tdh3 (77) has been established. The tdh3 probe is 18 bases long and its target encodes amino acids 122-128 of the tdh gene.

Vibrio vulnificus: Cytotoxin/hemolysin

V. vulnificus has been implicated as a cause of human infections and septicemia. The primary source of infection appears to be raw or undercooked seafood, especially raw oysters (71). This lactose-positive vibrio produces a cytotoxin/hemolysin which was implicated as a virulence factor (28), and the gene has been cloned (109). A 3.2 kb DNA fragment carrying the structural gene for this protein is a specific probe for V. vulnificus (72) and has been sequenced (112). One synthetic probe (VV6) exhibited 100% specific for 166 laboratory and environmental strains of V. vulnificus (FDA Contract No. 223-84-2031, Task XIII).

Yersinia pseudotuberculosis: Invasive gene (INV-3)

A chromosomal gene of Y. pseudotuberculosis, inv, which plays an integral part in Yersinia pathogenicity, has been cloned and sequenced (38,39). Oligonucleotide probe INV-3, based on published inv sequence (39) is 21 nucleotide bases long and targeted to a region 200 base pairs away from the 5' terminus of the inv gene of Y. pseudotuberculosis (19). Tests of INV-3 using Southern and colony hybridizations were compared with HeLa cell invasion studies and shown to be specific only for invasive Y. pseudotuberculosis isolates. Although there are homologous sequences between the inv genes of Y. enterocolitica and Y. pseudotuberculosis, this homology is not detectable by INV-3.

Yersinia enterocolitica: Chromosomal invasion gene (PF-13)

The genes responsible for mammalian cell invasion are also carried on the chromosome in Y. enterocolitica (66). Unlike Y. pseudotuberculosis, however, Y. enterocolitica has two loci that encode the invasion phenotype. The inv locus, homologous to the inv gene of Y. pseudotuberculosis, allows high level invasion of several tissue culture cell lines, whereas the ail gene shows more host specificity (66). Analysis of Yersinia serotypes and species using cloned probes from inv and ail showed that all disease-causing isolates are tissue culture-invasive; all these isolates reacted with the AIL gene probe (67). The INV probe reacted with both tissue culture-invasive and noninvasive isolates; however, recent evidence suggests that the inv in these noninvasive strains may not be expressed. The oligonucleotide probe PF13 is targeted specifically to a region 60 base pairs away from the 3' terminus of the ail gene of Y. enterocolitica. The probe is 18 nucleotides in length. A comparison of colony and Southern hybridization studies of 150 yersiniae and non-yersiniae isolates and HeLa cell invasion studies showed that PF13 hybridized only with invasive Y. enterocolitica isolates (19).

Yersinia enterocolitica: Plasmid gene (SP-12)

All pathogenic Yersinia species carry a 42-48 Mdal plasmid (pYV), which encodes for many of the virulence-associated phenotypes (84). These include Ca²⁺-dependent growth, mouse lethality, cytotoxicity, Sereny reaction, production of V and W antigens, serum resistance, and production of outer membrane proteins (YOPs). The pYV plasmid of Y. enterocolitica was subcloned and the region encoding for HEp-2 cell cytotoxicity and Sereny reaction was identified and sequenced (91). A 24 base oligonucleotide probe, SP12, targeted to this region was shown to be specific for the virulence plasmid. The use of SP12 for detecting pathogenic Y. enterocolitica isolates in artificially inoculated foods was also evaluated (65).

For additional information on specific probes, contact the authors as follows:

At FDA Seafood Research Center, 22201 23rd Dr., S.E., Bothell, WA 98021:

• Walter E. Hill - C.jejuni; ribosomal DNA; E. coli LT and ST enterotoxin genes; S. aureus entB; Vibrio spp. probes.

At FDA, 200 C Street, S.W., Washington, DC 20204:

• Atin R. Datta - L. monocytogenes iap and hly genes
• Peter Feng - Yersinia spp. probes and O157:H7 serotype-specific probe
• Keith Lampel - E. coli (EIEC) and Shigella invasive gene probes; Salmonella probes
• William L. Payne - E. coli (EHEC) and Shiga-like toxin genes

In the future it may be possible to carry out DNA probe tests with one standardized condition. Unfortunately, the inherent differences in the length and sequence composition of oligonucleotide probes and the variable susceptibility of microorganisms to lysing conditions require the use of several buffers and hybridization and washing temperatures. The types of microorganisms to be tested dictate the media and growth conditions. To minimize any confusion about similar but slightly different conditions to be used with various gene probes, each procedure is listed separately in this chapter. Although this results in much repetition, each protocol is complete and can be followed step by step. However, it is recommended that all the method sections be read, since some concepts and techniques discussed in the context of a particular probe may be applied in the use of others. In addition, for reference, a procedure for the end-labeling of oligonucleotides is presented.

Four different techniques can be used for colony hybridization tests:

1. Direct plating of samples for enumeration.
2. Direct plating of cultures after enrichment to determine presence/absence.
3. Spotting of individual colonies or pure cultures for an additional hybridization assay to confirm a positive result from colony hybridization with a mixed culture.
4. Returning to a "master" replica plate to make pure cultures of positive colonies for further study and analysis.

The first three techniques differ as to when solid media are inoculated. In the first, aliquots of the homogenized sample are plated immediately after blending. In the second, plates are inoculated after aliquots of the homogenized sample have been incubated under selective conditions. Samples from the first and second techniques are plated onto selective agar media whenever such appropriate media are available. For the third technique, individual positive colonies are re-streaked and an additional colony hybridization test is conducted to ensure that the initial positive or negative results can be repeated. With the last technique, a pure culture can be obtained without selective enrichment, and additional microbiological tests requiring a pure culture can then be performed.

Control cultures

Strains that are positive or negative for the various probe tests must be properly stored and periodically tested for the appropriate phenotypic characteristics. A test methodology other than a gene probe must be used to independently verify the genotype of the control microorganisms. Control cultures must also be stored appropriately to minimize the possibility of genetic change. Usually, freezing liquid cultures at -70°C in 10-25% glycerol will suffice, except for Vibrio species, which are particularly sensitive to cold. Appropriate control strains have been listed, but other strains can be used if they have been properly characterized.

Preparation of controls

The importance of running controls cannot be overemphasized. Perhaps the most rigorous (and time-consuming) procedure for preparing controls is the inoculation of a food sample with a known number of positive (or negative) control cells. The food sample should have been previously tested by conventional microbiological techniques and shown to be free of the pathogen currently being sought. To prepare controls, streak out positive and negative cultures (or spot them in an array if pure cultures are being tested) onto the same medium as that used for food samples. Process the controls and the sample unknowns in an identical fashion at the same time. Such controls will signal if the cell growth, lysis, and hybridization steps have been successful. Although not serving as filter preparation controls, filter test strips will control for hybridization conditions. They can be prepared in advance by spotting control cultures into a repeated array. After cell lysis, cut the support into strips and add a strip to the hybridization mixture to control for conditions.

1. Materials and equipment
   1. Whatman 541 filter paper (82-88 mm diameter)
   2. Absorbent paper for pads, such as Whatman 3 or 3MM
   3. Plastic-backed absorbent paper, such as Kaydry, Benchkote, or Labmat
   4. X-ray film, such as Kodak XAR-2 or equivalent and appropriate developer and fixer

2. Media and reagents
   1. Butterfield's buffer (R11)
   2. Normal physiological saline (R63)
   3. MacConkey agar (M91)
   4. 50X Denhardt's solution (R17)
   5. Sonicated calf thymus or salmon-sperm DNA (R75)
   6. Hybridization mixture (6X SSC) (R35)
   7. HC agar (M62)
   8. Trypticase soy broth (modified) (M156)
   9. Novobiocin solution (R50)
   10. 20X SSC, pH 7.0 (R77)
   11. SSC (6X, 3X, 2X) (R77)
   12. 0.5 M EDTA, pH 8.0 (R20)
   13. 10 N Sodium hydroxide (R74)
   14. LPM agar (M81)
   15. Trypticase soy agar with yeast extract (TSAYE) (M153)
   16. Trypticase soy broth with yeast extract (TSBYE) (M157)
   17. 10X Kinase buffer (R37)
   18. Scintillation fluid (R68)
   19. 4 M Ammonium acetate (R1)
   20. 0.25 M Ammonium acetate (R2)

PROCEDURES

Enterotoxigenic Escherichia coli: Heat-Stable Enterotoxin (Human), Heat-Stable Enterotoxin (Porcine), and Heat-Labile Enterotoxin

Growth

1. Aseptically add 25 g of sample to 225 ml Butterfield's buffer and blend according to BAM procedures.
2. Spread-plate 0.1 ml directly from blender onto each of 2 MacConkey agar plates.
3. Make tenfold dilution from blender in Butterfield's buffer and spread 0.1 ml on each of 2 MacConkey plates. (Additional dilutions may be plated, depending on level of microbial contaminants suspected and health hazard concern with the specific pathogen.)
4. Incubate plates for 18-24 h at 35-37°C.

Filter preparation--NOTE: If pure cultures are to be sought by picking colonies from original master plate after "positive" areas are found, use sterile Whatman 541 filters. Wrap filters in aluminum foil and autoclave on liquid cycle.

1. Mark filters (with pencil) and agar plates so that they may be oriented correctly with respect to each other.
2. Carefully apply filter over surface of colonies. Remove any air pockets, using gentle pressure on glass spreading rod.
3. Lyse cells by transferring filter (colony side up) to petri dish containing absorbent paper that has been thoroughly wetted (but not soaked) with normal saline. Very carefully remove any air pockets. Store agar plates at 4°C if confirmation is necessary.
4. Microwave for 30 s at 600-700 W.
5. Repeat step 3, using filters wetted with 0.5 M NaOH in 1.5 M NaCl (final concentrations). If possible, transfer filters horizontally to minimize DNA flow across filter. Let sit 10 min.
6. Neutralize NaOH by transferring filters to absorbent filter wetted with 1.0 M Tris-HCl (pH 7) in 2.0 M NaCl (final concentrations). Let filters sit 5-10 min.
7. Filters may be used immediately or air-dried and stored in vacuum desiccator at room temperature for several months.

Hybridization

1. Prepare 50 ml of fresh hybridization mixture (6X SSC, 5X Denhardt's solution, 10 MM EDTA, pH 8.0) in plastic tube.
2. Boil 1.0 ml sonicated calf-thymus DNA for 5 min and add to hybridization mixture.
3. Dispense 5-10 Ml into petri dish to thoroughly wet colony hybridization filter as prepared above.
4. Calculate volume required to contain 10⁶ cpm of radioactive probe, allowing for half-life of 14.2 days. (NOTE: Do not use probes labeled more than 15 days previously.) Add this amount to each filter and mix briefly.
5. Incubate overnight at 37°C. This temperature is not critical but should be between 35 and 45°C for LT and ST probes.

(NOTE: Several filters may be processed in the same petri dish. See Kaysner et al. (48) for protocol.)

Washing

1. Remove spent radioactive hybridization solution, preferably by using disposable pipet. Dispose of radioactive waste properly.
2. Transfer radioactive filter to fresh petri dish and add about 10 Ml 6X SSC containing 0.1% SDS that has been prewarmed to 50°C.
3. Incubate 20 min at 50°C with occasional gentle agitation. Drain.
4. Remove used wash liquid and repeat steps 2 and 3, but without transferring to new petri dish.
5. Rinse filters briefly in 2X SSC at room temperature.
6. Air-dry filters on absorbent paper.

Autoradiography

1. Carefully tape radioactive filters, colony side up, to paper support and cover with plastic sheet. Qualitatively estimate amount of radioactivity bound to filter with a Geiger counter. This will help estimate exposure time (see below).
2. In darkroom with appropriate safelight, place X-ray film on top of plastic and place in film cassette with intensifying screens.
3. If Geiger counter reads a significant level (more than 5 cps), a 4-h film exposure may be sufficient. If less radioactivity is present, consider exposing film at -70°C overnight.
4. Let cassette warm somewhat before opening it in the darkroom. Use correct safelight.
5. Develop film according to manufacturer's instructions. Development may be stopped after 30 s to 4 min, when spots appear on film. Dry the film.

Controls

These control strains must have been confirmed for enterotoxin production by an appropriate non-probe assay, such as the suckling mouse test for ST or the mouse Y1 adrenal cell test for LT. Because the genes for these toxins are usually found on plasmids, control strains should be stored frozen at -70°C in 10% glycerol.

The following strains are suitable for use as controls:

Interpretation of results

1. Compare intensity of control spots with filters from sample.
2. Record number of positive colonies from each dilution.
3. Calculate concentration of ETEC in food sample.

Confirmation

1. Place film under master plate used to make that filter and locate positive colonies.
2. Pick colonies that correspond to darkened areas of film and perform tests for identification of E. coli on cultures made from those colonies.

Invasive Shigella Species and Escherichia coli

Growth--enumeration method

1. Aseptically add 25 g sample to 225 ml Shigella broth without novobiocin and blend according to BAM procedures.
2. Spread-plate 0.1 ml directly from blender onto each of 2 MacConkey agar plates.
3. Make tenfold dilution from blender in Butterfield's buffer and spread 0.1 ml on each of 2 MacConkey plates. (Additional dilutions may be plated, depending on level of microbial contaminants suspected and health hazard concern with specific pathogen.)
4. Incubate plates for 18-24 h at 35-37°C.

Growth--presence/absence method

1. Aseptically add 25 g sample to 225 ml Shigella broth without novobiocin contained in sterile 500 ml Erlenmeyer flask.
2. Shake contents at 37°C for 24 h.
3. Withdraw 0.1 ml aliquot at 0, 4, and 24 h; dilute in diluent buffer, and spread 0.1 ml of diluted cultures onto MacConkey agar plates. If total aerobic plate count is desired, also plate onto trypticase soy agar (TSA) plates. Incubate at 37°C overnight. NOTE: For 0-h aliquot, shake flask for 2 min before dilution.

Filter preparation--see NOTE under PROCEDURES, above.

1. Mark filters (with pencil) and agar plates so that they may be oriented correctly with respect to each other.
2. Carefully apply filter over surface of colonies. Remove any air pockets, using gentle pressure on glass spreading rod.
3. Lyse cells by transferring filter (colony side up) to plastic-backed paper such as Kaydry, Benchkote, or Labmat (nonabsorbent side up) wetted with 2.0 ml 0.5 N NaOH for 7 min. Very carefully remove any air pockets by repositioning filter. Store agar plates at 4°C if confirmation is necessary.
4. If possible, transfer filters horizontally to minimize DNA flow across filter to a pad wetted with 2.0 ml 1.0 M Tris-HCl, pH 7.4, for 2 min.
5. Repeat step 4.
6. Place filters onto pads wetted with 2.0 ml 1.0 M Tris-HCl in 1.5 M NaCl (final concentrations) for 2 min.
7. Filters may be used immediately or air-dried and stored in vacuum desiccator at room temperature for several months.

To prepare filters using a microwave, use filter preparation method for E. coli SLT.

Hybridization

1. Prepare 50 ml of fresh hybridization mixture (6X SSC, 5X Denhardt's solution, 10 MM EDTA, pH 8.0) in a plastic tube.
2. Boil 1.0 ml sonicated calf-thymus DNA for 5 min and add to hybridization mixture.
3. Dispense 5-10 Ml into petri dish to thoroughly wet a colony hybridization filter as prepared above.
4. Calculate volume required to contain 10⁶ cpm of radioactive probe, allowing for half-life of 14.2 days. (NOTE: Do not use probes labeled more than 15 days previously.) Add this amount to each filter and mix briefly.
5. Incubate overnight at 37°C. (This temperature is not critical but should be between 35 and 45°C for the invasive probe.)

NOTE: Several filters may be processed in the same petri dish. See Kaysner et al. (48) for protocol.

Washing

1. Remove spent radioactive hybridization solution, preferably by using disposable pipet, and dispose of radioactive waste properly.
2. Transfer radioactive filter to fresh petri dish and add about 10 Ml of 6X SSC containing 0.1% SDS that has been prewarmed to 54°C.
3. Incubate 1 h at 54°C with occasional gentle agitation. Drain.
4. Remove used wash liquid and repeat steps 2 and 3, but without transferring to new petri dish.
5. Rinse filters briefly in 2X SSC at room temperature.
6. Air-dry filters on absorbent paper.

Autoradiography--see procedure under Enterotoxigenic E. coli, above.

Controls

The following strains have been used successfully as controls for the inv gene probe:

Interpretation of results

1. Compare intensity of control spots with filters from sample.
2. Record number of positive colonies from each dilution.
3. Calculate concentration of organisms with invasion gene in food sample.

Isolation of suspected colonies

Match spots present on autoradiogram to area on plate from which filter was made. Pick colony and establish pure culture. Follow procedure described in Chapter 6 to isolate and identify any Shigella spp.

NOTE: Compositing procedures may be applied to this method (see Chapter 1, on sample handling).

The most difficult problem is deciding what dilutions are necessary for plating. Because the bacterial flora in each food varies, a fixed number for the dilutions would be inadequate. For foods with a low microbial flora, dilutions of 10², 10³, and 10⁴ are adequate, whereas foods with a high bacterial background, e.g., alfalfa sprouts, require higher dilutions (10³-10⁶). Duplicate plates should be made for each dilution.

Confirm any positive cultures by using the Congo Red assay (94).

Escherichia coli Shiga-Like Toxin (SLT)

Growth--standard procedure

1. Blend, according to BAM procedures, 25 g of sample in 225 ml Butterfield's buffer.
2. Spread-plate 0.1 ml of sample on each of 4 HC agar plates.
3. Incubate plates inverted for 24 h at 43°C (humidity incubator); include a plate with positive and negative strains as controls.

Growth--low levels expected

If very low levels of EHEC are suspected, prepare filters from enriched food samples, although this will make it impossible to obtain quantitative results.

1. Blend 25 g sample in 225 ml modified trypticase soy broth (mTSB).
2. Incubate with shaking (about 100 rpm) for 18-24 h at 37°C.
3. Dilute enrichment cultures in Butterfield's phosphate buffer and spread 0.1 ml of each dilution on duplicate HC agar plates.
4. Invert and incubate at 43°C for 24 h.
5. Select 2 plates of the same dilution with approximately 10³ colonies for preparing filters.

Growth--control strains and pure cultures for screening

NOTE: Store isolates in 10% glycerol at -70°C if possible.

1. Inoculate 5 ml brain heart infusion (BHI) broth or TSB with 0.6% yeast extract (TSBYE) and incubate overnight at 37°C with shaking.
2. Dilute overnight cultures and either spread-plate 0.1 ml containing 10²-10³ colonies or streak onto HC agar; alternatively, spot cultures in orderly asymmetric pattern with a transfer device such as a Replaclone (LAO Enterprises, Gaithersburg, MD) or sterile toothpicks.
3. Incubate overnight at 43°C.

Filter preparation--see NOTE under PROCEDURES, above.

1. Mark filters (with pencil) and agar plates so that they may be oriented correctly with respect to each other.
2. Carefully apply filter over surface of colonies. Remove any air pockets, using gentle pressure on glass spreading rod. Let sit 5 min.
3. Lyse cells by transferring filter (colony side up) to glass petri dish containing absorbent paper (such as Whatman No. 3) that has been thoroughly wetted (but not soaked) with 4-5 ml 0.5 N NaOH in 1.5 M NaCl (final concentrations). Very carefully remove any air pockets. Let stand for 5 min. Store agar plates at 4°C if confirmation is necessary.
4. Microwave for 30 s at 30% maximum power.
5. Transfer filter to glass petri dish containing absorbent paper wetted with 4-5 ml 1.0 M Tris-HCl (pH 7.0) in 2.0 M NaCl (final concentrations) for 5 min.
6. Filters may be used immediately or air-dried and stored in a vacuum desiccator at room temperature for several months.

Hybridization

1. Prepare 50 ml of fresh hybridization mixture (6X SSC, 5X Denhardt's solution, 10 MM EDTA, pH 8.0) in plastic tube.
2. Boil 1.0 ml sonicated calf-thymus DNA for 5 min and add to hybridization mixture.
3. Dispense 8-10 Ml into petri dish to thoroughly wet colony hybridization filter as prepared above. Let filter set at room temperature for 15 min.
4. Calculate volume required to contain 10⁶ cpm of radioactive probe, allowing for half-life of 14.2 days. (NOTE: Do not use probes labeled more than 15 days previously.) Add this amount to each filter and mix briefly.
5. Incubate overnight with gentle shaking at 37°C.

NOTE: Several filters may be processed in the same petri dish; however, this method has not been tested with this probe. See Kaysner et al. (48) for protocol.

Washing

1. Remove spent radioactive hybridization solution, preferably by using disposable pipet. Dispose of radioactive material properly.
2. Transfer radioactive filter to fresh 20 mm petri dish and rinse for 10 s in 30 ml 3X SSC prewarmed to 56°C. Drain.
3. Add 40 ml 3X SSC prewarmed to 56°C and incubate 1 h at 56°C with gentle agitation. Drain.
4. Repeat step 3.
5. Rinse filters briefly in 2X SSC at room temperature.
6. Air-dry filters on absorbent paper. Store under vacuum at room temperature if not used immediately.

Autoradiography--see procedure given under Enterotoxigenic E. coli, above.

Controls

Store control strains (and other pure cultures to be tested) preferably at -70°C in 10% glycerol or on BHI slants at room temperature. The following strains have been tested for the presence of the SLT gene by cytotoxin-neutralization assays and by colony hybridization with gene probes.

Interpretation of results

1. Compare intensity of control spots with filters from sample.
2. Record number of positive colonies from each dilution.
3. Calculate concentration of cells with SLT gene in food sample only for sample where enrichment was not used.

Confirmation

Pick probe-positive colonies from master plates (which were stored at 4°C) and confirm as E. coli. Serotype these isolates (17,60) and test for production of SLT by cytotoxicity assay (27) as modified by O'Brien (80).

Perform toxin neutralization assay according to O'Brien and LaVeck (79).

Growth and filter preparation--see procedures under E. coli Shiga-like toxin (SLT).

Hybridization and washing--see procedures under Yersinia pseudotuberculosis INV-3, but use 60°C as the optimal washing temperature.

Autoradiography--see procedure under Enterotoxigenic E. coli, above.

Controls--Available from Peter Feng, Division of Microbiological Studies (HFS-516), FDA, 200 C Street, S.W., Washington, DC 20204.

Listeria monocytogenes: Combination of Invasion-Associated
Protein (iap) and Hemolysin (hly) Gene Probes - AD713

Growth--enumeration method

1. Homogenize 25 g sample in 225 ml Listeria enrichment broth (see Chapter 10) and make dilutions in the same medium.
2. Plate 0.1 ml of each dilution onto LPM agar on duplicate plates and incubate 48 h at 37°C.
3. Count total number of colonies and pick some "blue" (presumptive Listeria) colonies for future characterization.

Growth--pure cultures

1. Grow pure cultures in TSBYE at 37°C for 24 h.
2. Spot cultures in regular array on TSAYE plates, using sterile needle or loop; to facilitate production of multiple filters, transfer 200 1 of liquid culture to wells of sterile microtiter plate. Then, using sterile replicating device, such as Replica Plater (Sigma:R2383) transfer cultures onto duplicate TSAYE plates.
3. Incubate 24 h at 37°C.

Filter preparation

1. For best results, select LPM plate with not more than 300 colonies.
2. Place properly marked Whatman 541 filters onto LPM plates (enumeration method) or TSAYE plates (for pure cultures) and press gently with stirring rod.
3. Let sit 5 min. Carefully lift filter and place into plastic petri dish on paper wetted with saline (about 3 ml), colony side up. Place cover on dish.
4. Microwave for 30 s on high (about 700 W).
5. Transfer filter paper to absorbent paper wetted with 0.5 N NaOH in 1.5 M NaCl (final concentrations) for 5 min.
6. Neutralize 5 min on paper wetted with 1.0 M Tris (pH 7) in 2.0 M NaCl (final concentrations).
7. Air-dry and store under vacuum if not used immediately.

Hybridization

1. Prepare 50 ml of fresh hybridization mixture (6X SSC, 5X Denhardt's solution, 10 MM EDTA, pH 8.0) in plastic tube.
2. Boil 1.0 ml sonicated calf-thymus DNA for 5 min and add to hybridization mixture.
3. Dispense 10-15 ml into petri dish to thoroughly wet a colony hybridization filter as prepared above.
4. Calculate volume required to contain 2-5 x 10⁶ cpm of end-labeled radioactive AD713 probe, allowing for half-life of 14.2 days. (NOTE: Do not use probes labeled more than 15 days previously.) Add this amount to each filter and mix briefly.
5. Incubate overnight at 37°C with gentle shaking.

Washing

1. Remove spent radioactive hybridization solution, preferably by using disposable pipet. Dispose of radioactive material properly.
2. Transfer radioactive filter to fresh petri dish and add about 10 Ml 3X SSC that has been prewarmed to 50°C.
3. Incubate 1 h at 50°C with occasional gentle agitation. Drain.
4. Remove used wash liquid and repeat steps 2 and 3, but without transferring to new petri dish.
5. Rinse filters briefly in 2X SSC at room temperature.
6. Air-dry filters on absorbent paper.

Autoradiography--see procedure given under Enterotoxigenic E. coli, above.

Controls: Use filters inoculated with L. monocytogenes and L. innocua.

Interpretation of results

1. Compare intensity of control spots with filters from sample.
2. Record number of positive colonies (dark spots) from each dilution.
3. Calculate concentration of L. monocytogenes in sample by using number of dark spots on filters prepared by enumeration method.

Confirm positive cultures with BAM procedures recommended for Listeria.

Staphylococcus aureus entB

Growth

1. Aseptically add 25 g of sample to 225 ml Butterfield's buffer and blend according to BAM procedures.
2. Spread-plate 0.1 ml directly from blender onto each of 2 Baird-Parker agar plates.
3. Make tenfold dilution from blender in Butterfield's buffer and spread 0.1 ml onto each of 2 Baird-Parker plates. (Additional dilutions may be plated, depending on level of microbial contaminants suspected and health hazard concern with the specific pathogen.)
4. Incubate plates 18-24 h at 35-37°C.

Filter preparation--see NOTE under PROCEDURES, above.

With this probe, nylon supports, such as Nylon 66 (Micron Separations Inc., Westborough, MA 01581), generate less nonspecific background than do Whatman 541 filters.

1. Mark filters (with pencil) and agar plates so that they may be oriented correctly with respect to each other.
2. Carefully apply filter over surface of colonies. Remove any air pockets, using gentle pressure on glass spreading rod.
3. Lyse cells by transferring filter (colony side up) to petri dish containing absorbent paper that has been thoroughly wetted (but not soaked) with 0.5 M NaOH in 1.5 M NaCl (final concentrations). Very carefully remove any air pockets. Store agar plates at 4°C if confirmation is necessary.
4. Microwave for 30 s at 600-700 W.
5. As in step 3, transfer filters to pads wetted with 0.5 M NaOH in 1.5 M NaCl (final concentrations). If possible, transfer filters horizontally to minimize DNA flow across the filter. Let sit 10 min.
6. Neutralize NaOH by transferring filters to absorbent filter wetted with 1.0 M Tris-HCl (pH 7) in 2.0 M NaCl (final concentrations). Let filters sit 5-10 min.
7. Filters may be used immediately or air-dried and stored in vacuum desiccator at room temperature for several months.

Hybridization

1. Prepare 50 ml of fresh hybridization mixture (6X SSC, 10X Denhardt's solution, 10 MM EDTA, pH 8.0) in plastic tube.
2. Boil 1.0 ml sonicated calf-thymus DNA for 5 min and add to hybridization mixture.
3. Dispense 5-10 Ml into petri dish to thoroughly wet a colony hybridization filter as prepared above.
4. Calculate volume required to contain 10 cpm of radioactive probe, allowing for half-life of 14.2 days. (NOTE: Do not use probes labeled more than 15 days previously.) Add this amount to each filter and mix briefly.
5. Incubate overnight at 60°C.

Washing

1. Remove spent radioactive hybridization solution, preferably by using disposable pipet. Dispose of radioactive material properly.
2. Transfer radioactive filter to fresh petri dish and add about 10 Ml 6X SSC containing 0.1% SDS that has been prewarmed to 65°C.
3. Incubate 20 min at 65°C with occasional gentle agitation. Drain.
4. Remove used wash liquid and repeat steps 2 and 3, but without transferring to new petri dish.
5. Rinse filters briefly in 2X SSC at room temperature.
6. Air-dry filters on absorbent paper.

Autoradiography--see procedure given under Enterotoxigenic E. coli, above.

Interpretation of results

1. Compare intensity of control spots with filters from sample.
2. Record number of positive colonies from each dilution.
3. Calculate concentration of cells with gene for enterotoxin B.

Confirm toxin production by using a rapid method, as listed in Appendix 1.

Vibrio cholerae ctxA11

Growth--pure cultures

1. Transfer individual isolates onto TSA with 2% NaCl.
2. Incubate about 18 h at 37°C.

Filter preparation--see NOTE under PROCEDURES, above.

1. Mark filters (with pencil) and agar plates so that they may be oriented correctly with respect to each other.
2. Carefully apply filter over surface of colonies. Remove any air pockets by using gentle pressure on glass spreading rod.
3. Lyse cells by transferring filter (colony side up) to petri dish containing absorbent paper that has been thoroughly wetted (but not soaked) with 3 ml normal saline. Very carefully remove any air pockets. Store agar plates at room temperature if confirmation is necessary.
4. Microwave for 30 s at 600-700 W.
5. Repeat step 3, using filters wetted with 0.5 M NaOH in 1.5 M NaCl (final concentrations). If possible, transfer filters horizontally to minimize DNA flow across filter. Let sit 10 min.
6. Neutralize NaOH by transferring filters to absorbent filter wetted with 1.0 M Tris-HCl (pH 7) in 2.0 M NaCl (final concentrations). Let filters sit 5-10 min.
7. Filters may be used immediately or air-dried and stored in vacuum desiccator at room temperature for several months.

Hybridization

1. Prepare 50 ml of fresh hybridization mixture (6X SSC, 5X Denhardt's solution, 10 MM EDTA, pH 8.0) in plastic tube.
2. Boil 1.0 ml sonicated calf-thymus DNA for 5 min and add to hybridization mixture.
3. Dispense 8-10 Ml into petri dish to thoroughly wet a colony hybridization filter as prepared above.
4. Calculate volume required to contain 10⁶ cpm of radioactive probe, allowing for half-life of 14.2 days. (NOTE: Do not use probes labeled more than 15 days previously.) Add this amount to each filter and mix briefly.
5. Incubate overnight at 37°C. This temperature is not critical but should be between 35 and 45°C.

NOTE: Several filters may be processed in the same petri dish. See Kaysner et al. (48) for protocol.

Washing

1. Remove spent radioactive hybridization solution, preferably by using disposable pipet. Dispose of radioactive waste properly.
2. Transfer radioactive filter to fresh petri dish and add about 10 Ml 6X SSC, containing 0.1% SDS that has been prewarmed to 45°C.
3. Incubate 20 min at 45°C with occasional gentle agitation. Drain.
4. Remove used wash liquid and repeat steps 2 and 3, but without transferring to a new petri dish.
5. Rinse filters briefly in 2X SSC at room temperature.
6. Air-dry filter on absorbent paper.

Autoradiography--see procedure given under Enterotoxigenic E. coli, above.

Controls

Strain	Reaction	Reference
ATCC 14033	Positive	99
E. coli H10407	Negative (high stringency)	99
E. coli H10407	Positive (low stringency)	99

Interpretation of results: Compare intensity of control spots with sample spots. Confirm positive cultures with BAM procedures recommended for V. cholerae.

Vibrio parahaemolyticus tdh3

The colony hybridization protocol is identical to that of V. cholerae, including the 45°C washing temperature. Confirm by performing those tests required to identify V. parahaemolyticus.

Plating medium is modified CPC agar (M98). The colony hybridization protocol is identical to that for V. cholerae, except that washing temperature is 60°C. Confirm by performing those tests required to identify V. vulnificus.

Growth--contaminated foods

1. Follow general instructions for sampling and blending detailed in Chapter 8.
2. Spread-plate 0.1 ml from appropriate dilutions on MacConkey agar and incubate.

NOTE: If samples are suspected to be heavily contaminated with other microflora, this level may be reduced by brief treatment with alkali, as demonstrated by Jagow and Hill (42).

Growth--pure culture testing for invasiveness and pathogenicity

1. Spot cultures onto TSA plates in regular array, using sterile needle or toothpick.
2. Incubate overnight at 25°C.

Filter preparation--see NOTE under PROCEDURES, above.

1. Mark filters (with pencil) and agar plates so that they may be oriented correctly with respect to each other.
2. Carefully apply filter over surface of the colonies. Remove any air pockets by using gentle pressure on glass spreading rod.
3. Lyse cells by transferring filter (colony side up) to petri dish containing absorbent paper that has been thoroughly wetted (but not soaked) with normal saline. Very carefully remove any air pockets. Store agar plates at 4°C if confirmation is necessary.
4. Microwave for 30 s at 30% maximum power.
5. Repeat step 3, using filters wetted with 0.5 M NaOH in 1.5 M NaCl (final concentrations) to denature the DNA. If possible, transfer filters horizontally to minimize DNA flow across filter. Let sit 5 min.
6. Neutralize NaOH by transferring filters to absorbent filter wetted with 1.0 M Tris-HCl (pH 7) in 2.0 M NaCl (final concentrations). Let filters sit 5 min.
7. Filters may be used immediately or air-dried and stored in vacuum desiccator at room temperature for several months.

Hybridization

1. Prepare 50 ml of fresh hybridization mixture (6X SSC, 10X Denhardt's solution, 10 MM EDTA pH 8.0) in plastic tube.
2. Boil 1.0 ml sonicated calf-thymus or salmon-sperm DNA (5 mg/ml) for 5 min and add to hybridization mixture.
3. Dispense 5-10 Ml into petri dish to thoroughly wet a colony hybridization filter as prepared above.
4. Prehybridize filters for 3-4 h at 45°C.
5. Calculate volume required to contain 10⁶ cpm of radioactive probe, allowing for half-life of 14.2 days. (NOTE: Do not use probes labeled more than 15 days previously.) Add this amount to each filter and mix briefly.
6. Incubate overnight at 37°C.

NOTE: Several filters may be processed in the same petri dish. See Kaysner et al. (48) for protocol.

Washing

1. Remove spent radioactive hybridization solution, preferably by using disposable pipet. Dispose of radioactive waste properly.
2. Transfer radioactive filter to fresh petri dish and add about 10 Ml 6X SSC containing 0.1% SDS that has been prewarmed to 58°C.
3. Incubate 30 min at 58 ± 2°C with occasional gentle agitation. Drain.
4. Remove used wash liquid and repeat steps 2 and 3, but without transferring to new petri dish.
5. Rinse filters briefly in 2X SSC at room temperature.
6. Air-dry filters on absorbent paper.
7. Optimal washing temperature is 58°C but may vary 1 or 2°C.

Autoradiography--see procedure given under Enterotoxigenic E. coli, above.

Controls

Strain	Reaction
Yp188 isolate 82-599	Positive
Ye133 (8081)	Negative

Interpretation of results

1. Compare intensity of control spots with filters from sample.
2. Record number of positive colonies from each dilution.
3. Calculate concentration of cells with invasive gene.

Yersinia enterocolitica Chromosomal Probe PF-13

The colony hybridization protocol is identical to that described for INV-3, except that the optimal wash temperature for this probe is 47°C.

Controls

Strain	Reaction
Ye133 (8081)	Positive
Yp188	Negative

The colony hybridization protocol is identical to that described for INV-3,except that the optimal wash temperature for this probe is 57°C.

Controls

End-Labeling of Oligonucleotides

1. Rehydrate lyophilized preparation of synthetic oligonucleotides with 0.25-1.0 ml water to yield stock solution with A₂₆₀ between 1 and 10 units (50-400 g/ml).
2. Dilute preparation and read at A₂₆₀. A reading of 1 corresponds to about 33 g/ml. The molecular weight (MW) of a nucleotide is about 330 daltons.
3. Calculate concentration of stock solution and prepare 10 M working solution. For example, the MW of a 22 base oligonucleotide is about 7260; thus, a 10 M solution is 72.6 g/ml (10 pmoles/l).
4. Mix 5 l of working solution with 2.5 l kinase buffer, 15 l water, 1.5 l gamma ³²P ATP (3000-7000 Ci/mmol) and 1 l (20 units/l) T4 kinase in plastic 500 l conical microcentrifuge tube on ice.
5. Mix well and incubate at 37°C for 1 h.
6. Add 2 l 0.5 M EDTA to stop reaction.
7. Add 1.6 l 4.0 ammonium acetate solution to bring ammonium acetate concentration to 0.25 M.
8. Equilibrate NACS PREPAC column with 0.25 M ammonium acetate and load reaction mixture onto column.
9. Wash column, using gravity or gentle pressure, with a minimum of 4 ml 0.25 M ammonium acetate to remove free ATP.
10. Elute bound DNA with 200 l aliquots of 4 M ammonium acetate, but do not force through column.
11. Collect 3 fractions in 500 l conical plastic centrifuge tubes.
12. Determine amount of radioactivity in each fraction by spotting 2 l onto paper and count by any method suitable for detecting beta decay. Most of the radioactivity is eluted in fractions 1 and 2. Use fraction 1, but if more counts are needed, fractions 1 and 2 can be pooled. Usually 1-2 x 10⁸ cpm is obtained if ATP of a specific activity of 3000-7000 Ci/mmol is used.
13. Store at -20°C. For best results use probe within 15 days of labeling.

Acknowledgments

We thank Ashok Chopra, Department of Microbiology, University of Texas Medical Branch, Galveston, TX, for information on the use of probe ctxA11. Drs. Mary Trucksess and Kristina Williams, FDA, Washington, DC, kindly provided unpublished information on the probe for staphylococcal enterotoxin.

References

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