Nucleic acid sequences diagnostic for pathogenic E.coli...

Details Nucleic acid sequences diagnostic for pathogenic E.coli... Nucleic acid sequences diagnostic for pathogenic E.coli...

1998-10-27

2001-09-18

Myers, Carla J. (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S015000, C435S019000, C435S091200, C536S023100, C536S023700, C536S024320, C536S024330

Reexamination Certificate

active

06291168

ABSTRACT:

FIELD OF THE INVENTION
This invention pertains to the fields of microbiology and biotechnology, and particularly to the field of identification of a particular nucleotide sequence which is diagnostic for the presence of a particular microorganism.
BACKGROUND OF THE INVENTION
Escherichia coli
is a normal inhabitant of the lower gastrointestinal tract of animals, including humans, and is commonly found in fecal matter and raw sewage. Contamination of food with
E. coli,
often due to unsanitary conditions, can result in food poisoning, with clinical signs ranging from mild nausea and diarrhea, to severe illness and even death.
Fortunately, food poisoning with most strains of
E. coli
causes only mild illness. However, in recent years, a number of deaths have occurred in the United States and elsewhere due to outbreaks of illness following ingestion with food bearing
E. coli
bacteria. The causative organism of several of these outbreaks has been identified as a particular pathogenic strain of
E. coli
known
E. coli
O157:H7, unlike ordinary commensal
E. coli
is an enterohemorrhagic bacterium and causes enterohemorrhagic colitis, hemolytic uremic syndrome, and mesenteric adenitis. The O157:H7 serotype is the most frequent enterohemorrhagic
E. coli,
and the most common cause of these life threatening illnesses.
E. coli
O157:H7 infections can arise from a variety of sources but most commonly by the ingestion of contaminated foods or beverages. These foods, most commonly meat and dairy products, but also vegetables and fruits, such as berries, which are contaminated with
E. coli
O157:H7 are indistinguishable on visual or other sensory inspection from food which is not contaminated or which is contaminated with other strains of
E. coli.
Present methods for identifying
E. coli
O157:H7 are time consuming, expensive, and non-specific. Thus, these methods are not optimal for routine testing of food for the detection of the O157:H7 strain.
One such currently available method involves a multi-day test whereby a suspected fecal or food sample is placed in an enrichment medium to select for gram negative bacteria. The sample is then incubated on filter paper on which colonies of
E. coli
O157:H7 will grow and produce toxins called verotoxins. The toxins are trapped in the filter paper and are identified using antibodies which specifically bind to the verotoxins.
Another method, disclosed in U.S. Pat. No. 5,354,661 (1994), utilizes a monoclonal antibody in an ELISA test to detect serotype O157:H7 in a sample. This method, however, has several disadvantages. For one thing, a presumptive diagnosis of serotype O157:H7 is obtained after about 20 hours, with a definitive diagnosis within an additional two days. Second, the antibody of the '661 patent is not specific for serotype O157:H7, but rather detects one other
E. coli
serotype, thereby possibly resulting in false positive tests.
U.S. Patent No. 5,475,098 (1995) discloses the detection of enterohemorrhagic
E. coli,
including serotype O157:H7, by the presence of a signature DNA sequence which is common to the group. False positive results are associated with this method as enterohemorrhagic
E. coli
other than O157:H7 are detected.
A serious need exists for a method of detection of O157:H7 which is inexpensive, rapid, and specific for this serotype. It is only by such a method that the risk of disease due to foods contaminated with O157:H7, or that serotype O157:H7 itself, can ever be eliminated.
SUMMARY OF THE INVENTION
This invention relates to diagnostic tests that will specifically identify
E. coli
O157:H7. In one embodiment the invention is an isolated DNA molecule comprising all or a portion of the DNA sequence shown in SEQ ID NO:1 and SEQ ID NO:13. The term “portion”, as used in this specification and as it relates to SEQ ID NO:1 or SEQ ID NO:13, means a subset of contiguous bases of SEQ ID NO:1 or SEQ ID NO:13 which, when identified, is diagnostic of the presence of the entire sequence. In accordance with the present invention, a DNA sequence consecutively comprising 12 or more contiguous bases of SEQ ID NO:1 or SEQ ID NO:13 is considered to be a suitable “portion”.
The isolated DNA molecule is pathognomic of
E. coli
serotype O157:H7. That is, the DNA molecule is diagnostic of the O157:H7 serotype and is not found in strains other than O157:H7.
The presence of a DNA molecule represented by the DNA sequence of SEQ ID NO:1 or SEQ ID NO:13 is useful for the definitive diagnosis of serotype O157:H7 in a fecal or food sample, or a crude microbiological sample from any source. Typically, the presence of a portion of the DNA sequence of SEQ ID NO:1 or SEQ ID NO:13 provides a presumptive diagnosis of O157:H7 although, because of the virtual certainty that the portion is part of the DNA sequence of SEQ ID NO:1 or SEQ ID NO:13, the detection of the portion, as defined above, may be considered a definitive diagnosis of the serotype.
A second embodiment of the invention is a method for the identification and diagnosis of
E. coli
O157:H7 in a sample using polymerase chain reaction (PCR). In accordance with the method of the invention, the
E. coli
strain is diagnosed by obtaining a sample suspected of harboring
E. coli
O157:H7, and determining by PCR, the presence or absence in the sample of a DNA molecule having a nucleotide sequence comprising the sequence shown in SEQ ID NO:1 or SEQ ID NO:13, which DNA molecule consecutively comprises 12 or more contiguous bases of SEQ ID NO:1 or SEQ ID NO:13, in addition to the oligonucleotide primers used for the amplification reaction. In a preferred embodiment, the PCR amplification is carried out using a single oligonucleotide primer, N58, (SEQ ID NO:2) to produce a DNA molecule having a nucleotide sequence comprising SEQ ID NO:1, or a portion thereof.
A third embodiment of the invention is a method for the identification and diagnosis of
E. coli
O157:H7 in a sample by identifying by Southern blotting a restriction fragment comprising the sequence shown in SEQ ID NO: 1 or SEQ ID NO:13 or a portion thereof. The restriction fragment comprising SEQ ID NO:1 or SEQ ID NO:13 or portion is identified by digesting DNA from a sample suspected of harboring
E. coli
O157:H7 with an appropriate restriction enzyme, followed by the detection of said restriction fragment by hybridizing the digested DNA sample with a probe that anneals to SEQ ID NO:1 or SEQ ID NO:13 or a portion thereof.
A fourth embodiment of the invention is a method for the identification and diagnosis of
E. coli
O157:H7 in a sample by identifying by Southern blotting a restriction fragment comprising the IS2 insertion element represented by SEQ ID NO:3 and which also comprise the nucleotide sequences represented by SEQ ID NO:4 and SEQ ID NO:5, or a portion thereof, such restriction fragment being diagnostic of
E. coli
O157:H7. Unexpectedly, it has been discovered that the IS2 insertion element stably resides in a portion of the genome that is unique to, and therefore diagnostic of
E. coli
O157:H7. In this embodiment of the invention, restriction fragments comprising this unique nucleotide region of
E. coli
O157:H7 which comprise the IS2 insertion element are identified by Southern blotting and are detected by hybridization using the IS2 nucleotide sequence, or a portion thereof as a probe. In a preferred embodiment if the invention, the restriction enzymes EcoRI or EcoRV are used to digest the DNA of a sample suspected of harboring
E. coli
O157:H7, which generate diagnostic restriction fragments of about 8.5 kbp and about 10 kbp, respectively.
A fifth embodiment of the invention is a method for the identification and diagnosis of
E. coli
O157:H7 in a sample by PCR. In this embodiment, the PCR reaction is carried out with oligonucleotide primers that anneal to the nucleotide sequences which are unique to
E. coli
O157:H7 and which comprise SEQ ID NO:4 and SEQ ID NO:5. These primers therefore anneal to nucleotide sequences that flank the 5′ and 3′ ends of the IS2 insertion element, and gener

Affiliated with

Also associated with

Rating

Nucleic acid sequences diagnostic for pathogenic E.coli... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Nucleic acid sequences diagnostic for pathogenic E.coli..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Nucleic acid sequences diagnostic for pathogenic E.coli... will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2456143