Nucleic acid probe and method for the rapid detection of typhoid fever bacteria

This invention relates to a nucleic acid probe and method for the rapid detection of typhoid fever bacteria by use of a nucleic acid hybridization probe, equivalent to the DNA region encoding the Vi antigen of enteric bacteria such as Salmonella typhi, S. paratyphi C, or Citrobacter freundii, in a nucleic acid hybridization reaction with a clinical specimen containing typhoid fever bacteria.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
All publications or patents mentioned in this specification are herein 
incorporated by reference. 
This invention relates to a unique nucleic acid hybridization probe and 
method for the rapid detection of typhoid fever bacteria. 
2. Prior Disclosure 
Diarrheal diseases caused by enteric bacteria are still a major cause of 
illness and death worldwide, especially among infants and young children 
in developing nations. Also, these maladies are an important military 
problem in deployed soldiers. Although the incidence of diarrheal disease 
is highest in tropical countries, geography is not as important a factor 
as socioeconomic conditions; e.g. as manifested by drinking water purity, 
sewage disposal methods, and the availability of balanced diets. Some 
enteric diseases are short-lived, self-limiting and result in a mild 
gastroenteritis (e.g. certain Salmonella serotypes). In contrast, typhoid 
fever, caused by Salmonella typhi, is a prolonged, generalized, and 
usually serious infection of humans of all age groups. Similar enteric 
diseases are caused by related bacteria such as Salmonella paratyphi A, B, 
and C and by other Salmonella serotypes. 
All strains of Salmonella typhi and S. paratyphi C, as well as a few 
atypical but genetically related Citrobacter and Salmonella strains, are 
capable of synthesizing a capsular antigen termed Vi for virulence 
(Edwards, P. R., and W. H. Ewing, 1972, Identification of 
Enterobacteriaceae, 3rd Edition pages 146-207, Burgess Publishing Company, 
Minneapolis). This galactosamine uronic acid polymer (i.e. the Vi antigen) 
has been associated with the virulence of S. typhi (Felix, A., S. S. 
Bhatnagar, and R. M. Pitt, 1934, Observations on the Properties of the Vi 
Antigen of B. typhosus, Br. J. Exp. Pathol. 15:346-354; and Heyns, K., G. 
Kiessling, W. Lindenberg, H. Paulsen, and M. E. Webster, 1959, 
d-Galaktosaminuronsaure (2-Amino-2-desoxy-D-galakturonsaure) als Baustein 
des Vi-Antigens, Chem. Ber. 92:2435-2437). Two separate chromosomal loci 
necessary for Vi antigen expression, viaA and viaB, have been identified 
in genetic studies of S. typhi (Johnson, E. M., B. Krauskopf, and L. S. 
Baron, 1965, Genetic mapping of Vi and somatic antigenic determinants in 
Salmonella, J. Bacteriol. 90:302- 308; and Johnson, E. M. B., B. 
Krauskopf, and L. S. Baron, 1966, Genetic analysis of the viaA-his 
chromosomal region in Salmonella, J. Bacteriol. 92:1457-1463). The viaB 
region appears to encode the structural genes for this antigen (Johnson, 
E. M., B. Krauskopf, and L. S. Baron, 1965, Genetic mapping of Vi and 
somatic antigenic determinants in Salmonella, J. Bacteriol. 90:302-308). 
Analogous and presumably allelic chromosomal sites have been identified in 
S. paratyphi C (Snellings, N. J., E. M. Johnson, and L. S. Baron, 1977, 
Genetic basis of Vi antigen expression in Salmonella paratyphi C. J. 
Bacteriol. 131:57-62) and in some strains of Citrobacter freundii 
(Snellings, N. J., E. M. Johnson, D. J. Kopecko, H. H. Collins, and L. S. 
Baron, 1981, Genetic regulation of variable Vi antigen expression in a 
strain of Citrobacter freundii, J. Bacteriol. 145:1010-1017). Although the 
expression of the Vi antigen is relatively stable in S. typhi, Vi-positive 
Citrobacter strains exhibit a rapid, reversible transition between forms 
that express the Vi antigen and forms that appear not to express it, 
referred to as non-Vi or W forms (Baron, L. S., D. J. Kopecko, S. M. 
McCowen, N. J. Snellings, E. M. Johnson, W. C. Reid, and C. A. Life, 1982, 
Genetic and molecular studies on the regulation of a typical citrate 
utilization and variable Vi antigen expression in enteric bacteria, pages 
175-194, In Hollaender (Editor), Genetic Engineering of Microorganisms For 
Chemicals, Plenum Press, NY; and Snellings, N. J., E. M. Johnson, D. J. 
Kopecko, H. H. Collins, and L. S. Baron, 1981, Genetic regulation of 
variable Vi antigen expression in a strain of Citrobacter freundii, J. 
Bacteriol. 3 145 1010-1017). 
Proper chemotherapeutic treatment of typhoid or related enteric fever 
disease in many cases is only instituted following the proper 
identification of the causative agent. The standard biochemical and 
serological identification of enteric bacteria from fecal or blood 
specimens generally requires 24 to 48 hours even with the most up-to-date 
clinical microbiology facilities. The absence of these facilities in areas 
of military troop deployment and in underdeveloped countries prevents the 
proper epidemiological identification of diseases and the administration 
of appropriate chemotherapeutic regimens A rapid method, which could be 
utilized in remote, ill-equipped areas, for the identification of specific 
enteric bacteria would be of obvious benefit to mankind. Several 
scientific groups have developed deoxyribonucleic acid (i.e. DNA) 
hybridization techniques, disclosed in U.S. Pat. Nos. 4,358,535 (Falkow, 
et al.) and 4,139,346 (Rabbani), and specific immunological procedures for 
the rapid identification of bacteria viruses and other organisms in 
culture specimens. Thus, there are several basic concepts available around 
which one can design a rapid diagnostic detection tool. Notwithstanding 
these readily available data, it takes considerable ingenuity to develop a 
bacterial identification assay that is differentially specific, rapid and 
inexpensive, and which can he conducted in remote areas with little 
equipment. For these reasons, relatively few rapid diagnostic assays are 
broadly applicable. 
DNA Hybridization Procedures 
The bacterial chromosome is a double-stranded DNA molecule in which one DNA 
strand is chemically complementary and hydrogen-bonded to the other DNA 
strand. These strands can be separated and reannealed, to form a hybrid, 
with single DNA strands of another type. Nucleic acid hybridization is a 
term used to define the chemical reaction that occurs between two 
complementary and homologous DNA strands or between DNA and ribonucleic 
acid (i.e. RNA) as described in U. S. Pat. No. 4,358,535 
This biochemical methodology, which was developed over the past 20 years, 
has recently been applied to the detection of pathogenic bacteria in 
clinical specimens (Moseley, S. L., et al., J. Infect. Dis., 1982, 
145:863-869). This hybridization procedure requires: (1) a nucleic acid 
probe sequence that will specifically hybridize with a particular 
bacterial DNA sequence; and (2) clinical specimens to analyze for the 
particular pathogenic bacteria. The procedure involves: (1) preparing a 
labelled (i.e. detectable) nucleic acid probe; (2) inoculating the 
clinical specimens on nitrocellulose filters or other appropriate support 
material; (3) preparing the clinical specimens on the filter for 
hybridization; (4) conducting the hybridization reaction between the 
nucleic acid probe and the clinical specimen fixed on the support 
material; and, finally (5) detection of any specimens that bound the 
labelled probe DNA. These general steps are outlined in Table 1. 
The article (J. Infect. Dis. 145:863-869) referenced above employed 
radiolabeled probe DNA and used autoradiography to detect the reacted 
clinical specimens. Although this technique is very useful in the 
identification of certain pathogenic organisms, identification still 
requires 24 or more hours. There are presently available alternate methods 
to detect hybridized probe DNA; (e.g. one commercially available method 
employs a DNA probe labeled with biotinylated nucleotides which can be 
detected in a few hours. This methodology suggests that identification of 
S. typhi may be obtained within several hours, using a biotinylated DNA 
probe in conjunction with hybridization procedures. 
Basic Genetic Studies of the Vi Antigen 
The virulence (Vi) antigen is a capsular monosaccharide polymer of 
galactosamine uronic acid and it is produced by all strains of Salmonella 
typhi and Salmonella paratyphi C and by a few strains of Citrobacter 
freundii. This antigen appears to be essential for the intracellular 
survival of the bacterial host and, hence, it is an important virulence 
property. Previous genetic studies have been revealed that this antigen is 
encoded by two widely separated chromosomal loci designated viaA and viaB 
that are situated at analogous positions in the chromosomes of S. typhi, 
S. paratyphi C, and C. freundii. The ViaA locus is located near his 
(chromosomal minute 44) and the ViaB region is situated near mel (92 
minutes) on the chromosome. Certain strains of Citrobacter freundii 
exhibit an unusual, frequent, reversible expression of the Vi antigen 
(i.e. these cells undergo a reversible transition between full Vi antigen 
expression and no Vi antigen expression). Each cell is, thus, reversibly 
able to generate the alternate type. The basic genetic importance of this 
"expression switch" called for further study. Further genetic studies have 
demonstrated that the ViaB locus encodes the structural genes determining 
Vi antigen expression as well as the associated "expression switch" 
(Baron, L. S., et al., 1982, pages 175-194, in Genetic Engineering of 
Microorganisms for Chemicals, (Editor, A. Hollaender), Plenum Press, NY). 
These basic genetic studies, as described in the L. S. Baron, et al. 
article, were aimed only at studying the unusual expression switch and not 
at isolating a DNA probe for diagnostic detection. As mentioned 
previously, Vi antigen expression in enteric bacteria is controlled by two 
widely separated and distinct genetic regions termed viaA and viaB. In 
addition, the viaA gene region is normally present in some enteric 
organisms that do not synthesize a Vi antigen, e.g. E. coli and Salmonella 
typhimurium. DNA from this genetic region would not serve as a specific 
probe for Vi-expressing organisms. However, this point is not deemed 
obvious and is only known by a few scientists. Although one might guess 
that the viaB gene region might serve as a specific probe, the only way to 
be sure is to clone the appropriate DNA fragment and test it for 
specificity in DNA hybridization reactions, which involves extensive 
experimentation of the type disclosed by herein. 
SUMMARY OF THE INVENTION 
This invention is directed to a method for the rapid detection of typhoid 
fever bacteria by use of a unique nucleic acid hybridization probe, 
equivalent to the DNA region encoding the Vi antigen of enteric bacteria 
such as Salmonella typhi, Salmonella paratyphi C, or Citrobacter freundii, 
in a nucleic acid hybridization reaction with a clinical specimen 
containing typhoid fever bacteria. 
Another embodiment of this invention is directed to a nucleic acid probe, 
consisting of an 18 kilobase pair (kb; 1 megadalton equals 1.5 kb) nucleic 
acid segment, or any subset of these sequences) representing the viaB 
region of the Vi antigen encoding sequences, that can be used to detect 
diagnostically Salmonella typhi, the typhoid fever bacillus. All clinical 
isolates of Salmonella typhi are Vi-antigen-expressing and no other 
enteric bacterium is known to express the Vi antigen except very rare 
strains of Citrobacter freundii and strains of Salmonella paratyphi C, 
which are thought to be much less prevalent pathogens than S. typhi. Thus, 
reaction of a clinical specimen with a probe specific for the Vi antigen 
genes is highly diagnostic for typhoid fever bacilli. 
We have identified a DNA sequence that could be used to facilitate the 
diagnostic identification of Salmonella typhi, the causative agent of 
typhoid fever. All virulent S. typhi strains encode a relatively unique 
capsular antigen termed the virulence (Vi) antigen. Two distinct genetic 
loci, viaA and viaB, are involved in the synthesis of this antigen. The 
structural genes, located at viaB, were considered as a possible specific 
DNA probe. The viaB locus, contained in a recombinant cosmid, was 
subcloned to various plasmid vectors for this purpose. Selected 
viaB-region DNA fragments were then analyzed for specificity in DNA colony 
hybridization reactions with more than 170 strains representing a variety 
of enteric bacteria. An 8.6-kilobase EcoRl fragment was highly specific 
for the viaB gene region and was considered a good hybridization probe. 
This DNA probe should prove useful in rapid diagnostic assays set up to 
detect S. typhi in mixed bacterial samples (e.g., stools) within a few 
hours of specimen collection.

DETAILED DESCRIPTION OF THE INVENTION 
It is understood that the DNA probes described herein, for purposes of 
illustration, can h=converted to their corresponding RNA probes by well 
known techniques (e.g. the commercially available Riboprobe System.TM.. 
Materials and Methods 
Bacterial strains and plasmids. Bacterial strains and plasmids are listed 
in Tables 3 and 4. S. typhi WR4201 (ViaA.sup.+ ViaB.sup.+) expresses the 
Vi antigen; previously constructed derivatives WR4205 (Johnson, E M., B. 
Krauskopf, and L. S. Baron, 1965, Genetic mapping of Vi and somatic 
antigenic determinants in Salmonella, J. Bacteriol. 90:302-308) and WR4226 
(Snellings, N. J., E. M. Johnson, D. J. Kopecko, H. H. Collins, and L. S. 
Baron, 1981, Genetic regulation of variable Vi antigen expression in a 
strain of Citrbacter freundii, J. Bacteriol. 145:1010-1017) were used as 
DNA hybridization controls since they are ViaA-, ViaB.sup.+ and 
ViaAl.sup.+, ViaB.sup.-, respectively. Escherichia coli WR2376, a 
Vi-positive E. coli C600 recombinant carrying the viaB locus of C freundii 
WR7004 (Baron, L. S., et al., D. J. Kopecko, S. M. McCowen, N. J. 
Snellings, E. M. Johnson, W. C. Reid, and C. A. Life, 1982, Genetic and 
molecular studies on the regulation of atypical citrate utilization and 
variable Vi antigen expression in enteric bacteria, pages 175-194) was 
also used as a DNA hybridization control in some experiments. Salmonella 
strains from the Centers for Disease Control (CDC), Atlanta, GA, used to 
determine probe specificity included groups A, B, C.sub.1, C.sub.2, 
C.sub.3, D.sub.1, D.sub.2, E.sub.1 , E.sub.2, E.sub.3, E.sub.4, F, 
G.sub.1, G.sub.2, H, I, J, K, L, M, N, 0, P, Q, R, S, T, U, V, W, X, Y, Z, 
51, 52, 53, 54, 55, 66, and 67. Additional bacterial strains were obtained 
from the collection at the Walter Reed Army Institute of Research (WRAIR), 
Washington, DC. 
Media and culture conditions. Bacteria were grown at 37.degree. C. on 
nutrient agar or in Penassay or brain heart infusion broth (Difco 
Laboratores, Detroit, MI). Antibiotics were used at the following final 
concentrations: kanamycin, 20 mirograms per ml; tetracycline, 10 mirograms 
per ml; chloramphenicol, 20 micrograms per ml; spectinomycin, 25 
micrograms per ml; and ampicillin, 25 micrograms per ml. 
Vi antigen expression. Vi antigen-expressing bacterial colonies on agar 
media were identified microscopically by oblique illumination (Snellings, 
N. J., E. M. Johnson, D. J. Kopecko, H. H. Collins, and L. S. Baron, 1981, 
Genetic regulation of variable Vi antigen expression in a strain 
Citrobacter freundii, J. Bacteriol. 145:1010-1017). Vi antigen-expressing 
forms are seen as dense, bright, orange-tinted colonies which are readily 
distinguishable from the dull, translucent colonies of non-Vi forms. Vi 
antigen expression was verified by slide agglutination with rabbit 
antiserum prepared against Vi-encapsulated C. freundii WR7004 cells. An 
additional test for Vi antigen expression involved the sensitivity of Vi 
antigen-expressing cells to Vi-specific typing phage. A drop of Vi phage 
was spotted on an area of a nutrient agar plate that was heavily swabbed 
with a bacterial culture. After overnight incubation at 37.degree. C., 
cell lysis was observed in the spotted area only in the case of cells 
expressing the Vi antigen. 
Isolation and manipulation of DNA. Bacterial cells were grown at 37.degree. 
C. for 16 to 18 hours in Penassay broth. Plasmid DNA was isolated by a 
cleared lysis method with Triton X-100 detergent followed by plasmid 
purification on cesium chloride density gradients, (Kupersztoch, Y.M. and 
D.R. Helinski, 1973. A catenated DNA molecule as an intermediate in the 
replication of the resistance transfer factor R6K in Escherichia coli, 
Biochem. Biophys. Res. Commun. 54:1451-1459). Digestion of DNA with 
restriction endonucleases was carried out under the conditions specified 
by the vendor (New England Bio. Labs., Inc., Beverly, MA; International 
Biotechnologies, Inc., New Haven, CT). Plasmids and restriction 
endonuclease-generated DNA fragments were resolved and analyzed by 
horizontal gel electrophoresis (International Biotechnologies, Inc.) in 
0.7 to 2.0% agarose (SeaKem; FMC Corp., Maine Colloids Div., Rockland, ME; 
International Biotechnologies, Inc.) prepared in TBE buffer (89 mM Tris, 
pH 8.3, 2.5 mM EDTA, 89 mM boric acid). We visualized DNA bands by 
staining the gel in 0.5 micrograms of aqueous ethidium bromide per ml and 
then illuminating it with a 300-nm UV light source (Fotodyne, New Berlin, 
WI). 
Recombinant plasmids were constructed in vitro by ligation, with T4 DNA 
ligase, of endonuclease-linearized vector DNA to endonuclease-generated 
DNA fragments (New England Bio. Labs.) at 17.degree. C. for 16 to 18 hours 
with the buffer described by Maniatis, et al. (1982, Molecular cloning. A 
laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). 
E. coli HB101 cells were prepared for transformation with plasmid DNA by 
the method of Kushner, (Kushner, S. R., 1978. An improved method for 
transformation of Escherichia coli with ColEl derived plasmids, pages 
17-23. In H. W. Boyer and S. Nicosia (edition), Genetic engineering, 
Elsevier/North-Holland Biomedical Press. Amsterdam). 
Preparation of .sup.32 P-labeled DNA probes. To purify DNA fragments for 
use as probes in hybridization experiments, we digested plasmids with the 
selected restriction endonucleases and resolved the resulting fragments by 
agarose gel electrophoresis. After ethidium bromide staining of the gel, 
the appropriate DNA band was cut out and the DNA was electroeluted with 
(i) a concentrator (model 1750; ISCO, Lincoln, NE) or (ii) a dialysis 
membrane filled with the agarose slice and TE buffer (0.01 M Tris, pH 8.0, 
0.001 M EDTA) with 0.1 .times.TBE buffer surrounding the membrane (100 V 
for 2 hours followed by reversed current for 2 minutes), (Maniatis, T., E. 
F. Fritsch, and J. Sambrook, 1982, Molecular cloning. A laboratory manual. 
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Fragments were 
further purified by another round of agarose gel electrophoresis followed 
by electroelution. Vi antigen gene locus fragments were radiolabeled in 
vitro by nick translation, (Rigby, P. W. J., M. Dieckmann C. Rhodes, and 
4P. Berg, 1977. Labeling deoxyribonucleic acid to high specific activity 
in vitro by nick translation with DNA polymerase. I, J. Mol. Bio. 
113:237-251) with a kit from New England Nuclear Corp., Boston, MA. 
(Alpha-.sup.32 P dCTP, 3,000 Ci/mmol). After 1 hour at 14.degree. C., 6 
microliters of 0.3 M EDTA was added to terminate the reaction. 
Unincorporated nucleotides were separated from labeled DNA by 
centrifugation through a 1-ml Sephadex G-50 column equilibrated and run 
with 0.2% sodium dodecyl sulfate (SDS)-0.1 M NaCl in TE 16 buffer. 
Specific activity of the probe was usually 2 .times.10.sup.8 cpm/microgram 
of probe DNA. 
Filters for in situ colony hybridization. Pure bacterial cultures were 
grown overnight and transferred by toothpick to an 82-mm diameter 
nitrocellulose filter (BA 85; Schleicher & Schuyll, Inc., Keene, NH; HAHY 
082 50; Millipore Corp., Bedford, MA) layered on MacConkey agar. 
Generally, 15 to 50 cultures were inoculated on each nitrocellulose 
filter. After 3 to 6 hours of incubation at 37.degree. C., the filters 
were removed from the agar and the attached cells were lysed with 0.5 M 
NaOH and prepared by the method described by Moseley, et al., (Moseley, S. 
L., P. Echeverria, J. Seriwatana, C. Tirapat, W. Chaicumpa, T. 
Sakuldaipeara, and S. Falkow, 1982. Identification of enterotoxigenic 
Escherichia coli by colony hybridization using three enterotoxin gene 
probes, J. Infect. Dis. 145:863-669). These nitrocellulose filters were 
then transferred face up for one 1 minute each to a series of three paper 
filters each saturated with 1.0 M ammonium acetate and 0.02 M NaOH. After 
10 minutes on a fourth change of the latter solution, nitrocellulose 
filters were air dried and the DNA was fixed by incubation at 70.degree. 
C. for 2 hours in vacuo. 
In addition to nitrocellulose, 541 paper (Whatman, Inc., Clifton, NJ) was 
used as a solid support for DNA hybridizations. The Whatman 541 papers 
were prepared by the method described by Maas, (Maas, R., 1983. An 
improved colony hybridization method with significantly increased 
sensitivity for detection of single genes, Plasmid 10:296-298). An 82-mm 
circular piece of Whatman 541 paper was placed over colonies that had been 
inoculated onto a nutrient agar plate and incubated at 37.degree. C. 
overnight. After approximately 15 minutes, the Whatman 541 paper was 
peeled off and placed colony side up on a paper filter saturated with 0.5 
M NaOH-1.5 M NaCl (lysing solution), steamed for 3 minutes, immersed in 
fresh lysing buffer for 1 minute, immersed in 1 M Tris (pH7)-2 M NaCl 
(neutralization solution) for 4 minutes, and air dried. Prehybridization 
of Whatman 541 paper is not necessary; the hybridization experiments were 
carried out in the same manner as that described for nitrocellulose. In 
addition, the probe could be removed from Whatman 541 paper by washing in 
0.5 M NaOH for 30 minutes and then washing in 2 .times.SSC (1 .times.SSC 
is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% SDS for 30 minutes; after 
air drying, hybridization could be repeated as described above. 
Hybridization. The solution for prehybridization and hybridization 
consisted of 50% formamide, 5 .times.SSC 0.1% SDS, 1 mM EDTA, and 
1.times.Denhardt solution (0.02% Ficoll, Pharmacia Fine Chemicals, 
Piscataway, NJ; 0.01% polyvinylpyrrolidone; 0.02% bovine serum albumin). 
Nitrocellulose filters, prepared as described above, were incubated for 2 
to 4 hours in prehybridization solution containing 50 micrograms of 
heat-denatured, sonicated salmon sperm DNA per ml. The filters were then 
transferred to fresh hybridization solution containing labeled probe DNA 
(10.sup.6 cpm) and 50 micrograms of heat-denatured, sonicated salmon sperm 
DNA per ml. The probe DNA was denatured with alkali as described by Hill 
and Payne, (Hill, W. E., and W. L. Payne, 1984. Genetic methods for the 
detection of microbial pathogens. Identification of enterotoxigenic 
Escherichia coli by DNA colony hybridization:collaborative study, J. 
Assoc. Off. Anal. 
Chem. 67:801-807) or by boiling for 10 minutes. The filters were hybridized 
overnight at 37.degree. C.. Excess hybridization mixture was removed, and 
the filters were washed once in 5.times.SSC-0.1% SDS at room temperature 
for 15 minutes, then three times in 2.times.SSC-0.1% SDS at 65.degree. C. 
for 15 minutes each, and finally three times in 0.1 x SSC-0.1% SDS at 
65.degree. C. for 15 minutes each. Hybridized filters were air dried, and 
autoradiograms were exposed for 4 to 18 hours at -80.degree. C. with Kodak 
XAR film and regular intensifying screens. 
As a part of the plasmid mapping studies, the Southern blot hybridization 
technique (Southern, E. M., 1975. Detection of specific sequences among 
DNA fragments separated by gel electrophoresis, J. Mol. Biol. 98:503-517) 
was used to transfer DNA from an agarose gel onto a nitrocellulose filter 
in 6 x SSC. Probe hybridization to the Southern blots was carried out as 
described by Maniatis, et al. (Maniatis, T., E. F. Fritsch, and J. 
Sambrook, 1982. Molecular cloning. A laboratory manual, Cold Spring Harbor 
Laboratory, Cold Spring Harbor, NY). 
Studies of probe hybridization sensitivity. A minifold II apparatus 
(Schleicher & Schuell) was used to deposit 10-fold dilutions of overnight 
bacterial cultures onto nitrocellulose filters. We prepared dilutions in 
0.9% saline and plated them on nutrient agar to obtain viable counts. 
These filters were processed and hybridized in the same manner as for 
colony hybridization studies, as described above. In an attempt to 
increase sensitivity, 10% dextran sulfate or increased amounts of probe 
DNA (10.sup.7 to 108 total cpm) or both were added to the hybridization 
mixture in some experiments. Kodak XAR film was exposed at -80.degree. C. 
for various times from 18 to 72 hours. 
Construction of DNA Probes Specific for Vi Antigen Structural genes 
Utilizing basic genetic and epidemiological data obtained over the past 50 
years or so on Vi antigen organization and the absolute correlation of the 
Vi antigen with all strains of S. typhi, we decided to develop a rapid 
detection system for S. typhi using a DNA hybridization assay and a DNA 
probe specific for the Vi gene re9ion. -n a previous study, chromosomal 
DNA from E. coli WR2376, which contained the chromosomally integrated C. 
freundii WR7004 genes encoding Mel.sup.30 (melibiose utilization) and the 
adjacent Vi antigen structural genes (i.e., the viaB locus), was partially 
digested with endonuclease PstI and the resulting material was cosmid 
cloned into the vector plasmid pHC79. One recombinant cosmid, pWR75, 
contained a 31-kb insert and expressed both tetracycline resistance and 
the Vi antigen in E. coli HB101, which normally contains functional viaA 
sequences, Baron, L. S., D. J. Kopecko, S. M. McCowen, N. J. Snellings, E. 
M. Johnson, W. C. Reid, and C. A. Life, 1982, Genetic and molecular 
studies on the regulation of atypical citrate utilization and variable Vi 
antigen expression in enteric bacteria, pages 175-194. In Hollaender 
(Editor), Genetic engineering of microorganisms for chemicals, Plenum 
Press, NY. In further studies aimed at investigating the reversible nature 
of Vi antigen expression, we subcloned Vi antigen genes from pWR75 into 
the single-copy plasmid pDPT429 by using a partial EcoRl digest of both 
plasmids. One resulting recombinant plasmid, pWR80, was isolated, which 
has a 29-kb fragment from pWR75 inserted into pDPT429 (Baron, et al., 
Abstr. Annu. Meet. Am. Soc. Microbiol., 1983). We used Vi 
antigen-expressing plasmid pWR80 as our beginning material to identify and 
study potential Vi gene-specific DNA probes. Initially, we reduced the 
insert by partially digesting pWR80 with EcoRl and inserting an 18-kb 
fragment into the EcoR1 site of the broad-host-range vector pRK290 to 
generate pWR122, a Vi antigen-expressing recombinant plasmid. This 18-kb 
viaB DNA insert in pWR122 consists of two EcoRl fragments, which we 
designated EcoR1-A and EcoR1-B (8.6 and 9.4 kb, respectively). Since 
vector pRK290 was derived from Pseudomonas sp., Ditta, G., S. Stanfield, 
D. Corbin, and D. R. Helinski, 1980, Broad host range DNA cloning system 
for gram-negative bacteria; construction of a gene bank of Rhizobium 
meliloti. Proc. Natl. Acad, Sci, U.S.A. 77:7347-7351, it was hoped that 
this vector would not share homology with enteric bacteria. HoweYer, when 
.sup.32 P-labeled pWR122 was used as a probe, it hybridized weakly to DNAs 
of some E. coli and Shigella strains, and further cloning of the insert 
was necessary. The 18-kb viaB insert of pWR 122 was then cloned into 
pACKCl (a small, amplifiable ColEl derivative vector) by ligation of 
EcoR1-digested pWR122 and pACKCl, resulting in the construction of a Vi 
antigen-expressing recombinant plasmid, pWR127. We separately subcloned 
EcoR1-A and EcoR1-B, the two fragments of the viaB region, into the 
vectors pBR325 and pACKCl, respectively, to construct pWR141 and pWR137. 
Cells harboring plasmids pWR137 or pWR141 do not express the Vi antigen. 
In all of the cloning studies, we assessed Vi antigen expression by using 
the three methods described above. Before further subdividing these two 
viaB gene fragments, we attempted to assess their hybridization 
specificity. 
Our method for the detection of Salmonella typhi and other related bacteria 
capable of expressing the Vi antigen comprises, allowing a clinical 
specimen of the bacteria to react with a DNA probe, consisting of the ViaB 
portion of the DNA regions encoding the Vi antigen, in a DNA hybridization 
reaction with the clinical specimen. The DNA probe used in our method is 
typically a linear DNA fragment, having a molecular size equal to or less 
than 18,000 nucleotide base pairs, containing the ViaB gene sequences of 
Citrobacter freundii, Salmonella typhi, or other related enteric bacteria 
expressing the Vi antigen. It should be noted that virtually any plasmid 
vector could h=used to clone the Vi antigen gene sequences. The use of 
those vectors described above and listed in Table 3 are illustrative were 
selected for the convenience of applicants without any implied limitation 
on the practice of this invention. Also, one could isolate Vi gene 
specific sequences from either S. typhi, S. paratyphi C, or other rare 
isolates of Vi antigen-expressing enteric bacteria. We used C. freundii as 
a source of the Vi genes because of the experimental ease of this system. 
Alternatively, one could synthesize an oligonucleotide probe based on the 
specific DNA sequences within the 18 kb ViaB gene segment described above. 
Testing of Vi DNA Probe for Specificity 
The DNA of plasmids pWR80, pWR122, and the vector pRK290 was radiolabelled 
with 32P by the conventional nick translation procedure (Maniatis, T., et 
al., 1982, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor 
Labs., NY). Cells of tester bacterial strains were implanted onto 
nitrocellulose filter paper. The loaded test filters with positive and 
negative experimental control spots were reacted by standard DNA filter 
hybridization reactions (Maniatis, T., et al., 1982, Molecular Cloning--A 
Laboratory Manual, Cold Spring Harbor Labs., NY, see Table 1) to each 
above radiolabeled DNA probe. Finally, the hybridized filters were exposed 
to X-ray film to detect positive hybridization reactions. The results 
shown in Table 2 were obtained. Whole plasmid probe pWR80, containing 29 
kilobases of Citrobacter DNA, reacted with the appropriate ViaB.sup.+ 
control strains but also reacted undesirably and strongly with many 
Shigella strains. Next, the recombinant plasmid pWR122 (consisting of 
pRK290 plus 18 kb of ViaB gene sequences) was tested and proved to be a 
good hybridization probe. pWR122 reacted strongly with all Vi.sup.+ 
control strains, did not react with several control Vi.sup.- Salmonella 
typhimurium strains, and showed differentially (i.e. can distinguish from 
a strong reaction) minor reaction, with certain Shigella strains. Further 
studies (Table 2) showed that the pRK290 plasmid vector probe alone 
reacted slightly with these same Shigella strains. This result was 
unexpected since pRK290 is a Pseudomonas plasmid and was thought to have 
no homology to typical enteric bacterial strains. However, these latter 
data convincingly demonstrate that the ViaB gene sequences in pWR122 are 
hybridizing strongly with the analogous ViaB sequences in the tester 
bacteria and that the slight reaction between the pWR122 probe and certain 
Shigella strains is due to the pRK290 vector sequences. Thus, pRK290 can 
not be used together with the ViaB gene sequences as a whole plasmid 
probe. 
To circumvent this slight crossreaction, we assayed the EcoR1-A and EcoR1-B 
restriction fragments of the cloned ViaB gene sequences of pWR122 for 
their potential use as a highly specific DNA probe. Colony hybridization 
experiments, as described above, were conducted to determine if either the 
EcoR1-A or EcoR1-B fragments of the viaB region could be used as 
hybridization probes for detecting the presence of the Vi gene locus in 
the test bacterial strains. Bacterial DNA was fixed on nitrocellulose 
filters and probed with .sup.32 P-labeled DNA as described above. Several 
positive and negative controls were included on each filter. C. freundii 
WR7004, from which the probe Vi antigen genes were originally cloned, 
served as one positive control. S. typhi WR4201 was always included as a 
typical Vi antigen-expressing typhoid strain. S. typhi WR4205 contains a 
mutation in the viaA region but has an intact viaB locus. It, therefore, 
was used as a positive control for the presence of Vi structural genes in 
S. typhi. However, S. typhi WR4226 contains an intact viaA region, but the 
viaB locus has been replaced by S. typhimurium chromosomal DNA; this 
created a ViaB.sup.- phenotype, and thus, strain WR4226 served as a 
negative S. typhi control. The Vi-positive E. coli WR2376, (Baron, L. S., 
D. J. Kopecko, S. M. McCowen, N. J. Snellings, E. M. Johnson, W. C. Reid, 
and C. A. Life, 1982, Genetic and molecular studies on the regulation of 
atypical citrate utilization and variable Vi antigen expression in enteric 
bacteria, pages 175-194. In Hollaender (edition), Genetic engineering of 
microorganisms for chemicals, Plenum Press, NY) was used as another 
positive control. Strain 17-59 is a rare, Vi-positive isolate of S. dublin 
and was used as an additional positive control, (LeMinor, L., and P. 
Nicolle, 1964, Sur deux souches de Salmonella dublin possedant l'antigen 
Vi. Ann. Inst. Pasteur (Paris) 107:550-556). A representative sample of 
various enteric bacterial strains (e.g.,E. coli, S. typhimurium, S. 
sonnei, and S. flexneri) was used to test the specificity of the various 
probes. Table 4 summarizes the results of these studies. 
Both EcoR1-A and EcoR1-B were tested as hybridization probes with 
nitrocellulose filters as well as Whatman 541 paper. The EcoRI-A probe 
only hybridized with DNA samples containing the viaB locus, whether or not 
the Vi antigen was expressed (Table 4). Of 140 various Salmonella strains 
(Table 4) obtained from the CDC, the EcoR1-A probe hybridized only to DNA 
from colonies of S. typhi, S. paratyphi C, and Vi-positive S. dublin, as 
one would expect of a highly specific probe. No hybridization of EcoR1-A 
was detected against DNA from 16 Citrobacter strains obtained from the 
CDC. 
The EcoR1-B probe was less specific. Although EcoR1-B hybridized strongly 
to DNA samples containing the viaB locus, a weak hybridization signal was 
detected against many Salmonella and Citrobacter strains. Furthermore, an 
unexpected strong hybridization of EcoR1-B to Citrobacter strain 4182-83 
was observed. 
In recently reported hybridization experiments, cloned K1 capsular antigen 
genes exhibited homology with DNA from strains of E. coli capsular types 
K92, K7, and K100, (Echarti, C., B. Hirschel, G. J. Boulnois, J. M. 
Varley, F. Waldv.COPYRGT.9el, and K. N. Timmis, 1983, Cloning and analysis 
of the K1 capsule biosynthesis genes of Escherichia coli; lack of homology 
with Neisseria meningitidis group B DNA sequences, Infect. Immun. 
41:54-60). Therefore, we probed several E. coli strains that produce 
capsular antigens with EcoR1-A and EcoR1-B to see if any hybridization 
could be detected. After hybridization of Whatman 541 paper, 
autoradiograms resulting from a 4-hour exposure were identical when 
EcoR1-A or EcoR1-B was used as a probe. Strong hybridization was observed 
with DNA from positive control strains with viaB sequences, but 
hybridization was not detected against the other strains tested, which 
included negative controls and strains of E. coli that produce common 
capsular antigens. Although hybridization with EcoR1-A was not detected 
even with longer exposure, weak hybridization between EcoR1-B and these E. 
coli strains was observed when the filters were autoradiographed 
overnight. Thus, one example of an operable subset of the cloned 18 kb 
ViaB region is the8.6 kb EcoR1-A fragment made by us and shown here to 
serve as an absolutely specific probe. 
Restriction mapping of EcoR1-A. Since the EcoR1-A Fragment of the viaB gene 
region appeared to serve as a highly specific DNA probe, we decided to map 
its sites for endonuclease cleavage with several restriction enzymes. 
Plasmid pWR141 contains the EcoR1-A of pWR127 incorporated into pBR325. 
Single and double restriction endonuclease digests of pWR141 were resolved 
by electrophoresis on agarose gels, and we analyzed the DNA fragments by 
size and Southern blot hybridization to construct a restriction map (FIG. 
1). 
Study of probe sensitivity. To determine the fewest number of bacteria that 
could be detected with the EcoR1-A probe and the radiolabeling procedure, 
we performed the following study. The DNA from 10.sup.1 to 10.sup.5 cells 
of each of three test bacterial strains was fixed on a nitrocellulose 
filter and probed with EcoR1-A. When dextran sulfate and additional probe 
(10.sup.7 10.sup.8 cpm) were used in the hybridization mixture, 10.sup.3 
Vi-positive cells could be clearly detected (Table 5). In some 
experiments, as few as 100 to 500 Vi-positive cells were detected, but 
detection was made difficult because of increased nonspecific reactivity. 
CONCLUSION 
Rapid identification tests for microbial pathogens are currently being 
developed by recombinant DNA technology combined with radio- or 
enzyme-linked immunoassay techniques. DNA probe detection systems have 
been reported for the following enteric bacteria:enterotoxigenic E. coli 
(Hill, W. E., and W. L. Payne, 1984, Genetic methods for the detection of 
microbial pathogens, Identification of enterotoxigenic Escherichia coli by 
DNA colony hybridizaion: collaborative study, J. Assoc. Off. Anal. Chem. 
67:801-807; Moseley, S. L., P. Echeverria, J. Seriwatana, C. Tirapat, W. 
Chiacumpa, T. Sakuldaipeara, and S. Falkow, 1982, Identification of 
enterotoxigenic Escherichia coli by colony hybridization using three 
enterotoxin gene probes, J. Infect. Dis. 145:863-869; Vibrio spp., Kaper, 
J. B., R. K. Campen, R. J. Seidler, M. M. Baldini, and S. Falkow, 1984, 
Cloning of the thermostable direct or Kanagawa phenomenon-associated 
hemolysin of Vibrio parahaemolyticus, Infect. Immun. 45:290-292; Kaper, J. 
B., and M. M. Levine, 1981, Cloned cholera enterotoxin genes in study and 
prevention of cholera, Lancet, ii:1162-1164; Yersinia enterocolitica, 
Hill, W. E., W. L. Payne, and C. C. G. Aulisio, 1983, Detection and 
enumeration of virulent Yersinia enterocolitica in food by DNA colony 
hybridization, Appl. Environ. Microbiol. 46:636-641; Salmonella spp., 
Fitts, R., M. Diamond, C. Hamilton, and M. Neri, 1983, DNA-DNA 
hybridization assay for detection of Salmonella spp. in foods, Appl. 
Environ. Microbiol. 46:1146-1151; and Shigella spp. Boileau, C. R., H. M. 
d'Hauteville, and P. J. Sansonetti, 1984, DNA hybridization technique to 
detect Shigella species and enteroinvasive Escherichia coli, J. Clin. 
Microbiol. 20:959-961). 
Typhoid fever remains a serious public health problem in developing 
countries and continues to be endemic in many areas of the world. 
Currently, microbiological identification of S. typhi from clinical 
specimens generally requires 36 to 48 hours. To simplify identification, 
we assessed the Vi capsular antigen ViaB structural gene region for use in 
the development of a rapid detection DNA probe system for S. typhi. 
We have provided sufficient detailed disclosure to enable one skilled in 
the art to make the viaB gene probe. Either S. typhi, C freundii or any 
other Vi-antigen expressing enteric bacterium could be used as a source of 
the viaB gene region. As discussed herein, chromosomal DNA containing the 
viaB gene region was cleaved with the Pst 1 restriction endonuclease and 
inserted into the cosmid cloning vector pHC79 by standard cloning 
techniques (see Maniatis, T , et al., 1982. Molecular cloning - a 
laboratory Manual, Cold Spring Harbor Labs., NY). E. coli C600 mel.sup.- 
recipient cells infected with the recombinant cosmids generated above were 
examined and several Mel+, Vi antigenexpressing clones were isolated 
Techniques for testing the melibiose character and ensuring Vi antigen 
expression are clearly presented in the Snellings article (J. Bacteriol. 
145:1010-1017, 1981) which describes procedures for testing Vi antigen 
expression. The cloned viaB genes were reduced in size from 31 kb to 18 kb 
and eventually to 8.6kb by the subcloning procedures described herein. 
Although the information presented by us in the specification is enabling 
to one skilled in the art to make a probe according to this invention, the 
18 kb DNA probe defined in the specification has been deposited in the 
ATCC and designated WR3007, which is Escherichia coli HB101 (pWR122), 
having the assigned ATCC No. 67096. Additionally, the experiments 
conducted to test probe specificity have been discussed herein and the 
results are summarized in Tables 2 and 4. The pWR80 is a recombinant 
plasmid containing 29 kilobase pairs of inserted DNA including the viaB 
gene region of C. freundii. When pWR80 was used as a probe, it 
cross-reacted with some bacterial strains that did not contain the Vi 
antigen genes (see Table 2 under column for pWP80). Thus, it was 
unsuitable as a specific probe. However, recombinant plasmid pWR122 
consists of the vector plasmid pRK290 containing the 18 kilobase pair DNA 
insert including the ViaB gene region and this plasmid reacts strongly 
only with bacteria containing Vi antigen genes. Unfortunately, pWR122 
reacted very weakly and nonspecifically with several Shigella strains. 
However, as shown in the right hand column of Table 2, the vector plasmid 
pRK290 alone reacts weakly and nonspecifically with these same Shigella 
strains. These data convincingly demonstrate that the weak, nonspecific 
reaction of the recombinant plasmid pWR122 with Shigella strains is due 
entirely to the pRK290 vector component of pWR122. In other words, the 18 
kilobase pair region cloned into plasmid vector pRK290 to form recombinant 
plasmid pWR122 is specific for bacteria carrying the viaB gene region, as 
demonstrated in Table 2. Thus, these data show that the viaB gene region 
probe of 18 kilobase pairs in length will serve as a specific probe for 
diagnostic detection of enteric bacteria expressing the Vi antigen (i.e. 
mainly S. typhi). As disclosed herein, the viaB gene region fragments must 
be separated from pRK290 in order to be used as a specific probe, i.e. to 
differentiate only those organisms expressing the Vi antigen. It should be 
noted that hybridization strength is discussed in the legend to Table 2. A 
strong hybridization reaction (4+) can be easily distinguished visually 
from a weak (1+) reaction (i.e. the stronger hybridization reaction gives 
a more intense color signal; a 4+ reaction would generate an intense dark 
spot whereas a 1+ reaction would generate a very light grey shading). 
Also, it should be realized that no available diagnostic probes are 100% 
specific, but at times give up to 5% false positive crossreaction. One to 
five percent crossreaction is an acceptable level of nonspecificity for 
most purposes. Thus, weak crossreaction of a probe does not eliminate the 
practical usefulness of a DNA segment as a diagnostic probe. The 18 kb 
probe is itself and the 8.6 kb fragment thereof are considered to be 
specific for bacteria carrying viaB gene sequences and serve as useful 
diagnostic probes. Since the 8.6 kilobase EcoR1-A fragment has been shown 
to act as an absolutely specific nucleic acid hybridization probe, any 
subset of sequences within this region will be highly specific for the 
ViaB gene region. Similarly, any subset of sequences within the larger 
cloned 18 kilobase viaB region will also serve as a differentially 
specific probe. Current nucleic acid hybridizaion reactions require a 
nucleic probe of a minimum size of approximately 10 nucleic acid base 
pairs. Thus, any subset sequence from about 10 base pairs to 18 kilobase 
pair of the cloned viaB region will be used as a specific nucleic acid 
probe in accordance with this invention. We consider our disclosure to be 
sufficiently enabling to allow on skilled in the art to construct a 
similar probe and to use the 18 kb probe in standard hybridization 
reactions (pages 387 to 389 of the Maniatis Molecular Cloning Manual or U. 
S. Pat. No. 4,358,535, describe readily available techniques) for 
detecting Vi antigen expressing enteric bacteria. 
Our approach for development of the DNA probe for the detection of S. typhi 
involved cloning the viaB region of C. freundii. The smallest recombinant 
clone that expresses the Vi antigen contained an 18-kb DNA insert. 
Digestion of this 18-kb cloned insert with EcoR1 restriction endonuclease 
produced two fragments, which were designated as EcoR1-A (8.6 kb) and 
EcoR1-B (9.4 kb). Each of these fragments was tested as a possible probe 
for detecting S. typhi. When used to probe a variety of enteric strains, 
including highly related Salmonella and Citrobacter strains, EcoR1-B was 
not absolutely specific. Weak hybridization of EcoR1-B was observed with 
many Salmonella strains, some Citrobacter strains, and E. coli strains 
producing capsular antigens. EcoR1-A, however, was absolutely specific for 
strains containing viaB gene sequences and is considered to be a highly 
specific DNA probe for rapid diagnostic detection of S. typhi. 
Using various enteric bacterial strains, we tested probe specificity with 
nitrocellulose and Whatman 541 paper and found Whatman 541 paper to have 
several advantages when used in the colony hybridization protocol. As a 
paper with high wet strength, it is easier to handle than nitrocellulose. 
In addition, the papers do not have to be baked in a vacuum oven to fix 
DNA to the solid support. Another advantage of Whatman 541 paper is that 
prehybridization is not necessary (S. Moseley, personal communication). 
Finally, hybridized probe can be removed easily and the samples can be 
tested sequentially with different probes. 
In sensitivity studies, the EcoR1-A probe detected 10.sup.4 Vi-expressing 
cells with the standard hybridization solution as described above. Since 
dextran sulfate has been shown to increase sensitivity (Totten, P. A., K. 
K. Holmes, H. H. Handsfield, J. S. Knapp, P. L. Perine, and S. Falkow, 
1983, DNA hybridization technique for the detection of Neisseria 
gonorrhoeae in men with urethritis, J. Infect. Dis. 148:462-471; Wahl, G. 
M., M. Stern, and G. R. Stark, 1979, Efficient transfer of large DNA 
fragments from agarose gels to diazonbenzyloxymethyl-paper and rapid 
hybridization by using dextran sulfate, Proc. Natl. Acad. Sci. U.S.A. 
76:3683-3687) we reexamined the sensitivity of our probe with this reagent 
included in the hybridization solution. A 10-fold increase in sensitivity 
was observed when added (Table 4). We expect that the EcoR1-A probe can be 
placed into a nonradioactive labeled system in which detector signals can 
be amplified, resulting in a further increase in sensitivity as well as 
rapid identification of S. typhi. 
TABLE 1 
__________________________________________________________________________ 
Outline of General Method for Genetic Identification of Pathogens 
A. Preparation of DNA probe 
B. Preparation of Clinical Specimen 
C. Colony Hybridization 
__________________________________________________________________________ 
Growth of bacteria 
Grow bacterial cultures or 
Pre-incubate filters to 
isolate specimens eliminate nonspecific 
hybridization 
Amplify plasmid Inoculation of above culture 
DNA Hybridization reaction 
or specimen on filter or 
other support material 
Label DNA fragment 
Lyse colonies and fix (i.e. 
Wash filters thoroughly 
single stranded DNA to the 
support material such as 
nitrocellulose filter) 
Purify plasmid Detection of bound probe 
nucleic acid 
Digest plasmid with Interpretation 
restriction endonculeases 
Purify labelled DNA probe fragment 
__________________________________________________________________________ 
TABLE 2 
__________________________________________________________________________ 
Testing of Vi DNA Probes for Specificity 
ViaB gene 
Hybridization reactions obtained 
Bacterial region 
with probes 
Source.sup.(a) presence.sup.(b) 
pWR80 
pWR122 
pRK290 
__________________________________________________________________________ 
Citrobacter freundii 7004 
+ 4+ 4 + - 
Citrobacter freundii 7011 
+ 4+ 4+ - 
Salmonella typhi 643 
+ 3+ 3+ - 
Salmonella typhi 643 W 
+ 3+ 3+ - 
Salmonella typhi 643 viaB.sup.- 
- - - N.D. 
Escherichia coli C600 viaB.sup.+ 
+ 3+ 3+ N.D. 
Salmonella typhimurium C-5 
- - - N.D. 
Salmonella typhimurium TML 
- - - N.D. 
Salmonella typhimurium Fisher 
- - - N.D. 
Shigella sonnei form I 
- 4+ + N.D. 
Shigella sonnei form II 
- 4+ + + 
Shigella flexneri M25-8A 
- + + + 
Shigella flexneri M42-43 
- 4+ + + 
Shigella flexneri serotype 3 
- 4+ + + 
Shigella flexneri serotype 4 
- 4+ + N.D. 
Shigella flexneri serotype 5 
- 4+ + + 
Shigella flexneri serotype 6 
- 4+ + N.D. 
Escherichia coli AB313 
- + + + 
Escherichia coli HB101 (pRK290) 
- N.D. N.D. 4+ 
__________________________________________________________________________ 
.sup.(a) Cells of each bacterial source were spotted on nitrocellulose 
filters and hybridized with one of the three indicated probes. 
.sup.(b) (+) indicates presence of ViaB gene region and (-) indicates 
absence of this DNA region. 
c. Strength of hybridization reactions was measured by autoradiography. 
(4+) = highly positive, (-) = negative. N.D. = not determined. 
TABLE 3 
______________________________________ 
Plasmid Cloning Vectors 
Plasmid Size (kb) Relevant characteristics.sup.a 
Source 
______________________________________ 
pHC79 6.5 Ap.sup.r (PstI), Tc.sup.r 
B. Hohn.sup.e 
pDPT429.sup.b 
8.7 Cm.sup.r (EcoRI), Sp.sup.r 
D. Taylor.sup.c 
pRK290 20.0 Tc.sup.r (EcoRI) 
D. Helinski.sup.f 
pACKCl 4.0 Cm.sup.r (EcoRI), Km.sup.r 
V. Burdett.sup.d 
pBR325 6.0 Cm.sup.r (EcoRI), Ap.sup.r, Tc.sup.r 
F. Bolivar.sup.g 
______________________________________ 
.sup.a Single restriction sites that inactivate drug resistance in the 
vectors are included in parentheses next to the appropriate antibiotic 
resistance. Ar.sup.r, ampicillin resistant; Tc.sup.r, tetracycline 
resistant; Cm.sup.r, chloramphenicol resistant; Sp.sup.r, spectinomycin 
resistant; and Km.sup.r, kanamycin resistant. 
.sup.b Single-copy vector derived from plasmid R100. 
.sup.c SmithKline Beckman Corp., Philadelphia, PA. 
.sup.d Duke University, Durham, NC. 
.sup.e B. Hohn and J. Collins, 1980, A small cosmid for efficient cloning 
of large DNA fragments, Gene, 11:291-298. 
.sup.f G. Ditta, S. Stanfield, D. Corgin, and D. R. Helinski, 1980, Broad 
host range DNA cloning system for gramnegative bacteria; construction of 
gene bank of Rhizobium meliloti. Proc. Natl. Acad. Sci. U.S.A., 
77:7347-7351. 
.sup.g F. Bolivar, 1978, Construction and characterization of new cloning 
vehicles, III, Derivatives of plasmid pBR322 carrying unique EcoRI 
generated recombinant molecules, Gene, 4:121-136. 
TABLE 4 
______________________________________ 
Summary of in situ Colony Hyridization Experiments 
Bacterial Response to probe:.sup.a 
species Strains tested 
Source 
##STR1## 
##STR2## 
______________________________________ 
C. freundii 
WR7004 Vi.sup.+ 
WRAIR ++++ ++++ 
C. freundii 
4182-83 CDC - +++ 
C. freundii 
Five strains 
CDC - - 
C. diversus 
Five strains 
CDC - + 
C. amalonaticus 
Five strains 
CDC - + 
S. typhi WR4201 WRAIR ++++ ++++ 
(ViaA.sup.+ 
ViaB.sup.+) 
S. typhi WR4205 WRAIR ++++ ++++ 
(ViaA.sup.- 
ViaB.sup.+) 
S. typhi WR4226 WRAIR - - 
(ViaA.sup.+ 
ViaB.sup.-) 
S. typhi Ty 2 WRAIR ++++ ++++ 
S. typhi Six strains 
CDC ++++ ++++ 
S. paratyphi C 
Two strains 
CDC ++++ ++++ 
S. dublin Vi.sup.+ 
17-59 and a 
L. LeMinor 
++++ ++++ 
CDC strain 
S. typhimurium 
C-5 WRAIR - - 
TML WRAIR - - 
CDC strain CDC - - 
Salmonella spp. 
130 CDC CDC - +.sup.c 
strains.sup.b 
E. coli K-12 
AB313 E. Adel- - - 
berg 
HB101 H. Boyer - - 
52 R137 WRAIR - - 
(LT.sup.+) 
E. coli 218 (O18:K1) 
R. Silver - + 
437 (O4:K12) 
R. Silver - + 
439 (K92) R. Silver - + 
440 (O86:K2) 
R. Silver - + 
441 (O15:K7) 
R. Silver - + 
442 (K15) R. Silver - + 
501 R. Silver - + 
(O75:K100) 
S. flexneri 
Serotype 1b, 
WRAIR - - 
M25-8A 
Serotype 2a, 
WRAIR - - 
M4243 
Serotype 3, 
WRAIR - - 
J17B 
Serotype 4, 
WRAIR - - 
Willis 
Serotype 5, 
WRAIR - - 
M90T 
Serotype 6, 
WRAIR - - 
CCH060 
S. sonnei 53G form I WRAIR - - 
53G form II 
WRAIR - - 
______________________________________ 
.sup.a ++++, Very strong hybridization; +++, strong hybridization; +, wea 
hybridization; -, no hybridization observed. 
.sup.b Hybridization data in this row exclude the following CDC strains; 
S. typhi, S. paratyphi C, and S. dublin Vi.sup.+. 
.sup.c Weak hybridization was detected in 27% of Salmonella strains probe 
with the 
##STR3## 
TABLE 5 
______________________________________ 
Sensitivity of EcoRI-A Probe 
Hybridization reaction with:.sup.a 
No. of C. freundii E. coli S. typhi 
bacterial cells 
WR7004 HB101 WR4201 
______________________________________ 
10.sup.5 ++++ - ++++ 
10.sup.4 ++ - ++ 
10.sup.3 + - + 
10.sup.2 -(+).sup.b - -(+).sup.b 
10.sup.1 - - - 
______________________________________ 
.sup.a Hybridication observed with addition of dextran sulfate and probe 
DNA (10.sup.7 cpm) to the hybridization mixture. 
.sup.b In some experiments with probe DNA (10.sup.8 cpm), as few as 100 t 
500 cells could be detected.