Note: Descriptions are shown in the official language in which they were submitted.
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DETECTION OF SPORE FORMING BACTERIA
FIELD OF THE INVENTION
This invention relates to methods for the detection of spore forming bacteria.
The invention is particularly useful in detecting bacteria in paper products
and paper
manufacturing streams. Detection of spore forming bacteria according to the
present
invention includes methods involving polymerase chain reaction. Primers
particularly
suitable for use in detection of spore forming bacteria are disclosed as well.
BACKGROUND OF THE INVENTION
Paper products used in the packaging of food should be free from the presence
of
microorganisms which adversely affect the hygiene of the food. The most common
route of contamination by these microorganisms is during the manufacturing of
the paper
products, where the microorganisms can grow and flourish. Commonly, such
contamination is dealt with through the use of biocides or heat. However,
biocide usage
may be limited by risks at both the paper mill, and in the final paper
product.
Additionally, some microorganisms are able to avoid eradication by their
inherent
protection mechanism - sporulation.
In the paper industry, one of the more costly and persistent problems is
control of
spore forming bacteria (SFB). Unlilce most bacteria, SFB can pass through
dryer
sections of a mill to pose a contamination threat when the paper product is
used, for
example, in food packaging. Also, spore forming bacteria are frequently
resistant to all
but the most toxic of biocides. A number of SFB have been identified as
problematic in
papennaking, and have been described by Pirttijarvi and others in Journal of
Ar~blied
Bacteriolo~v 81, 445-458 (1996), the entire contents of which are hereby
incorporated by
reference.
A number of industry trends have generated even more concern over the
microbiological quality of paper used for food packaging. Recycled fiber which
often
contains starch and coating material can support microbial growth. As the
fraction of
recycled material going into production increases, so will the chance for
contamination
of the finished product. Coinciding with this increase in recycled fiber is a
desire to
decrease the use of biocides for control of microbial growth. Fast, reliable,
simple and
cost-effective monitoring of product quality will increase overall production
efficiency
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by allowing problematic populations to be controlled while at the same time
permitting
biocides to be applied when, and at the specific location needed.
The current industry standard for food paclcaging grade material in the United
States is 250 spores per gram of paper. This is determined by the Dairyman's
method, a
plate count enumeration technique which requires a 48-hour incubation period.
A more
rapid diagnosis of a contamination problem would result in significantly less
wasted
product and an overall increase in mill productivity.
The need to rapidly detect spore forming bacteria is not limited to paper
making
processes. For example, the heat resistant spores formed by members of
Bacillus,
PaefZbacillus, and Clostridium, for example, can be problematic in food,
pharmaceutical,
and medical product processing, where heat sterilization under pressure is not
appropriate. In these processes, special care must be taken to avoid
contamination and to
evaluate sources of contamination when present. A rapid identification of a
contaminating source material can often prevent unnecessary production
stoppages, and
may save thousands of dollars.
The need to identify spore forming bacteria also arises in medical treatment.
Occasionally, for example, in the treatment of a bacterial infection, e.g.,
bronchitis, upper
respiratory tract infection, earache, etc., the antibiotic selected is
effective against the
organism causing the infection but fails to kill a population of bacteria such
as a
Clostridium strain (a spore forming bacteria). While the Clostf°idium
is normally not
problematic, in the absence of competition from other organisms (which are
killed by the
original course of antibiotics), the Clostridium thrives, causing a
potentially serious
infection. Thus, there is a need for detecting the presence of such species in
a biological
sample.
SUMMARY OF THE INVENTION
The present invention is directed to methods for detecting the presence of
bacteria.
More particularly, the present invention is directed to methods for detecting
the
presence of bacteria using nucleotide primers and probes.
In particular, the present invention is directed to detecting spore forming
bacteria
with such primers. Detection methods according to the present invention
include the use
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of polymerase chain reaction in conjunction with electrophoresis, or
fluorescence
techniques.
The present invention is further directed to nucleotide primers, and more
particularly, to sets of nucleotide primers, which are used in the detection
of spore
forming bacteria.
These and other aspects of the present invention are achieved by the provision
of
methods for the systematic identification of sporulation genes in spore
forming bacteria
comprising amplifying a portion of a gene from total cellular DNA of the spore
forming
bacteria by using a primer group comprising 5 -AGTATCATTCATGAAATTGG-3
(SEQ ID NO. 1), 5 -AAAAAAGCAGTTGACT-3 ( SEQ ID NO. 2), 5 -
CGGCTTGCCGTTGTATT-3 (SEQ ID NO. 3), 5 -GAAGATGTGACGAAAAAG-
3 (SEQ ID NO. 4), 5 -CAAGAAGATGTGACGAAA-3 ( SEQ ID NO. 5), 5 -
GTTGTATTATATTTCTTTGC-3 (SEQ ID NO. 6), and 5 -
GTTGTGTTAAATTTTTTGGC-3 (SEQ ID NO. 7), and 5 -
AGTATCATTCATGAAATTGGCGTTCC-3 ( SEQ ID NO. 8); and detecting the
presence of the amplification product. Spore forming bacteria include, but are
not
limited to, Bacillus naegatef°ium, Bacillus lichenfof°nais,
Bacillus cer~eus group, Bacillus
pumilus, as well as Paenbacillus rnacer~aras, Paenbacillus polymyxa,
Paenbacillus pabuli,
Bacillus flexus, Bacillus subtilis, Bacillus anthyacis, Bacillus
spot°oth.er-moduf°ans,
Bacillus splaaef°icus, Clostridium pe~fi~ingens, Clostridium buty~icum,
Closty°idiuna
pasteu~ianurn, Clostridium cochleaniuyn, Clostf~idiunz scatologenes,
Clostf~idium
so~dellii, Clostridium litusebunense, ClostJ°idium paradoxum,
Clostridium thermocellum,
They°moanae~°obacter bf°ockii, Moos°ella
thenmoautotnophica, Sporomusa ovate,
Thermobraclaiuna celere, Bacillus acidocalda~us, Bacillus amyloliquefaciens,
Bacillus
brevis, Bacillus tlauringiensis, Bacillus stea~othermoplailus,
Clostf°idium dificile,
Clostridium cellulolyticum, Clostridium bifermentans, and Clostridium
acetobufylicum.
Amplifying may include the use of polymerase chain reaction, and detecting may
include
electrophoresing the amplification product and visualizing an electrophoresis
substrate
with staining. In some embodiments, the electrophoresis substrate comprises
agarose
gel; in some embodiments, staining comprises applying ethidium bromide.
Another aspect of the present invention includes a primer pair comprising a
member selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ
ID
NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO.
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8, and another aspect of the present invention includes a nucleotide sequence,
which may
be a primer or probe, comprising a sequence selected from the group consisting
of SEQ
ID NO. l, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO.
6, SEQ ID NO. 7, and SEQ ID NO. 8. The present invention also includes primers
selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO.
3,
SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8.
The present invention is still further directed to a composition comprising at
least
one cellulose-containing material and at least one primer comprising a
sequence selected
from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID N0. 3, SEQ ID
NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8. The
cellulose-containing material may comprise paper pulp.
Also within the scope of the present invention are kits for testing for the
presence
of spore forming bacteria, wherein the kits comprise at least one primer
comprising a
sequence selected from the group consisting of SEQ ID NO. l, SEQ ID NO. 2, SEQ
ID
NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO.
8, and at least one media supportive of spore forming bacterial growth. The
kits may be
designed for use in testing paper-making processes, or may be generic to
testing any
material. Methods of using such kits are also within the scope of the present
invention.
The present invention is still further directed to methods for testing a
sample for
the presence of spore forming bacteria. One method comprises a) combining at
least two
nucleotide primers with a sample, wherein said nucleotide primers i) are
complimentary
to at least one forward and at least one reverse nucleic acid sequence from
the total
cellular DNA of the bacteria, ii) are able to hybridize spoOA gene conserved
regions of
spore forming bacteria, but not those of non-spore forming bacteria, and iii)
are such that
amplification of a portion of the spoOA gene from cellular DNA of such spore
forming
bacteria using such primers results in the generation of a 346-365 nucleotide
long DNA
product; b) amplifying cellular DNA of bacteria in the sample with primers;
and c)
detecting the presence of amplified DNA. The sample may be a cellulose-
containing
sample and may be a sample taken from a paper making process. Such samples
include,
but are not limited to, samples from white water, head box, broke, additive
storage tanlc,
and coated calender. Other samples include air, soil, water, blood, fecal
matter, starch,
protein, or an epichlorohydrin reaction product. Any of the nucleotide
sequences
disclosed in the present application may be used for the primer pairs, and
such sequences
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include SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5,
SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8.
The present invention also provides methods for testing cellulose-containing
samples f~r the presence of spore forming bacteria, wherein the methods
comprise
combining at least one primer comprising a sequence selected from the group
consisting
of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ
ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8 with a cellulose-containing sample.
The
present invention is also directed to methods for testing a cellulose-
containing sample for
the presence of spore forming bacteria. One such method comprises a) combining
at
least two nucleic acid primers of the invention, complimentary to at least one
forward
and at least one reverse nucleic acid sequence from the total cellular DNA of
the bacteria
with a cellulose-containing sample; and b) visualizing hybridized primers. The
at least
two nucleic acid primers preferably comprise at least one of SEQ ID NO. 1, SEQ
ID NO.
2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and
SEQ ID NO. 8.
The present invention also provides methods for controlling a population of
spore
forming bacteria in an industrial process stream, the methods comprising a)
detecting
bacteria in the process stream using a primer comprising a sequence selected
from the
group consisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4,
SEQ
ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8; and b) adjusting a
biocide
concentration in the process stream sufficient to reduce the number of
bacteria. The
industrial process stream may be, for example, a paper making process stream,
or a food
processing stream.
Other aspects of the present invention include methods for the systematic
identification of sporulation genes in spore forming bacteria, the methods
comprising: a)
amplifying a pouion of a gene from total cellular DNA of the spore forming
bacteria by
using at least one of SEQ ID NO. l, SEQ ID NO. 2, SEQ ID NO. 3, SEQ TD NO. 4,
SEQ
ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8; and b) detecting the
presence of an amplification product.
In another aspect, this invention provides a probe for detecting the presence
of
spore forming bacteria in a sample, the probe comprising a nucleic acid
sequence able to
form a detectable hybrid with highly conserved regions of the spoOA gene of
the spore
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forming bacteria Bacillus and Clostridium bacteria species set out in either
(a) or (b)
below:
a) Bacillus cereus, Bacillus naegateriuna, Bacillus anthracis,
and Clostridium pasteurianum;
b) Bacillus cereus, Bacillus naegateriuna, Bacillus splaaericus, and
Clostridium pasteuriaraum,
said nucleic acid sequence being unable to form a detectable hybrid with
genetic material
of non-spore forming bacteria. Preferred highly conserved regions of the spore
forming
bacterium species set out in (a) and (b) are those shown in Table II and Table
III of
Example 2 herein below. Preferred probes of this aspect of the invention are
those
wherein amplification of a portion of the spoOA gene from the cellular DNA of
a spore
forming bacteria by a polymerase chain reaction using such probe as one member
of a
primer set results in the generation of a detectable 346-365 nucleotide long
DNA
product. Additional preferred Bacillus and Clostridium bacteria species for
(a) above are
Bacillus subtilis and Clostridiufra they°ynoaceticum. Additional
preferred Bacillus and
Clostridium bacteria species for (b) above are Bacillus
steal°otlaernaoplailus and
Clostridium thermoaceticum. Preferred highly conserved regions for these
species are
also set out in Tables II and III of Example 2 herein below.
In another aspect, this invention provides a probe for detecting the
presence of spore forming bacteria in a sample, the probe comprising a nucleic
acid sequence able to form a detectable hybrid with highly conserved regions
of
the spoOA gene of the spore forming bacteria Bacillus and Clostridium bacteria
species set out in either (a) or (b) below:
a) Bacillus cereus, Bacillus megaterium, Bacillus subtilis, and
Clostridium pastern°ianurra;
b) Bacillus cereus, Bacillus megaterium, Bacillus sphaei°icus, and
Clostridium pasteurianum,
said nucleic acid sequence being unable to foam a detectable hybrid with
genetic material
of non-spore forming bacteria. Preferred highly conserved regions of the spore
forming
bacterium species set out in (a) and (b) are those shown in Table VI and Table
VII of
Example 3 herein below. Preferred probes of this aspect of the invention are
those
wherein amplification of a portion of the spoOA gene from the cellular DNA of
a spore
forming bacteria by a polymerase chain reaction using such probe as one member
of a
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primer set results in the generation of a detectable 346-365 nucleotide long
DNA
product. Additional preferred Bacillus and elosty~idium bacteria species for
both (a) and
(b) above are Bacillus thuringiensis and Clost~idiu~rt the~ssaoaceticurra.
Preferred highly
conserved regions of these species are those set out in Tables VI and VII of
Example 3
herein below.
The present invention also provides probes for detecting the presence of spore
forming bacteria in a sample, the probe comprising a nucleotide sequence able
to form a
detectable hybrid with spoOA gene of spore forming bacteria and unable to form
a
detectable hybrid with genetic material of non-spore forming bacteria, wherein
the
nucleotide sequence consists essentially of adenine, guanine, cytosine, and
thymine.
Such nucleotide sequences may comprise SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO.
3,
SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, or SEQ ID NO. 8.
Another embodiment of the present invention is a probe for detecting the
presence of spore forming bacteria in a sample, the probe comprising a
nucleotide
sequence able to form a detectable hybrid with spoOA gene of spore fonning
bacteria and
unable to form a detectable hybrid with genetic material of non-spore forming
bacteria,
wherein the nucleotide sequence is able to form a detectable hybrid to bases
76 to 93 of
the spoOA gene of Bacillus cereus, corresponding to GenBank accession number
gb
U09972. This nucleotide sequence may comprise SEQ ID NO. 4 and SEQ ID NO. 5.
The present invention also provides a probe for detecting the presence of
spore
forming bacteria in a sample, the probe comprising a nucleotide sequence able
to form a
detectable hybrid with spoOA gene of spore forming bacteria and unable to form
a
detectable hybrid with genetic material of non-spore forming bacteria, wherein
the
nucleotide sequence is able to form a detectable hybrid to bases 403 to 422 of
the spoOA
gene of Bacillus cereus, corresponding to GenBank accession number gb U09972.
The
nucleotide sequence may comprise SEQ ID NO. 3, SEQ ID N0.6 or SEQ ID NO. 7.
The present invention is also directed to compositions comprising at least one
primer comprising a sequence selected from the group consisting of SEQ ID NO.
1, SEQ
ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO.
7, and SEQ ID NO. 8. The composition may also include a cellulose-containing
material, such as paper pulp.
Aspects of the present invention include methods for testing samples for the
presence of spore forming bacteria, the methods comprising a) combining a
tagged or
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labeled probe of the invention with a sample, b) hybridizing the tagged or
labeled probe
to the target spore fomning bacteria spoOA gene, and c) detecting the
hybridized product.
SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID
NO. 6, SEQ ID NO. 7, or SEQ ID NO. 8 may used as tagged or labeled probes.
Samples
which may be tested include, but are not limited to, samples of air, soil,
water, blood,
fecal matter, starch, protein, and/or an epichlorohydrin reaction product.
Another aspect of the present invention includes probes for detecting the
presence
of spore forming bacteria in a sample, the probes comprising a nucleotide
sequence able
to form a detectable hybrid with spoOA gene of spore forming bacteria and
unable to
form a detectable hybrid with genetic material of non-spore forming bacteria,
wherein
the nucleotide sequence is able to form a detectable hybrid to bases 70 to 427
of the
spoOA gene of Bacillus cereus, the nucleotide sequence consisting essentially
of guanine,
cytosine, adenine, and thymine.
Another aspect of the present invention includes probes for detecting the
presence
of spore forming bacteria in a sample, the probes comprising a nucleotide
sequence able
to form a detectable hybrid with spoOA gene of spore forming bacteria and
unable to
form a detectable hybrid with genetic material of non-spore forming bacteria,
wherein
the nucleotide sequence is able to form a detectable hybrid to bases 70 to 427
of the
spoOA gene of Bacillus cer~eus, or to bases 570 to nucleotide 930 of the spoOA
gene of
Bacillus subtilis (gb M10082). Such nucleotide sequence preferably comprise at
least
one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5,
SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8.
Preferred probes of the foregoing aspects of the invention are those wherein
amplification of a portion of the spoOA gene from the cellular DNA of a spore
forming
bacteria by a polymerase chain reaction using such probe as one member of a
primer set
results in the generation of a detectable 346-365 nucleotide long DNA product.
Another aspect of the present invention includes methods of making nucleotide
sequences for detecting the presence of a conserved gene in spore forming
bacteria, the
methods comprising a) determining conserved regions of the conserved gene from
at
least two strains of spore forming bacteria; and b) preparing nucleotide
sequences able to
hybridize to the conserved regions, wherein the nucleotide sequences consist
essentially
of adenine, guanine, cytosine, and thymine. The conserved gene may comprise
spoOA,
ssp, and/or dpaAlB, but is preferably spoOA.
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The present invention is also directed to systems for identifying spore
forming
bacteria, the systems comprising: a) means for rendering DNA of the spore
forming
bacteria susceptible to hybridization with at least one nucleotide primer; b)
at least one
nucleotide primer; and c) means for detecting the hybridization of the DNA of
the spore
forming bacteria to the at least one nucleotide primer. The DNA of the spore
fomning
bacteria may comprise the spoOA gene, and the at least one nucleotide primer
may
consist essentially of adenine, guanine, cytosine, and thymine. The at least
one
nucleotide primer may comprise a sequence selected from the group consisting
of SEQ
ID NO. l, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO.
6, SEQ ID NO. 7, and SEQ ID NO. 8. The means for rendering DNA of the spore
forming bacteria susceptible to hybridization may comprise a growth step in
which the
bacteria are placed in an environment which encourages growth, followed by a
lysis step
in which the bacteria are lysed. The lysis step may comprise heating. The
means for
detecting the hybridization may comprise polymerase chain reaction. The means
for
detecting the hybridization may comprise a fluorescence detection technique.
DETAILED DISCLOSURE OF THE INVENTION
The present invention is directed to methods for detecting bacteria, and in
particular, spore forming bacteria (SFB). Spore forming bacteria are those
bacteria
which have the ability to form spores, and such bacteria are well known in the
art.
Examples of such SFB include, but are not limited to, Bacillus megaterium,
Bacillus
lichenfof°mis, Bacillus cereus group, Bacillus purnilus, as well as
Paenbacillus mace~arzs,
Paenbacillus polynayxa, Paenbacillus pabuli, Bacillus flexus, Bacillus
subtilis, Bacillus
anthy°acis, Bacillus spot°othe3°moduy-ans, Bacillus
splZaey-icus, Closts°idiurn perfringejZS,
Clostr~idiuna butyricum, Clostridium pasteurianum, Clostf~idiuna cochlearium,
Clostr°idiurn scatologenes, Clostridium sof°dellii, Clostridium
litusebuy~ense, Clostridium
pa~adoxum, Clostridium thermocelluf~a, The~moanae~obacte~ br~ockii, Mooy~ella
tlaef°r~aoautoty-oplzica, Spo~omusa ovata, Ther~naob~achiurn cele~e,
Bacillus acidocaldar~us,
Bacillus arnyloliquefaciens, Bacillus br~evis, Bacillus thuningiensis,
Bacillus
steal°othef°mophilus, Clostridium dificile, Clostf°idium
cellulolyticurn, Clostridium
bifer~rneratans, and Clostridium acetobu~ylicum.
The present invention is useful in the detection of SFB in paper making
processes, but is not limited to such processes. (As used herein, the term
"paper" is to be
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used in the generic sense. That is, "paper," as in a "paper making process,"
is meant to
include paper, paperboard, cardboard, etc.) When used for testing in paper
making
processes, the process water itself may be tested. The process water may be
tested
anywhere in the process, but is preferably tested in head boxes or storage
tanlts. Such
storage tanks may contain paper malting additives which are to be tested for
the presence
of SFB. Such additives include starch, latex, clays, proteins, and
epichlorohydrin
reaction products, including but not limited to reaction products of
poly(adipic acid-co-
diethylenetriamine) and epichlorohydrin, sold under the trade name Kimene. In
addition
to testing process water in the paper malting process, the paper making
machine may be
tested for the presence of SFB. Frequently, it is preferable to test shower
head deposits
for the presence of SFB.
The present invention may also be used in detecting SFB in air, soil, food,
and
water, including waste water, industrial process water, and drinking water.
The present
invention may be used in the detection of SFB in protein-containing samples.
The
present invention may be used in the detection of SFB in medical diagnostic
applications, including, for example, testing for at least one SFB in blood or
fecal matter.
The methods for detecting bacteria in these other media are similar to those
for detection
in paper making, as described herein.
The present invention focuses on the evolutionary conservation of genes
mediating the process of sporulation. A subset of phylogenetically diverse
bacteria are
able to form spores. Most commonly found spore forming bacteria are members of
the
genus Bacillus (aerobic bacteria) and Clost~idiuna (anaerobic bacteria).
Sporulation is a
complicated developmental process, responsive to adverse environmental
conditions and
under strict physiological control of the cell. Heat, starvation, and chemical
perturbation
include some but not all of the factors that may induce the sporulation
pathway. Genes
involved early in the sporulation process are highly homologous across species
boundaries. SpoOA, one such gene, may be considered a "master switch" in the
sporulation process.
The spoOA gene encodes a kinase responsible for signaling, via
phosphorylation,
other genes in the process to become active. The phosphorylation state of the
spoOA
kinase dictates its activity in the cell. Due to this central role in
triggering sporulation,
spoOA is a highly conserved gene and hence a good target gene for detection.
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The present invention is based on the discovery that spore forming bacteria
have
some conserved genetic material that may be targeted in their detection. The
conserved
genetic material targeted in accordance with the present invention is the
spoOA gene, or a
gene homologous thereto. By targeting this gene (or a homologous gene), the
present
invention is able to detect a very broad range of bacteria. Each of the
bacteria detectable
according to the present invention is believed to have the spoOA gene, or a
gene
homologous thereto, which may be involved in sporulation. Other genes which
may be
targeted in accordance with the present invention include the ssp gene and the
ilpaAlB
gene, each of which is present in sporogenic bacteria and absent in
asporogenic bacteria.
The concept underlying the present invention is the discovery that specific,
short
chains of nucleotides, can bind to the genetic material of the targeted
bacteria. Through
a number of different techniques, this binding can be visualized or even
quantified. The
basic underlying technology of the use of nucleic acid probes, or primers, to
identify
target genetic material is well known in the art, and has been described
elsewhere. For
their discussion of spore forming bacteria, and methods for their detection
using the
spoOA gene, Brill and Wiegel (Journal of Microbiological Methods 31 (1997) 29-
36),
and Brown et al. (Molecular Microbiolo~v 14(3) (1994) 411-426), are hereby
incorporated by reference. For their discussion of the use of probes and
primers for
identifying bacteria, U.S. Patent Nos. 5,747,252, 5,969,122, 5,430,137,
5,714,321, and
5,958,679, are hereby incorporated by reference.
Thus, the present invention is directed to the use of nucleotide sequences for
targeting specific portions of the spoOA gene. These nucleotide sequences can
bind, or
hybridize, to target portions of the SFB genetic material. The target portion
of the spoOA
gene spans bases beginning at about 70 and ending at about 427 of Bacillus
cei°eus,
GenBank accession #gb U09972. The nucleotide sequences of the present
invention can
also target homologous sequences from other SFB.
Obviously, the numbering of the bases will differ from strain to strain.
However,
using the CLUSTAL alignment program (Baylor College of Medicine Nucleotide
Search
Launcher) to search for homologous sequences in the GenBanlc database, one of
skill in
the art can easily determine other SFB, and their corresponding genetic
material. (Of
course, other alignment programs may be used.) By way of non-limiting example,
Bacillus subtilis (gb M10082) would be targeted at nucleotide 570 to
nucleotide 930.
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The polymerase chain reaction (PCR) is one technology which may be used to
visualize the presence of sporulation genes. This method is based on the base
complimentarity of DNA. DNA is composed of two anti-parallel strands composed
of
nucleotide "bases." These bases, adenine, guanine, cytosine and thymine, form
specific
hydrogen bonds with one another. Adenine pairs with thymine and guanine pairs
with
cytosine. Strands of DNA can be denatured or converted to a single strand form
by
alkali or heat treatment. When conditions are favorable DNA will reassociate
to its
double stranded conformation.
The polymerase chain reaction (Mullis, U. S. Pat. Nos. 4,683,195, 4,683,202,
and 4,800,159, the entire contents of each of which is incorporated by
reference) is a
commonly used method to amplify target DNA segments to detectable levels. It
is
currently being employed to detect many pathogenic bacteria. In this process,
DNA
primers of specific sequence, complementary to flanking regions of the target
area, are
used to prime enzymatic synthesis of DNA using a DNA polymerase. DNA
polymerase
requires a primer to initiate synthesis of a complementary DNA strand.
A number of different types of apparatuses and systems are available for
performing PCR. Common apparatuses include Mini Cycler (MJ Instruments), Delta
Cycler I System (EriComp), and Smart Cycler (Cepheid). Other systems may be
used in
accordance with the present invention as well. Examples are described in U.S.
Patent
Nos. 5,882,496, 5,674,742, 5,646,039, 5,589,136, 5,639,423, each to NORTHRUP
et al.,
5,527,510, to ATWOOD et al., and 5,958,349, to PETERSEN et al. For their
discussion
of PCR systems, U.S. Patent Nos. 5,882,496, 5,674,742, 5,646,039, 5,589,136,
5,639,423, each to NORTHRUP et al., 5,527,510, to ATWOOD et al., and
5,958,349, to
PETERSEN et al. are incorporated herein by reference.
Primers are shoat (usually about 15-22 bases) stretches of nucleotides.
Priming
during PCR is controlled at the annealing step by temperature. Annealing
conditions are
experimentally determined for each primer set to allow for specificity.
Following
annealing, polymerization occurs as the polymerase synthesizes a complementary
DNA
strand. After polymerization, the PCR reaction is heated to denature all
double stranded
DNA. The use of a thermostable DNA polymerase, isolated from the
hyperthermophile
The~fnus aquaticus, allows for repeated cycles of annealing, polymerization
and
denaturing to occur without loss of enzymatic activity. The process of PCR
amplification is a routine laboratory process carried out in automated
thermocyling units.
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The result is an exponential amplification of the targeted DNA segment. The
amplified
target may then be detected. Preferred primers, primer pairs and primer sets
of the
invention are those wherein such amplification results in the generation of a
detectable
346-365 nucleotide long DNA product. The nucleotide sequences of the present
invention, including SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4,
SEQ
ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8., may be used in
primers.
One method for detecting the presence of the amplified product is agarose gel
electrophoresis, followed by staining. Other detection methods include, but
are not
limited to, fluorescence detection techniques. In one fluorescence-based
technique, an
intercalating dye such as Syber Green or ethidium bromide binds to double
stranded
DNA and then fluoresces. Incorporation of these dyes into PCR reactions result
in an
increase in fluorescence as the PCR reaction proceeds and double stranded DNA
is
synthesized. Thermal denaturation of the generated products can be used to
ascertain the
size and %GC (%GC is the number of G or C bases divided by the total number of
bases) content of the PCR products generated.
One corninercially available PCR based fluorescent detection system is the
TaqManTM system. Examples of reporter dyes for this system are 6-
carboxyfluorescein
(FAM), tetra-6-carboxyfluorescein (TET), and hexachloro-6-carboxyfluorescein
(HEX).
See P. M. Holland, R. D. Abramson, R. Watson, S. Will, R. K. Sakai and D. H.
Gelfand,
1992. Detection of specific polymerase chain reactions product by utilizing
the 5'-3'
exonuclease activity of Thenmus aquaticus DNA polymerase. Clin. Chem., 38:462-
463.
Another coimnercially available detection system uses molecular beacons. See
S. Tayagi
and F. R. Kramer, 1996. Molecular Beacons: Probes that fluoresce upon
hybridization.
Nature Biotechnology 14:303-308.
In another technique, a tagged or labeled nucleotide sequence is used to
detect
hybridization. For example, a fluorescently tagged oligonucleotide sequence
derived
from an internal region of the spoOA PCR product can be used to detect the
presence of
the target in samples. As the PCR reaction proceeds the fluorescent tag is
cleaved from
the probe and fluorescence is observed. Increasing fluorescence is directly
correlated
with increased target in the test sample. Two examples of such sequences are 5
-
AGTATCATTCATGAAATTGG-3 ( SEQ ID NO. 1) and 5 -
AGTATCATTCATGAAATTGGCGTTCC-3 ( SEQ ID NO. 8). These sequences are
presented for illustrative purposes; other stretches of conserved sequences
within spoOA
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may also be targeted. Other nucleotide sequences of the present invention,
including
SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, and SEQ
ID NO. 7 would be useful in this regard.
Detection of hybridization between the nucleotide sequences of the present
invention and the target may be achieved in a number of manners, in addition
to those
already mentioned. Especially included in such other methods are those not
requiring a
polymerase chain reaction or primer pair to obtain a detectable hybrid. For
example, it is
envisioned that the nucleotide sequences of the present invention may be
tagged or
labeled and used to detect the target sequences using oligonucleotide probing.
The
sequences could be tagged or labeled with a fluorescent or radioactive
molecule. In the
case of fluorescent Labeling, the hybridized nucleotide sequence emits a
different energy
spectra than in non-hybridized form, which is detected by means well known in
the art.
With the radioactive probe, the hybridized sequence may be detected by
autoradiography
using exposure to a radiation-susceptible film. See Maniatis, T., E. F.Fritsch
and J.
Sambrook. 1982 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor
Laboratories, Cold Spring Harbor, New York. The nucleotide sequences of the
present
invention, including SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4,
SEQ
ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8, are useful in this
regard as
probes.
The nucleotide sequences of the present invention were generated by sequence
comparison of the spoOA gene from a broad spectrum of spore forming bacteria.
This
process entailed using a nucleic acid sequence alignment software program to
elucidate
highly conserved regions of the gene. From these regions, specific priming
sites were
chosen and appropriate primers were synthesized. Determination of the optimal
sequences for primer selection is done by trial and error. Preferred primers
meet all of
the following criteria:
i. detection of spoOA from a characterized set of spore
forming bacteria;
ii. negative results when testing non-SFB; and
iii. detection of spoOA from uncharacterized spore forming
bacteria isolated from paper or paper manufacture samples.
It should be noted that the inventive nucleotide sequences disclosed herein
are
selected based on their ability to hybridize target genes of SFB. In
particular, the instant
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nucleotide sequences are directed at conserved target genetic material of SFB.
In
considering which sequences will target the SFB, the instant nucleotide
sequences of the
present invention should be considered highly preferred. However, it is
recognized that
absolute identity to the sequences of the present invention may not be
necessary to
achieve a satisfactory result. That is, it is recognized that substitution of
one or more
bases may still allow hybridization to the target genes of SFB. Identity to
the instant
sequences is most preferred, and homologous or conservative substitutions are
less
preferred, but may still be acceptable. The trade-off will likely be a lower
level of
"inclusiveness," that is, fewer species of SFB will be identified by the
sequences in
which substitutions have been made. In some applications, e.g., where
identification of
only one species of SFB is needed, this lower level of inclusiveness may be
acceptable.
The methods of the present invention have been optimized to provide for
detection of spore forming bacteria. In accordance with the present invention,
spores
may be detected at levels as low as 200 spores per gram of paper (and possibly
even
lower). The following steps allow for optimal detection:
a. 10 ml of 1 % pulp sample ( 1 g pulp in 100 ml sterile water) is combined
with 40 ml of tryptic soy broth medium (Difco Laboratories) and placed
at 37 C for 7 hours.
b. 4 ml of this sample are spun down to a pellet in a microcentrifuge tube.
c. The centrifuged pellet is washed in 100 ~1 sterile water (deionized) and
centrifuged again.
d. The pellet is resuspended in 30 ~1 sterile water and boiled for 5 minutes.
e. 5 ~l of the boiled solution is used for PCR and results are visualized on
an
agarose gel.
Note that a shorter incubation time in step a) may be used where there are
higher
concentrations of SFB. Also, some samples may require an even longer
incubation
period in step a). For example, a 16-hour incubation period may be used (but
only 1 ml
of the sample is centrifuged in step b) for samples that are problematic. For
example,
longer incubation times may be used where very low numbers of SFB are believed
present, or if a PCR amplification inhibitor is present. Thus, with a longer
incubation
period, detection to levels as low as approximately 100 spores/g paper may be
achieved.
Additionally, in step a), a process water, additive, or stock sample may be
used instead of
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pulp as the starting sample. In step e), other visualization methods, e.g.,
fluorescence
methods, maybe used.
Without further elaboration, it is believed that one skilled in the art can,
using the
preceding description, utilize the present invention to its fullest extent.
The following
preferred specific embodiments are, therefore, to be construed as merely
illustrative, and
not limitative of the remainder of the disclosure in any way whatsoever.
The entire disclosures of all patents and publications, cited above and below,
are
hereby incorporated by reference as though set forth in full herein.
The following examples further illustrate the practice of the invention and
should
not be construed as limiting.
EXAMPLE 1 - PRIMER SET NUMBER 1
A set of spoOA sequences is accessed through GenBank and aligned using the
CLUSTAL alignment program. From the sequence alignment, oligonucleotide
priming
sites are selected and a preliminary primer set is chosen. A forward primer, 5
-
AAAAAAGCAGTTGACT-3 (SEQ ID NO. 2), and a reverse primer, 5 -
CGGCTTGCCGTTGTATT-3 (SEQ ID NO. 3), are synthesized. PCR products using
this primer set are expected to be in the range of about 300 to 400 base
pairs.-PCR
reaction conditions are optimized using "Ready to Go" PC R beads (Pharmacia
Biotech, Piscataway, N.J.) and different annealing temperatures for the
thermocycling
program. Any PCR apparatus may be used for this step, and the Mini Cycler and
Delta
Cycler are non-limiting examples thereof. Characterized SFB, as well as a set
of
uncharacterized SFB, isolated from a paper mill, are included in this test.
Uncharacterized samples (samples from a paper mill) testing positive are later
tested and are shown to be positive for the presence of the spoOA gene. The
results from
the characterized SFB are shown in TABLE I.
TABLE I
STRAIN PCR PRODUCT?
Bacillus cepeus +
Bacillus subtilis +
Bacillus megaterium +
Clostr~idiuna pe~f~iugeus
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The results show that all SFB except Clostridium perf °iragens showed a
band with
molecular weight size just under about 369 base pairs. To confirm that spoOA
is being
targeted, the agarose gel from the PCR is Southern blotted to nylon. As
predicted, all
SFB except Clostridium perfringens hybridized labeled amplification products
from
Bacillus cef°eus. However, even though Clostridium perfringens was not
detected using
this primer set, the positive results from uncharacterized paper mill samples
indicate that
this primer set is useful for its intended purpose.
EXAMPLE 2 - PRIMER SET NUMBER 2
Based on the results of Example 1, above, a second primer set is generated by
comparison against a larger data set. TABLE II shows the data considered for
the
forward primer and TABLE III the data for the reverse primer of the refined
primer set.
TABLE II
Bacillus cejeus G A A G A T G T G A C G A A A A A A G
Bacillusmegaterium G A A G A C G T A A C G A A A A A A G
Bacillus stearothe~mophilusG A A G A C G T G A C G A A A A A G G
Bacillus tlzuringiensisG A A G A T G T G A C G A A A A A A G
Bacillussphaericus G A A G A T G T A A T G A A A C A G G
Bacillus ar~tlaraeis G A A G A T G T G A C G A A A A A A G
Clostridium pasteurianumG A C A A A A T T A C T C A A A G A G
Clostridium ih~r.ocuumG A T C T C A T C G T G G C A G G T G
Clostridium thernaoaceticumG A G A G T A T G A C C A T G C G G T
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TABLE III
Bacillus cereus A A T A C A A C A G C A A G C C G
Bacillus naegaterium A A T A C A A C G G C A A G C C G
Bacillus stearothermophilusA A C A C A A C G G C A A G C C G
Bacillus thuringiensis A A T A C A A C A G C A A G C C G
Bacillus splaaericus A A T A C A A C A C C G T C A C G
Bacillus anthracis A A T A C A A C A G C A A G C C G
Clostridium pasteurianumA A T A C T A C T G C A A G C C G
Clostridium innocuum G C A A C C A C G G C A T C C C G
Clostridium thermoaceticumA T G A C T A C T C C C A G T C G
As can be seen from TABLES II and III, there is considerable sequence identity
and
homology in the genetic material of the SFB. Using the information from the
sequence
aligmnent, a new forward primer, 5 -GAAGATGTGACGAAAAAG-3 (S E Q ID NO.
4) is synthesized. This primer, together with 5 -CGGCTTGCCGTTGTATT-3 (S E Q
ID NO. 3), described in Example 1 above, comprise the new primer set.
This primer set (SEQ ID NOS. 4 and 3) is tested individually against known SFB
and non-SFB. This primer set yields spoOA products from characterized SFB and
no
products from non-SFB. Positive results are indicated by the presence of a
band of 346-
365 base pairs in size on an agarose gel, following PCR. TABLE IV shows the
results
from the characterized SFB which are tested.
TABLE IV
STRAIN PCR PRODUCT?
Bacillus cereus +
Bacillus sz~btilis +
Bacillus megaterium +
Clostfidiunz perfringens+
Staplaylococcus aureus -
Staphylococcus epidermis-
Pseudomonas aeruginosa -
Kleibsiella pneurnoniae-
Bacillus stearothermophilus+
Bacillus lichenformis +
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As can be seen from TABLE IV, Closty~idiurn perf ~ingens, in addition to the
other
species, is detected using the new primer set. As can be seen from the
results, this
primer set exhibited the desired characteristics: hybridization to SFB, and no
hybridization to non-SFB. Also, as with the previous primer set (from Example
1),
positive results from uncharacterized paper mill samples confirms that this
primer set
works for its intended purpose.
Once it is determined that the primer set did perform its intended function,
additional tests are performed to determine how sensitive the primer set is.
The
following procedure is therefore performed to determine the "detection limits"
for the
primer set. Although this procedure used paper samples as test materials, the
procedure
is adaptable to testing all mariners of samples, including air, soil, food,
and water,
including but not limited to, waste water, industrial process water, and
drinking water. It
should be noted that in Example 3, below, this procedure is further refined
and
optimized.
Determining Detection Limits
1. A 100 ml culture of Baeillus cef°eus is grown to lag phase and then
placed at
80 C to induce sporulation.
2. This culture is diluted in 10-fold increments in phosphate buffered saline
and
0.1 ml of the dilutions are spotted onto 0.5 g paper samples of different
types and grades
including A) Draft liner board, recycled, B) alkaline kraft paper, and C) acid
fine paper.
3. Paper samples are then placed in 10 ml phosphate buffered saline (PBS) and
vortexed for 2 minutes.
4. The samples are then placed at 80 C for 10 minutes and 1 (one) ml of the
sample is placed into 9 ml PBS to obtain another 10-fold dilution.
5. 0.1 ml of the sample is added to a sterile microfuge tube containing 0.1 ml
tryptic soy medium (0.1 ml of the sample and sample dilution are plated to
correlate PCR
result with colony forming units).
6. The samples are incubated at 37 C for 45 minutes to allow for germination.
[Note: this step is optimized in Example 3. A longer incubation time may be
necessary
for lower bacterial concentrations.]
7. The microfuge tubes are boiled for 5 minutes and 5 ~1 are used for PCR
using
"Ready to Go" P CR beads (Pharmacia).
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8. The thermocycler program is set as follows:
a. 5 minutes at 94 C
b. 30 cycles of: 0.5 minutes at 94 C, 0.5
minutes at 52 C, 0.5 minutes at 72 C
c. 3 minutes at 72 C
The detection limits established for this primer set are set forth in TABLE V.
TABLE V
Sample Spores/O.Sg Paper#spoOA PCR Product?
no paper* 171 ~ 6.0 +
no paper 22 ~ 1.0 -
A 114.5 ~ 1.5 +
A 1.5 ~ 0.5
B 59 ~ 6.0 +
B 77.0 -
C 149 + 1.0 +
C 19.5 ~ 18.5 -
# determined from plate counts
* broth culture of Bacillus cej°eus spores, no paper present
In TABLE V, A is kraft liner board, recycled, B is alkaline kraft paper, and C
is acid fine
paper.
EXAMPLE 3 - PRIMER SET NUMBER 3
The primer set from Example 2 rnay inconsistently detect Bacillus sphae~icus.
In
order to address this problem, a new primer set is prepared. The data
considered in
preparing the refined primer set is shown in TABLE VI (forward primer) and in
TABLE
VII (reverse primers).
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TABLE VI
Bacilluscereus C A A G A A G A T G T G A C G A A A
Bacillus megaterium C A A G A A G A C G T A A C G A A A
Bacillus stearothernaophilusC A G G A A G A C G T G A C G A A A
Bacillus thuringiensisC A A G A A G A T G T G A C G A A A
Bacillus splaaericus C A A G A A G A T G T A A T G A A A
Bacillus anthracis C A A G A A G A T G T G A C G A A A
Bacillus subtilis C A G G A A G A T G T C A C G A A A
Clostfidiurn pasteurianumC A A G A C A A A A T T A C C A A A
Clostridium innocuuna A A C G A T C T C A T C G T G G C A
Clostfidium thenmoaceticumC A G G A G A G T A T G A C C A T G
Clostridium perfringensC A A G A C A A A A T T A C T C A A
TABLE VII
Bacillus cereus G C A A A G A A A T A T A A T A C A A C
Bacillusmegaterium G A A A A A A A A T A T A A T A C A A C
Bacillus steayothernaoplailusG C C A A A A A A T A C A A C A C A A C
Bacillus thuringiensisG C A A A G A A A T A T A A T A C A A C
Bacillus sphaericus G C A A A G A A A T T C A A T A C A A C
Bacillus anthfacis G C G A A G A A A T A T A A T A C A A C
Bacillussubtilis G C C A A A A A A T T T A A C A C A A C
Clostridium pasteurianun2G C A A A A A A A T A T A A T A C T A C
Clostridium innocuum G C C A A G A A A T A T G C A A C C A C
Clostridium thejmoaceticumG C C C G C A A G T A T A T G A C T A C
Clostridium peyfringensG C A G G C A T G C A A G G C T T T
Using the information from the sequence alignment, a new primer set is
prepared. The
new set comprises one forward primer and two reverse primers. The new set is:
5 -
CAAGAAGATGTGACGAAA-3 (SEQ ID NO. 5) (forward), 5 -
GTTGTATTATATTTCTTTGC-3 (SEQ ID NO. 6) (reverse), and 5 -
GTTGTGTTAAATTTTTTGGC-3 (SEQ ID NO. 7) (reverse).
This primer set yields spoDA products from characterized SFB and no products
from
non-SFB. Positive results are indicated by the presence of a band of 347-356
base pairs
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in size on an agarose gel, following PCR. TABLE VIII shows the results from
the
characterized SFB which are tested.
TABLE VIII
STRAIN PCR PRODUCT?
Bacillus cereus ~ +
Bacillus subtilis (ATCC+
6051)
Bacillus subtilis (ATCC+
23059)
Bacillus megaterium +
Bacillus stealothe~mophilus+
Bacillus liclzenformis +
Bacillus sphaericus +
Clostridium perfringens+
Staphylococcus aureus -
*
Staphylococcus epidermis-
*
Staphylococcus pyogenes-
*
Pseudomonas aeruginosa -
*
Kleibsiella pneumoniae -
*
* Non-SFB
Once it is determined that the new primer set performed as intended, tests are
preformed to determine the limits of detection. The procedure for determining
the limits
of detection is similar to that in Example 2 above, with some exceptions.
1. 10 ml of 1% pulp sample (food-grade packaging board) is
combined with 40 ml tryptic soy broth medium and placed
at 37 C for 7 hours.
2. 4 ml of sample are centrifuged to a pellet in a
microcentrifuge tube.
3. The pellet is washed in 100 ~1 sterile water and centrifuged
again.
4. The pellet is resuspended in 30 ~.l sterile water and boiled
for 5 minutes.
5. 5 ~1 of the boiled solution is used in the polymerase chain
reaction.
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As noted above, some samples may require incubation time as long as 16 hours
to
optimize detection. (Longer periods are believed to be needed when the sample
has a
high concentration of clay, or other contaminant.) When a 16-hour incubation
is
employed, only 1 ml of sample is pelleted. The longer period may improve
detection to
as low as 100 spores/g paper.
The detection limits determined for the Example 3 primer set are shown in
TABLE IX below.
TABLE IX
Sample Spores/O.Sg Paper#spoOA PCR Product?
A 605 +43 +
B 590 +90 +
C 520 X50 +
D 340 +0 +
E 255 X55 -
F 175 X15 -
" determined from plate counts
In TABLE IX, samples A-F are all food-grade packaging board samples of the
same
type, spiked with different levels of SFB.
EXAMPLE 4 - DETECTING SPORE FORMING BACTERIA IN PAPER MAKING
PROCESS
Samples of 10 ml are taken from process water in the head box area of the
paper
mill. The samples are separately mixed with 40 ml tryptic soy broth medium.
Following
a 7-hour incubation period, samples are centrifuged to concentrate bacterial
contents.
The supernatant is decanted and the pellet resuspended.
The resuspended sample is boiled to lyse the bacteria, and the lysed sample
cooled and mixed with primers prior to placing the test mixture in a PCR
thermocycler.
The thermocycler is run and the PCR results are electrophoresed on an agarose
gel,
stained with ethydium bromide, and visualized under an ultraviolet light.
If spore forming bacterial counts are shown to be unacceptably high, biocide
is
added to kill the bacteria.
EXAMPLE 5 - DETECTING SPORE FORMING BACTERIA IN A FOOD MAKING
PROCESS
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Samples of 10 ml are taken from milk being processed both prior to, and after,
pasteurization. Samples are also periodically checked in the packaged product
as well.
Each lOml sample to be tested is separately mixed with 40 ml tryptic soy broth
medium.
Following a 7-hour incubation period, samples are centrifuged to concentrate
bacterial
contents. The supernatant is decanted and the pellet resuspended.
The resuspended sample is boiled to lyse the bacteria, and the lysed sample
cooled and mixed with primers prior to placing the test mixture in a PCR
thermocycler.
The thermocycler is run and the PCR results are electrophoresed on an agarose
gel,
stained with ethydium bromide, and visualized under an ultraviolet light.
Based upon the results of the testing, appropriate measures may be taken to
eradicate the spore forming bacteria at the appropriate stage in the process.
EXAMPLE 6 - DETECTING SPORE FORMING BACTERIA IN A BIOLOGICAL
SAMPLE
Samples of 100 mg are talcen from fecal matter to be tested. Each 100mg sample
to be tested is separately mixed with 50 ml tryptic soy broth medium.
Following a 7-
hour incubation period, samples are centrifuged to concentrate bacterial
contents. The
supernatant is decanted and the pellet resuspended.
The resuspended sample is boiled to lyse the bacteria, and the lysed sample
cooled and mixed with primers prior to placing the test mixture in a PCR
thermocycler.
The thermocycler is run and the PCR results are electrophoresed on an agarose
gel,
stained with ethydium bromide, and visualized under an ultraviolet light.
Based upon the results of the testing, an antibiotic which is effective at
treating a
spore forming bacterial infection is prescribed.
In each of Examples 4, 5, and 6 (and in other embodiments as well),
contaminants may interfere with the ability of the test method to detect spore
forming
bacteria. For example, the presence of clays, or some enzymes, in a sample may
result in
an interference with polymerase chain reaction. In such cases, it is
recommended that
dilution of the original sample be performed until the contaminants are no
longer present
at an interfering concentration.
Also, with regard to each of Examples 4, 5, and 6, it is noted that while PCR
is
taught as a method for detecting hybridization of the probes to the target
sample, other
methods may be used. For example, a probe may be linked to a fluorescent
(other
detectable) molecule prior to mixing with the sample. Upon hybridization, and
under the
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proper conditions, the tagged molecule will give off a detectable energy,
e.g.,
fluorescence.
From the foregoing descriptions, one skilled in the art can easily ascertain
the
essential characteristics of this invention, and without departing from the
spirit and scope
thereof, can make various changes and modifications of the invention to adapt
it to
various usages and conditions.
For example, the inventive nucleotide sequences disclosed herein are selected
based on their ability to hybridize target genes of SFB. In particular, the
instant
nucleotide sequences are directed at conserved target genetic material of SFB.
Thus, it is
believed that other nucleotide sequences which bind to the target area of SFB
genes are
within the scope of the present invention.
However, it is recognized that substitution of bases within the inventive
nucleotide sequences may still result in hybridization to the target genes.
Such
substitutions are believed to be within the scope of the present invention,
and should
amount to an insubstantial difference therefrom.
Additionally, as has been shown, the inventive nucleotide sequences can be
combined with other nucleotide sequences and still achieve the same result.
This effect
is demonstrated in Examples 1 and 2, where modifying only one of the two
primers
resulted in improved detection. Thus, it is believed that the combinations of
the present
inventive nucleotide sequences with other nucleotide sequences is within the
scope of the
present invention.
-25-
CA 02443441 2003-10-08
WO 02/092853 PCT/USO1/15793
SEQUENCE LISTING
<110> Hercules Incorporated
<120> DETECTION OF SPORE FORMING BACTERIA
<130> B1113P-PCT
<160> 8
<170> Patentln version 3.0
<210> 1
<211> 20
<212> DNA
<213> Bacillus cereus
<400> 1
agtatcattc atgaaattgg
<210> 2
<211> 16
<212> DNA
<213> Bacillus cereus
<400> 2
aaaaaagcag ttgact
16
<210> 3
<211> 17
<212> DNA
<213> Bacillus cereus
<400> 3
cggcttgccg ttgtatt
17
<210> 4
<211> 18
<212> DNA
<213> Bacillus cereus
Page 1
CA 02443441 2003-10-08
WO 02/092853 PCT/USO1/15793
<400> 4
gaagatgtga cgaaaaag
18
<210> 5
<211> 18
<212> DNA
<213> Bacillus cereus
<400> 5
caagaagatg tgacgaaa
18
<210> 6
<211> 20
<212> DNA
<213> Bacillus cereus
<400> 6
gttgtattat atttctttgc
<210> 7
<211> 20
<212> DNA
<213> Bacillus cereus
<400> 7
gttgtgttaa attttttggc
<210> 8
<211> 26
<212> DNA
<213> Bacillus cereus
<400> 8
agtatcattc atgaaattgg cgttcc
26
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