Abstract:
The present invention relates to species-specific DNA probes specific for  Vibrio vulnificus  and  Vibrio cholerae . The DNA probes of the present invention specifically detects  Vibrio vulnificus  or  Vibrio cholerae  in a mixed bacterial sample based on unique ribosomal RNA nucleotide sequences. When the DNA probes of the present invention are tagged with a labeled molecule such as a fluorescent label, it affords direct and immediate visualization of individual bacterial cells, and a rapid method of detection of bacterial infection in humans and shellfish without culturing.

Description:
This is a continuation of application Ser. No. 08/474,828 filed on Jun. 7, 1995 abandoned, which is a division of Ser. No. 07/891,987, filed May 28, 1992, U.S. Pat. No. 5,426,025. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to species-specific oligonucleotide probes for binding specifically to ribosomal RNA of bacterium  Vibrio Vulnificus  or  Vibrio cholerae . The present invention further relates to methods and kits for identifying the bacteria  Vibrio vulnificus  or  Vibrio cholerae  with such probes in a single day, without the need for culturing the bacteria. 
     BACKGROUND OF THE INVENTION 
       Vibrio vulnificus  and  Vibrio cholerae  are small organisms called bacteria that live in the marine environment.  Vibrio cholerae  can also survive in fresh water. By drinking water, eating fruits and vegetables, fish or shellfish that are contaminated with this bacterium (one bacteria), a person can become very ill or may even die from the disease cholera which causes severe diarrhea and dehydration.  Vibrio vulnificus  can cause serious illness and even death within three days in people who eat raw or improperly cooked fish or shellfish that are infected with this microorganism. 
     There are many types of bacteria, both good and bad, in food and water. To find out whether water or food contains these harmful Vibrio bacteria, or if a person is infected with them, laboratory tests must be performed. The first step is to culture, or grow, the bacteria in a special liquid. Then a series of tests are done to identify the bacteria based on whether or not they use certain sugars and other compounds in order to grow. It may take as long as one week to do these tests and by that time a person may die if not given the proper medicine. What is needed is a rapid and easy way to detect and identify  Vibrio cholerae  and  Vibrio vulnificus.    
     A faster way to check for the presence of these bacteria is to use a molecule called an antibody that recognizes then binds to a specific part of the bacterium. See, for example, Tamplin, M. L. et al.:  Applied and Environmental Microbiology , 57:1235-1240 (April 1991), which reports an enzyme immunoassay for the identification of  Vibrio vulnificus  in seawater, sediment and oysters. However, this method also requires growing the bacteria first and still takes several days to do. Also, antibodies may be too specific and detect only those bacteria from one source but not from another. 
     Another method involves the use of a “gene probe” to identify the bacteria. Every living organism has molecules of DNA (deoxyribonucleic acid) which contains instructions for each cell to make the things it needs to sustain life. A gene is a piece of DNA that instructs the cell to make one particular type of protein molecule. A gene probe is also made of DNA. See, for example, Amann, R. et al.:  Nature . 351:161-164 (May 9, 1991); and Amann, R. et al.:  J. Bacteriology . 173:762-770 (February 1990). 
       Vibrio vulnificus  and  Vibrio cholerae  have specific genes that make proteins which are responsible for causing sickness. A gene probe that specifically seeks out the genes for these toxic proteins can be used to find out whether these bacteria are in food, water or people. However, there is a problem with using a gene probe. Bacteria are one-celled organisms. Each bacterium has only one gene for each kind of protein it makes. It is difficult to detect the one and only toxic protein gene in each cell. 
     One way to solve this problem is to use a method called PCR to amplify (make many copies of) the gene in a test tube and then use the gene probe to find the gene copies. This method, however, is sophisticated and time-consuming and requires a sufficient number of bacteria to perform the method. 
     With the current outbreak of Vibrio cholerae in South America, it is apparent that a rapid means of detection is warranted. Consequently, there is a need in the industry for simple, but quick and accurate tests for the reliable and early detection of the bacteria  Vibrio vulnificus  or  Vibrio cholerae  in, for example, water, food, blood, feces, and the like, contaminated with such bateria. 
     SUMMARY OF THE INVENTION 
     In brief, the present invention alleviates and overcomes certain of the above-referenced problems and shortcomings of the present state of the art through the discovery of novel oligonucleotide probes which are species-specific for the 23S rRNA of  Vibrio vulnificus  and  Vibrio cholerae . Generally speaking, the DNA probes of the present invention seek out and hybridize with specific, unique RNA regions of each 23S ribosome in every cell of either the bacteria  Vibrio vulnificus  or the bacteria  Vibrio cholerae . The probes of the present invention are uniquely made of very short pieces of DNA, so small that they can easily enter a  Vibrio vulnificus  or  Vibrio cholerae  cell, respectively. 
     The DNA probes of the instant invention are amazingly versatile in that they can be mixed with for example, water, food such as fish or shellfish, blood, feces or other type of samples to be tested. To identify the bacteria, a labeled moiety, such as a dye, a biotin or a radioisotope molecule, is attached to one end of each DNA probe. When a fluorescent dye is selected and a light is shone on the sample (food, water, fish, shellfish, blood, feces, etc.), the fluorescent dye will glow only if the bacteria for which the DNA probe is specific are present. Quite amazingly, it is actually possible to see each tiny bacterium glowing by using an epifluorescent microscope. And, because there is no need to culture or grow the bacteria beforehand or to amplify their genes, the tests of the present invention are simpler, much quicker and more reliable than the other kinds of test which have been available heretofore for detecting  Vibrio vulnificus  and  Vibrio cholerae.    
     The methods of the present invention involve fixation of whole bacterial cells from a mixed culture sample on, for example, membrane filters or microscope slides followed by hybridization with tagged species-specific probe, and washing to remove excess or non-specifically bound probe. The presence or absence of  Vibrio vulnificus  or  Vibrio cholerae  cells is directly determined by, for example, epifluorescence microscopy when the DNA probes are labeled with a fluorescent dye. 
     Each probe of the instant invention is a single-stranded oligodeoxynucleotide sequence, approximately 15-20 bases in length, which specifically targets the ribosomal RNA (rRNA) component of the ribosomes within  Vibrio cholerae  or  Vibrio vulnificus  cells. 
     While the DNA probes of the present invention typically include between about 15 and about 20 nucleotides, it should nevertheless be understood by those skilled in this art that the present invention contemplates DNA probes of any size so long as the objectives of the present invention are not defeated, i.e., the DNA probes are of a size sufficient so that they can enter the bacteria cells, and the DNA probes include an effective number of nucleotides so that they can hybridize specifically with the ribosomal RNA of either the bacteria  Vibrio vulnificus  or the bacteria  Vibrio cholerae , respectively. 
     Accordingly, it can now be appreciated that the present invention is believed to provide a valuable and worldwide solution to the  Vibrio vulnificus  and  Vibrio cholerae  art that has long sought effective and inexpensive means to quickly and reliably detect such pathogenic bacteria. As a result, the present invention can help to protect the public health, and perhaps prevent future epidemics, by its ability to detect infection in humans as well as in contaminated water and food at an early state. 
     The above features and advantages will be better understood with reference to the Detailed Description set out hereinbelow. It will also be understood that the biological materials of this invention are exemplary only and are not to be regarded as limitations of this invention. 
    
    
     DETAILED DESCRIPTION 
     By way of illustrating and providing a more complete appreciation of the present invention and many of the attendant advantages thereof, the following detailed description is provided concerning the novel oligonucleotide probes, methods and kits. 
     The DNA probes of the present invention have been developed by PCR amplification and sequencing of the genes for the 16S and 23S rRNA of  Vibrio vulnificus  and  Vibrio cholerae . The gene sequences were then aligned with available sequences for other Vibrio and non-Vibrio rRNA genes in order to locate regions of the genes which are unique to  Vibrio vulnificus  or  Vibrio cholerae . The DNA probes of the instant invention work by specifically binding to their complementary rRNA sequence on the ribosome to form a DNA-RNA hybrid molecule. 
     The  Vibrio vulnificus  DNA probe sequences include: 
     SEQ ID NO:1 
     CGCTTCATTGAGCTA 
     SEQ ID NO:2 
     CGCTTCATTGAGCTAT 
     SEQ ID NO:3 
     CGCTTCATTGAGCTATG 
     SEQ ID NO:4 
     CGCTTCATTGAGCTATGT 
     SEQ ID NO:5 
     TGGCTTCATTGAGCTA 
     SEQ ID NO:6 
     TGGCTTCATTGAGCTAT 
     SEQ ID NO:7 
     TGGCTTCATTGAGCTATG 
     SEQ ID NO:8 
     TGGCTTCATTGAGCTATGT 
     SEQ ID NO:9 
     TTCGCTTCATTGAGCTAT 
     SEQ ID NO:10 
     TTCGCTTCATTGAGCTATG 
     SEQ ID NO:11 
     TTCGCTTCATTGAGCTATGT 
     The  Vibrio cholerae  DNA probe sequences include: 
     SEQ ID NO:12 
     GATTCCTAGGTTGAGCCCA 
     SEQ ID NO:13 
     GATTCCTAGGTTGAGCCCAG 
     The DNA probes of the present invention can be made in an automated DNA synthesizer. An amino linker is attached to the 5′ end of the probe to allow covalent coupling of a fluorescent dye. Dye molecules that can be attached to the probes include the isothiocyanate or sulfonyl halide forms of fluorescein, a rhodamine and other fluorephores which are commercially available. Fluorescently-tagged probes are purified on a Sephadex G-25 size exclusion column followed by HPLC. At this point, the probes are ready for use. 
     In addition to fluorescent dyes, the probes can be labeled with, for example, a biotin, a radioisotope, or other tag molecules and the hybridization between the probe and its target rRNA molecule is detected by means other than epifluorescence microscopy. 
     A method for detecting and identifying  Vibrio vulnificus  or  Vibrio cholerae  using the fluorescently-tagged or labeled DNA probes involves fixation of whole bacterial cells from a mixed culture sample (food, water, etc.) on a membrane filter or glass microscope slide followed by hybridization with the appropriate fluorescently-tagged, species-specific probe, and washing to remove excess or non-specifically bound probe. The presence or absence of  Vibrio vulnificus  or  Vibrio cholerae  cells is directly determined by epifluorescence microscopy. 
     Samples of environmental or clinical origin (e.g. water, shellfish, blood) to be tested for the presence of these bacteria are adhered to either glass microscope slides coated with gelatin or to non-fluorescent, inorganic, 0.2 micron membrane filters and air dried. The sample is then fixed in a formaldehyde:phosphate buffered saline solution (9:1), fixative solution A, for 20 minutes. This fixation step is repeated with fresh fixative solution. (These fixation steps can be omitted if the original sample is fresh.) Next, the sample is fixed in methanol:formaldehyde (9:1), fixative solution B, for 20 minutes, rinsed briefly in distilled water and air dried. 
     A small volume of a hybridization solution or buffer containing the probe is added to the fixed, dried sample. The hybridization solution or buffer includes about 0.2% sodium dodecyl sulfate (SDS), sodium chloride, sodium phosphate, EDTA (SSPE) (0.9 Molar sodium chloride, 0.06 Molar sodium phosphate, 0.006 Molar EDTA), Denhardt&#39;s solution—1×, and about 4 mcg/ml of double stranded heterologous DNA salmon sperm. When slides are selected, the hybridization solution or buffer should include enough probe so that approximately 50 nanograms of probe available per smear per slide. When filters are selected, the hybridization solution or buffer should include enough probe so that approximately 15 nanograms of probe is available per filter. The slides/filters are incubated in the dark at a specific temperature for about one to about two hours to allow hybridization between the probes and target rRNA. The hybridization temperature for the Vibrio vulnificus probe is about 51° C. The hybridization temperature for the  Vibrio cholerae  probe is about 57° C. 
     Washes to remove excess or non-specifically bound probes are performed at room temperature for a total of about 12 minutes followed by about a 20 minute wash at the hybridization temperature with a solution of lower salt content (wash solution B) than that of the first wash solution (wash solution A). Wash solution A includes 0.2% SDS and 6×SSPE (0.9 Molar sodium chloride, 0.06 Molar sodium phosphate, and 0.006 Molar EDTA). Wash solution B includes 0.2% SDS and 1×SSPE (0.15 Molar sodium chloride, 0.01 Molar sodium phosphate and 0.001 Molar EDTA). Slides/filters are rinsed briefly with distilled water, air dried in the dark, and viewed with an epifluorescence microscope. 
     To summarize a procedure of the present invention: 
     1. fix cells about (20-60 minutes); 
     2. hybridize with fluorescently-labeled probe solution (about 1-2 hours); 
     3. Wash to remove non-specifically bound probe (about 35 minutes); and 
     4. view cells with epifluorescence microscope. 
     A typical kit of the present invention includes: fixative solutions A &amp; B; fluorescently-labeled oligonucleotide DNA probe (5 nanograms/ml water); hybridization buffer; wash solutions A &amp; B; and non-fluorescent filters or Teflon-coated microscope slides (with sample wells). 
     The DNA probes of the present invention have been laboratory tested and are believed to be species-specific. Fluorescently-tagged species-specific DNA probes of the present invention, which target rRNA, have been successfully used for both environmental research and clinical diagnostic purposes. The advantages of using such DNA probes for detecting and identifying  Vibrio vulnificus  or  Vibrio cholerae  in either environments or clinical samples include the following: 1. rapid (one day) diagnosis; 2. elimination of the need for prior culturing of the bacteria in test sample; 3. the probes are small enough to penetrate whole, intact, fixed bacterial cells (in situ hybridization); 4. elimination of the need to extract or amplify DNA; 5. detection of target organisms in a mixed bacterial sample; 6. fluorescent label allows direct and immediate visualization of individual cells; 7. fluorescently-labeled probes are stable, sensitive and safe; and 8. methods utilize standard clinical and research laboratory equipment and requires minimal technical expertise. 
     Applications of the probes contemplated by the present invention include, for instance, clinical usage to rapidly diagnose human infections of  Vibrio vulnificus  or  Vibrio cholerae , regulatory agency use to detect bacteria in water and food (especially important in oyster meat), and research usage for ecological studies of the seasonality and distribution of these bacteria in the environment. 
     The present invention may, of course, be carried out in other specific ways than those herein set forth without departing from the spirit and essential characteristics of the invention. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive and all changes coming within the meaning and equivalency range of the appended claims are intended to be embraced herein. 
     
       
         
           
             13 
           
           
             
               15 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             
               not provided 
             
             1
CGCTTCATTG AGCTA                                                      15 
           
           
             
               16 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             
               not provided 
             
             2
CGCTTCATTG AGCTAT                                                     16 
           
           
             
               17 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             
               not provided 
             
             3
CGCTTCATTG AGCTATG                                                    17 
           
           
             
               18 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             
               not provided 
             
             4
CGCTTCATTG AGCTATGT                                                   18 
           
           
             
               16 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             
               not provided 
             
             5
TGGCTTCATT GAGCTA                                                     16 
           
           
             
               17 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             
               not provided 
             
             6
TGGCTTCATT GAGCTAT                                                    17 
           
           
             
               18 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             
               not provided 
             
             7
TGGCTTCATT GAGCTATG                                                   18 
           
           
             
               19 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             
               not provided 
             
             8
TGGCTTCATT GAGCTATGT                                                  19 
           
           
             
               18 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             
               not provided 
             
             9
TTCGCTTCAT TGAGCTAT                                                   18 
           
           
             
               19 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             
               not provided 
             
             10
TTCGCTTCAT TGAGCTATG                                                  19 
           
           
             
               20 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             
               not provided 
             
             11
TTCGCTTCAT TGAGCTATGT                                                 20 
           
           
             
               19 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             
               not provided 
             
             12
GATTCCTAGG TTGAGCCCA                                                  19 
           
           
             
               20 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             
               not provided 
             
             13
GATTCCTAGG TTGAGCCCAG                                                 20