Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-04-24
2001-01-09
Whisenant, Ethan (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100, C536S024300
Reexamination Certificate
active
06171789
ABSTRACT:
TECHNICAL FIELD
This invention relates to a novel hybrid insertion sequence found in virulent isolates of
Burkholderia cepacia
, and to methods of diagnosis and identification based on the hybrid insertion sequence. The invention also relates to uses of the hybrid insertion sequence obtained from the indicated isolates.
BACKGROUND ART
Burkholderia cepacia
(formerly known as
Pseudomonas cepacia
) is an aerobic gram-negative bacillus commonly found throughout the environment and as a phytopathogen causing soft rot in onions (1) (the numbers in brackets used throught this disclosure refer to the articles identified in the section entitled “References” provided later in the specification). Over the past decade, however, strains have been encountered with increasing frequency which cause opportunistic infections in humans, most notably in cystic fibrosis (CF) patients leading to an increase in morbidity and mortality (12, 38). Among non-CF patients, extrapulmonary nosocomial infections in compromised individuals have more recently been reported (21).
Although the mechanism of virulence of
B. cepacia
has not been elucidated (20), isolates from CF patients have been shown to adhere to mucin (26) and buccal epithelial cells (27). There may also be a correlation between the source of
B. cepacia
isolates (e.g. environmental, CF-associated epidemic and non-epidemic isolates) and the particular class of pili expressed (9). In addition, epidemic foci in Canada have been found associated with a suite of enzyme alleles characterized as electrophore type 12 (ET12) (Johnson et al., 1994).
The implications of being colonized with
B. cepacia
are a growing concern in the CF community and markers of strain virulence are eagerly sought. Enhanced transmissibility and virulence appear to be strain dependent and epidemic lineages are being defined anecdotally and genetically (10, 13, 17, 33, 34, 35, 36). To date, studies have indicated cross-infection between patients (10, 17, 25, 31) and nosocomial acquisition (19) as important parameters of transmission.
In attempts to limit the spread of
B. cepacia
, many clinical centres now segregate colonized and non-colonized CF patients. This has proved to be successful but is limited by the social contacts between patients outside the hospital setting that is the norm for CF patient groups, especially adults (10, 17, 31), and by the likelihood that not all
B. cepacia
strains are virulent.
Many studies involving
B. cepacia
have focused on its truly extraordinary potential to metabolize a wide variety of organic compounds. It is currently thought that this metabolic versatility may, in part, be the result of the genomic complexity (24) comprising three chromosomes and a large plasmid with possibly a large number of insertion sequence (IS) elements (7, 15). IS elements have the ability to promote genomic rearrangement, recruit foreign genes and cause insertional gene activation. Indeed, most of the IS elements in
B. cepacia
have been identified by observing these features (16) that dramatically modify the activity of isolates (8). It is conceivable that they may act genetically to increase transmissibility and pathogenicity of certain strains of
B. cepacia.
The inventors named in the present application originally identified the strains obtained by Govan et al. in 1993 and by themselves (13, 25), from the United Kingdom and Canada respectively, as having an identical enzyme electrophoretic allotype (ET12), the first direct evidence that the anecdotal association of Canadian
B. cepacia
strains currently endemic in Ontario and those causing an epidemic in the United Kingdom were the same.
However, while
B. cepacia
has recently been the subject of much research, not a great deal of information is available about why some strains are particularly virulent, and to what factors the difference in virulence can be attributed. In practice, there is a need for a simple diagnostic way of identifying particularly virulent isolates of
B. cepacia
so that carrier patients can be suitably treated and non-carrier patients can be protected.
DISCLOSURE OF THE INVENTION
A principal object of the present invention is to utilize a unique and newly discovered hybrid insertion sequence (IS402/IS1356—[SEQ ID NO:1]) for a variety of important diagnostic and identification purposes.
Another object of the invention is to provide a diagnostic method suitable for rapid and precise identification of virulent isolates of
B. cepacia
and possibly other bacteria.
Another object of the invention is to provide a test kit suitable for testing for a virulent strain of
B. cepacia.
According to one aspect of the invention, there is provided an insertion element characteristic of a virulent strain ET12 of
Burkholderia cepacia
, said element being a hybrid of IS402 [SEQ ID NO:2] and IS1356 [SEQ ID NO:3].
The identified insertion element has the sequence of SEQ ID NO:1, but sequence variation may occur in nature and the present invention includes minor sequence changes that do not change the essential character of the parental sequence. It is highly improbable that any sequence variation of the IS402/IS1356 hybrid would be so extensive as to render the sequence biologically or genetically unrecognizable from the parental IS402/IS1356.
According to another aspect of the invention, there is provided a method of testing for the presence of ET12/cblA isolates of
Berkholderia cepacia
in a sample, comprising testing for a hybrid insertion sequence of IS402 [SEQ ID NO:2] and IS1356 [SEQ ID NO:3] characteristic of said isolates, and indicating the presence of the sequence if the sequence is detected.
The identification of the stated hybrid sequence normally indicates the presence of the virulent isolates of
B. cepacia
as indicated, but this hybrid insertion element may be passed to other organisms transferring virulence factors to such organisms. Accordingly, a positive indication of the presence of the hybrid sequence may indicate virulent isolates of organisms other than those of
B. cepacia.
Any suitable method of testing for the presence of a known sequence may be used in the above method, for example PCR, ELISA-based methods, and other DNA sequence identification techniques. Such procedures are well known to persons skilled in the art and can be conducted relatively quickly and inexpensively compared to more conventional tests procedures for pathogenic organisms, such as metabolic discriminators and in vivo testing.
As an example, a method of testing involving PCR may involve: amplifying an insertion element sequence characteristic of strain ET12 of
Berkholderia cepacia
in a sample by polymerase chain reaction to form an amplified sequence, said insertion element being a hybrid of IS402 [SEQ ID NO:2] and IS1356 [SEQ ID NO:3]; and testing for the presence of said amplified sequence.
The basic techniques of the polymerase chain reaction are known, for example, from the following U.S. Pat. Nos. 4,683,202; 4,683,195; 4,683,188; and 5,075,216, among others. The disclosures of these patents are incorporated herein by reference.
The amplified sequence may be detected by any one of several known methods of identifying DNA segments. For example, this may be carried out by identifying the size (length of the DNA) using agarose gel electrophoresis, by designing labeled primers used for the PCR amplification, which primers are incorporated into the amplified sequences (labeling may be either radioactive or non-radioactive), or by designing a labeled (radioactive or non-radioactive) DNA probe for annealing with a known part of the amplified sequence, followed by Southern blot analysis or the like.
According to another aspect of the invention, there is provided a kit for the detection of virulent isolates of
B. cepacia
, comprising ingredients required for carrying out polymerase chain reaction and detection of a DNA sequence characteristic of the isolates, wherein the kit includes primers capable of amplifying, by
Johnson Wendy M.
Rozee Kenneth R.
Tyler Shaun D.
Her Majesty the Queen in right of Canada as represented by the
Whisenant Ethan
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