Note: Descriptions are shown in the official language in which they were submitted.
~2~
Boehringer Mannheim GmbH . 3468/00/
Det~tio~ o~ b~cteria u~ing ~ ~uolei~ ci~_a~ ication
The present patent application concerns a method for the
speci~ic d~tection of bacteria using a nucleic acicl
ampli~ication.
~he detection of bacteria in clinical diagnostic~ by
means of nucleic acids and in molecular biology has
recently become more and more important compared to the
classical immunological tests. This is related to the
fact that advances have been made in the investigation
of the nucleotide sequence of nucleic acids ~rom a
variety o~ sources.
The introduction of amplification methods has led to a
considerable increase in the sensitivity of nucleic acid
tests, ~or example in testing ~or sickle-cell anaemia.
Such a method is described for example in
EP-A-O 200 362. The amplification effect is based mainly
on the fact that with the aid oP a primer and
mononucleotide triphosphates, an extension product of
the primer is formed from a so-called target nucleic
acid as a template and this extension product is either
detected or can itself again be used as a template nucleic
acid. In this process a detectable nucleotide can also
be incorporated into the extension product. The
extension product formed can be detected
electrophoretically.
2~2~7~
- 2 -
.
However, this method of detection is disadvantageous
because of the complicaked and time-consuming
electrophoresis step.
The non-labelled extension produ~t can also be detected
according to EP~A-0 201 184 by hybridization with a
detectably labelled nucleic acid probe. ~owever, the
separation of the hybrid of extension product and probe
from the non-reacted probe using the method described in
this application is either inef~icient because of non-
specific interactions or involves many washing steE~s
which reduce the sensitivity.
A method is proposed in W0 89/11546 in which amplified
nucleic acids bound to a solid phase are reacted with a
primer and labelled mononucleotides whereby the labelled
nucleic acids which are formed in this process are
detected. A disadvantage of this method is that all
relevant reactions proceed on the solid phase which
impairs the reaction rate. Such a method is also
proposed in EP-A-0 324 474 in which labelled
mononucleotides are incorporated and these labelled
nucleic acids are captured with immobilized nucleic
acids which are complementary to the nucleic acid to be
detected. Nothing is stated about the method for the
denaturation of the amplified nucleic acids or about the
hybridization conditions. A further disadvantage of this
method is that the production of nucleic acids which are
efficiently bound to a solid phase is laborous.
A method is described in EP-A-0 357 011 in which two
primers are used in the extension reaction one of which
is detectably labelled and the other is suitable for
binding to a solid phase. A disadvantage of this method
is that it is more laborous to separate detectably
2~62~78
labelled oligonucleotides from extension products which
include these oligonucleotides. If a separation is not
carri~d out, a reduced sensitivity would be expected
because of competing reactions.
A method is described in EP-A-0 297 379 and EP-A-0 348
529 in which an immobilized or immobilizable primer is
extended with a detectable mononucleotide using the
target nucleic acid as template to form an immobilized
nucleic acid which is at the same time d~tectably
labelled. One disadvantage of this method is among
others that the specificity of the test is not very
high. The method, which is also described in
EP-A-0 297 379 in which only one immobilized or
immobilizable primer is used whereupon the extension
products are reacted with a labelled oligonucleotide,
has the drawback described in EP-~-0 357 011 that the
oligonucleotide is difficult to separate.
A method is described in WO 89/09281 in which both
primers have the same chemical group which is used for
immobilization as well as for detPction. This adds ~o
the aforementioned disadvantage of poor ssparability.
A method is also described in Proc. Natl. Acad. Sci.
USA, Vol. 86, pp 6230-6234 in which a detectably
labelled primer is extended. The detection is carried
out after binding the extension product to a capture
probe. In this case it is also not possible to separate
the detectably labelled primer without additional steps
and this therefore interferes with the detection
reaction~
_ 4 _ 2~2~7~
A method for detecting bacteria cells is known from
EP-~-0 131 052. In ~his method the bacterial nucleic
acids are reacted with a detectably labelled nucleic
acid probe which is shorter than the nucleic acid to be
detected and subsequently the hybrid which forms is
detected. A disadvantage of this methocl ls that it is
relatively insensitive and therefore requires an
extensivs removal of other cell components and the
nucleic acids to be detected have to be concentrated.
The present invention seeks to
avoid the disadvantages of the state of the art met:hod
and in particular to provide a detection method for
bacteria which combines high specificity or selectivity
with a low background signal.
The invention concerns a method for the speci~ic
detection of a bacterium in a sample which comprises the
following steps:
a) lysing th~ sample to release bacterial nucleic
acids,
b) xeacting the lysed sample with one or several
labelled mononucleoside triphosphates and one or
several enzymes which catalyze the production of
a labelled nucleic acid B which contains this
nucleotide,
c) reacting the sample with a nucleic acid probe C
which is specific for the bacterium and is
sufficiently complementary to the nucleic
acid B,
7 ~
d) detecting the nucleic acid hybrid D formed ~rom
the labelled nucleic acid B and nucleic acid
probe C,
e) whereby the nucleic acid probe contains at least
one immobilizabl~ group,
f) subjecting the reaction mixture to a thermal
denaturation directly before step c),
g) contacting the nucleic acid hybrid D with a
solid phase which can specifically bind the!
immobilizable nucleic acid probe C,
h) separating the liguid from the solid phase and
i) detecting the detectable cJroup bound to the
solid phaseO
The invention i~ not intended to encompass methods for
the detection of bacteria according to DE-A-39 29 030.
Thus methods are excluded in which the bacterial nucleic
acids are reacted with at least two adaptors per nucleic
acid strand, at least one of which contains a nucleotide
sequence which is specific ~or a replication system, to
~orm a nucleic acid which is essentially complementary
to the nucleic acid to be detected which in addition
contains at least one adaptor. Detection methods are
preferably excluded which are based on a protein-primed
replication system.
The method according to the present invention is a
special way of carrying out the so-called hybridization
7 ~
- 6 ~
tests, the main features of which are known to one
skilled in the area of nucleic acid diagnostics. As far
as experimental details are concerned which are not set
forth in the following, reference is made in its
entirety to "Nucleic Acid Hybridization", edited by B.D.
Hames and S.3. Hi~gins, IRL Press, 1986, in chapters 1
(Hybridisation Strategy), 3 (Quantitative Analysis of
Solution Hybridisation) and 4 (Quantitative Filter
Hybridisation), Current Protocols in Molecular Biolo~y,
Edt. F.M. Ausubel et al., J. Wiley and Son~ 1987r ~.9.1.
- 2.9.10 and Molecular Cloning, Edt. J. Sambrook et al~,
CSH, 1989, 9.4.7. - 9.5.8. The known methods also
include the production o~ labelled nucleoside
triphosphates such as those described in EP-A-0 324 474,
the chemical synthesis of modified and unmodified
oligonucleotides, the cleavage of nucleic acids by means
of restriction enzymes, the choice of hybridization
conditions by which means a specificity can be achieved
which depends on the extent of homology between the
nucleic acids to be hybridized, on their GC content and
their length, as well as the ~ormation of nucleic acids
from nucleoside triphosphates using polymerases, if
desired using so-called primers~
A label within the scope of the present invention is
comprised of a directly or indirectly detectable group
L. Examples of directly detectable groups are
radioactive (32p), coloured, or fluorescent groups or
metal atoms. Examples of indirectly detectable groups
are immunologically or enzymatically active ~ompounds
such as antibodies, antigens, haptens or enzymes or
enzymatically active parts of enzymes. These are
detected in a subsequent reaction or reaction sequence.
Haptens are particularly preferred since nucleoside
triphosphates which are labelled with them can generally
7 ~
be used very well as substrates for pol~nerases and a
subsequent reaction with a labelled antibudy against the
hapten or against the haptenized nucleosicle can be
easily carried out. Examples of such nucleoside
triphosphates are bromonucleoside triphosphates or
nucleoside triphosphates coupled to digoxigenin, digoxin
or fluorescein. The steroids mentionecl in EP-A-O 324 474
and the detection thereof have proven to be particularly
suitable. Reference is made in this connection to
EP-A-O 324 474 for their incorporation into nucleic
acidsO
.
Nucleoside triphosphates (NTP) are ribonucleoside
triphosphates (rNTP) or deoxyribonucleoside
triphosphates (dNTP)o
A target nucleic acid is understood as a bacterial
nucleic acid which is the target for the test and the
starting material for the method according to the
present invention.
A template nucleic acid A is a nucleic acid on which a
nucleic acid strand is newly formed which is essentially
complementary to it. ~ith reference to the sequence
information, the template nucleic acid serves as a
template and contains the sequence information which is
transcribed in reaction b~ into the nucleic acid B. The
nucleic acid A is either the ~arget nucleic acid or a
nucleic acid derived therefrom. It can for example be a
part of the target nucleic acid or contain a part of the
target nucleic acid in addition to other parts, for
example highly complex nucleic acids. It can also
contain a part of the strand which is complementary to
the target nucleic acid.
2 ~ '7 ~
Denaturation means separation of nucleic acid double
str~nds into single strands. A multitude of variants are
available to one skilled in the art.
A specific detection is understood as a method by means
of which certain bacteria can, if desired, be detected
selectively even in the presence of other bacteria.
However, it is also possible to detect a group of
bacteria using nucleic acids with partially
corresponding or similar nucleotide sequences. Either of
the two complementary strands can be used to detect
double-stranded nucleic acid~.
A nucleic acid or nucleic acid sequence which is
essentially complementary to a nucleic acid is
understood as nucleic acids or sequences which can
hybridize to the corresponding nucleic acid and have a
nucleotide sequence in the hybridizing region which is
either exactly complementary to the other nucleic acid
or differs by a few bases from the exactly complementary
nucleic acid. The specificity of this depends on the
degree of complementarity as well as on the
hybridization conditions.
Liquid phases are the aqueous phases which are usually
used in nucleic acid tests with dissolved organic or
inorganic constituents, e.g. hybridization buffer,
excess nucleotides, nucleic acids of further bacteria
which are not to be detected, prokeins etc.
The method according to the present invention for the
detection of bacteria is based on the specific detection
of a nucleic acid which is specific for the bacterium to
be detected. These nucleic acids are preferably present
v7 ~
_ 9 _
in solution. The reaction sequence is usually initiated
by making khe bacterial nucleic acid accessible using
appropriate reagents, the so~called lysis. For this,
physical methods, such as the us~ of shearing forces
(e.g. hydrodynamic shaaring forces, realized by a French
press, homogenizer), ultrasound tformation o~ cavities~,
osmosis (hydrostatic pressure which is directed against
the cell membrane), heat, repetition of extreme changes
in temperature (thawing/freezing) and ionizing radiation
as well as chemical methods such as khe inhibition of
cell wall synthesis (e.g. by antibiotics such as
penicillin), enzymatic degradation of the cell wall (by
enz~mes which specifically attack the cell wall
structure, such as lysozyme, lysostatin, proteases) and
bacteriophag~ lysis can be used. Such methods are
described for example in Methods in Microbiology,
Academic Press, Inc. ~.Y., Vol. 5 B, 1971 and Manual of
Methods for General Bacteriolo~y, American Society for
Microbiology, chapter 5, 1981.
Since the method according to the present invention is
very sensitive and selective it is possible to even
detect small amounts of nucleic acids in the presence of
other materials such as proteins, cells, cell fragments,
as well as nucleic acids which are not to be detected. A
purification of samples can therefore he omitted if it
can be assured that the nucleic acids to be detected are
su~ficiently accessible to the reagents used.
When examining ~oods it is preferable to carry out the
release in a multi-step process. Firstly, the sample to
be examined is disintegrated in order to release the
bacteria to be detected. If the bacteria occur in very
small numbers then these are propagated in a culture in
a subsequent step. This is not necessary for other
2 ~ 7 8
- 10 -
bacteria. The bacterial nucleic acids are released hy
lysis from the bacteria obtained in this way in a known
manner as described above. The nucleic acids can be
pretreated. The known pretreatments include for example
cDNA synthesis from RNA. In order that the advantages of
tha method according to the present invention ~ully come
to bear it has proven to be expedient that the nucleic
acid has a size of at least 40 bp.
In addition the nucleic acids can be the product of a
previous specific or unspecific nucleic acid
amplification. Such nucleic acid amplification methods
are disclosed for example in
EP-A-0 201 184, EP-A-0 272 098, DE~A~37 26 934,
EP-A-0 237 362, Wo 88/10315, W0 90/0106g, W0 a7/06270,
EP-A-0 300 796, EP-A-0 310 229, W0 89l09835,
EP-A-0 370 694, EP-A-0 356 021, EP-A-0 373 960,
EP-A-0 379 369, W0 89/12~96 or EP-A-0 361 983.
The (target) nucleic acids to be detected can be used
directly as ~emplate nucleic acids A in reaction b) if
they fulfil the required conditions for the selected
enzyme system. For some enzymes this requires that the
nucleic acids are single-stranded, for other enzymes it
is necessary that they include recognition sites or
promoters for the enzyme system.
If this is not the case then the target nucleic acids
must be converted into such template nucleic acids A in
a step prior to reaction b).
A can he a ribonucleic acid or a deoxyribonucleic acid.
Deoxyribonucleic acids are particularly preferred as
nucleic acid A.
~i2~37~
Within the scope of the invention the enzyme E is an
enzyme or enzyme system which cataly~es the template-
dependent synthesis o~ nucleic acids from mononucleoside
triphosphates. Preferred enzymes are polymerases an
transcriptases, which act to link mononucleotides. Such
enzymes are known to one skilled in the art. It i5
preferable to exclude protein-primed replicases.
In step b~ a labelled nucleic acid B is produced from
the template nucleic acid A. In principle, this can be
carried o~lt in any way or manner provided that the
specific information of the nucleotida sequence o~
nucleic acid A or a part theraof is essentially
preserved.
Since the bacterial detection according to the present
invention preferably uses ribonucleic acids as target
nucleic acids, it is generally not necessary to make
them single-stranded before or during reaction b). If a
strand separation is desired then this can be carried
out by treatment with alkali or thermally~ enzymatically
or by means of chaotropic salts.
The use o~ reactions b) which are dependent on the
presence of a specific initiator is particularly
preferred because of the increased specificity. Sllch
specific initiators have the effect that the enzyme only
acts on the nucleic acid which has bourld this initiator.
The initiator is preferably a so-called specific primer
p1 or a promoter.
Specific primers P1 are specially modified or unmodified
oligonucleotides which have a nucleotide sequence S
which is specially complementary to the nucleic acid A.
2~2~8
- 12 -
Modi~ied oligonucleotides can ~or example contain groups
which do not substantially impair the hybridization of
the primer with the nucleic acid. Such oligonucleotides
can be prepared by chemical or enzymatic means. The
target nucleic acid can be used diractly as the template
nucleic acid A.
The use of such primers P1 thare~ore requires that at
least a part of the sequence of the nucleic acid is
known. Moreover the sequence of the primer is selected
so that one of its ends, preferably the 3' end, is
shorter than the nucleic acid. This therefore has a
single-stranded part which extends beyond the 3' end o~
~he primer.
Thus it is possible to select primer sequences S on the
basis of the published nucleotide sequences of
individual bactaria (Salmonella: Inf. Immun. 58: 2651
tl990); Res. Microbiol. 140: 455 (1989), J. Bacteriol.
173: 86 (1991~; Listeria monocytogenes: Mol. Microbiol.
4: 1091 (1990); Infect. Immun. 55: 3225 (1987).
In reaction step b), one or several specific primers can
be used per nucleic acid single strand to be detected.
In the case of several primers, the regions on the
nucleic acid with which the primer~ can hybridize
preferably do not overlap and it is particularly
preferred that there are single-stranded regions of the
nucleic acid A between these regions. In addition to a
part which is essentially complementary to the template
nucleic acid A, the primer can also include another
nucleotide sequence Sl at the 5' end which cannot
hybridize with the nucleic acid in particular not with
the reyion which is adjacent to the complementary
region.
2~2~7~
This sequence S1 can for example be single-stranded or
double~stranded and also contain a recognition se~uence
for an enzyme. This can for example be a restrickion
cleavage site. With regard to e~eckive priming, the
primer can for example also contain a protein in bound
form which is recognized by an enzyme E which is
preferably a polymerase.
In order that the reaction b) can proceed, the
complem~ntary region of nucleic acid A and of primer Pl
must firstly be separated from complementary strands
which may be present. This separation can be carried out
by known methods, fo~ example thermally or ~on~
thermally. The non-thermal denaturation is preferr~d.
Reaction a) is then started using conditions under which
A and Pl can hybridize with one another.
If a primer is used as the initiator, an enzyms E is
also added to the sample that c~n synthesize a nucleic
ac:id complementary to A in a primer-dependent reaction.
These include in particular polymerases. Their substrate
specificity depends on khe nucleic acid A. If A is ~NA,
then RNA-dependent DNA polymerases such as reverse
transcriptase ~e.g. from AMV or M-MuLV~ come into
special consideration. If A is DNA, then DNA-dependent
DNA polymerases, such as Klenow enzyme or Taq DNA
polymerase are preferred.
. .
Deoxyribonucleoside triphosphates (dNTP) are also added
to the sample, at least one of which is labelled.
The product of the polymerase reaction is a labelled
deoxyribonucleic acid B in which the primer P1 as well
as the labelled deoxyribonucleoside triphosphate are
2 0 ~ 2 ~ r7 8
- 14 -
incorporated. This nucleic acid B is of the sama size as
or smaller than the template, but has a region which is
at least in part essentially complementary to the
template.
This nucleic acid B can now be used diractly in step c).
~owever, it is preferable to subject it again as a
template nucleic acid to a reaction analogous to step
b). For this, a primer P2 is added to the sample which
is essentially complementary to a part of nucleic acid
B/ preferably to a part of the new region formed from
dNTPs. The primer pair P1 and P2 preferably ~ulfil the
conditions described in EP-A-0 201 184. It is
particularly preferred that their 3' ends to be extended
are 100 % complementary to A or B. P1 and P2 are
preferably added simultaneously to the template A.
In order to achieve an even higher sensitivity, it is
possible to carry out reaction b) several more times,
preferably 1-60, particularly preferably 20-60 times
whereby each time the products of the reackion are used
again in reaction b). This results theoretically in an
almost exponential increase in the number of labelled
nucleic acids. In this process both nucleic acid strands
are formed whereby the length is determined by the non-
extendable ends of the primers P1 and P2. It is possible
in analogy to EP-A-0 201 18~ to also use so-called
nested primers in the later cycles of reaction b) whose
sequence is chosen so that the nucleic acids which form
are smaller than those produced first. Before each cycle
of reaction b) it is expedient to separate the double
strands formed from A and B in reaction b). This can be
carried out thermally or non-thermally. The thermal
separation is preferred. In each case primers P1 and P2
must again be present.
2 ~ 8
- 15
The initiator can also be a promoter. A promoter is a
nucleotide sequence which is recognized by a RNA
polymerase and causes this to synthesize a complementary
nucleic acid strand B on the nucleotide sequence which
follows ~he promoter. In this process the labelled NTP
is incorporatad in addition to non-labelled NTPs into
the nucleic acid strand which ~orms.
Suitable promoters are known, for example RNA-polymerase
binding sites from bacterial phages such as T3, T7 or
SP6 (Melton et al.: NAR 12, 1984, 7035-56; Pfeiffer
Gilbert: Protein Sequences and DNA Analysis I, 1988,
269-2~0; Uhlenbeck et al.: Nature 328, 1987, 596-600).
In a preferred embodiment of the test procedure, the
promoter is part of a specific primer for the production
of tha template nucleic acid A ~rom the target nucleic
acid. This primer has a nucleotide sequence S which is
essPntially complementary to a part of the target
nucleic acid and a nucleotide sequence S1 which is
r~cognized by a RNA polymerase and includes at least one
promoter se~uence. In a first reaction, a nucleic acid
strand A1 which is at least partially complementary to
the target nucleic acid and which includQs the primer
with the promoter is formed using the primer and a DNA
polymerase which is dependent on the type of target:
nucleic acid and dNTPs. If it is not already joinecl to
the primer, the newly ~ormed piece of nucleic acid is
linked covalently to the primsr by addit;on of a further
enzyme, preferably a ligase e.g. E. coli DNA ligase. The
ligase can also be thermally stable. Al is preferably
shorter than the target nucleic acid. In this
transcription reaction a second primer P3 complementary
to the target nucleic acid strand is preferably used and
the gap between both primers is closed, pre~erably by a
2 ~
-~ 16 -
gap filling reaction. The use of labelled dNTPs is
possible but not necessary in this case since this
measure only results in a slight amplificatiorl of the
measurement signal in the method according to the
present invention~
If the target nucleic acid is ~NA, then this is
preferably selectively degraded in a subsequent step.
Known methods are suitable for this, ~or example
treatment with alkali or RNAases. Subsequently, a
nucleic acid strand A2 is formed which is complementary
to A1. For this a primer P2 is preferably added to the
reaction mixture that is complementary to a part,
preferably to a newly formed part, of the strand A:L. The
primer P2 pre~erably also contains the promoter sequence
S2. This is ext~nded as described above for A1 to form
A2. Such procedural steps are described for example in
EP-A-0 329 822, DE~A-37 26 g34, WO 88/10315,
WO 87/06270, EP~-A-0 310 229 and EP-A-0 373 9~0 which is
why reference is made to these disclosures in their
entirety.
Reference is made to these disclosures in particular
with regard to details which are useful and necessary
for the reverse transcription of RNA or the
transcription of DNA.
In this embodiment it is particularly preferred that the
sample additionally contains the strand complementary to
the target nucleic acid whereby P1 and P2 are extended
simultaneously.
In the said embodiment, a DNA-dependent RNA polymerase
under promoter-specific control is subsequently added
21~2~78
- 17 -
-
according to the present invention to the sample
pretreated in this way. Such polymerases are e.g. T3, T7
or SP6 RNA polymerase. Using them, labelled nucleic
acids B are formed from NTPs and the labelled NTP. In
this case the double-stranded nucleic acid Al~A2 serves
as the template nucleic acid, preferably several times.
Preferred NTPs are ribonucleotide triphosphates. Since
in this variant of reaction b) single-stranded nucleic
acids B are formed, a denaturation is not absolutely
necessary to separate the strands but it i~ possible.
A preferred manner of carrying out the invention is the
procedura according to DE-A-4010465, however, usinlg
detectably labelled ribonucleotide triphosphates.
After the last cycle of step b), the reaction mixture is
subjected to a thermal denaturation (reaction f ~ in the
method according to the present invention. Even if step
b~ is carried out several times, a thermal denaturation
is carried out directly before step c). In this process,
especially nucleic acid double strands are separated
from one another. Thermal denaturation means in
particular denaturation in a t~mp~rature range of ca.
50-95C, preferably 85-95C. The denat-lration is
preferably carried out for 1-15 min.
An advantage of the thermal denaturatioll is that no
additional reagents, such as sodium hydroxide solution
have to be used. Thus pipetting steps can be omitted,
the method is simplified and the reproducibility of the
test is increased.
7 ~
- 18 -
In the subsequent reaction step c), nucleic acid B is
reacted with the probe C in such a way that they
together form a nucleic acid hybrid D~
Oligonucleotides or polynucleotides having a length of 6
to 5000, prPferably 15 to 2000, come lnto particular
consideration as nucleic acid probe C. The probe C can
be a plasmid, a nucleic acid fragment or an
oligonucleotide. It can be RNA or DNA. Nucleic acid
probe C is added in excess of the expected amount of
nucleic acid B to the reaction mixture which is
preferably in the form of an aqueous solution. The probe
C has a nucleotide seguence which is essentially
complemantary to B and which is specific ~or B, and
it does not hybridize with or only to a very slight
extent with nucleic acids present in the sample or which
are newly formed that are not intended to be detected.
The combination of the use of specific pximers and
hybridiæation with a specific probe makes the method
according to the present invention particularly
selective.
C can be a double-stranded nucleic acid, one strandL C1
of which is complementary to a part of B. The other
strand C2 of the nucleic acid probe is pre~erably
complementary to other nucleic acids B, in particular to
those which are formed when carrying out reaction b)
with B as tha template nucleic acid. In this case the
denaturation of C can be carried out separately ~rom B.
It is, however, preferred that C be denatured together
with B.
It is preferred that C is a single-stranded nucleic acid
probe Cl and that the strand C2 complementary to Cl is
not added. It is then also possible to add Cl be~ore
2~2~
. - 19 -
denaturing B. The solution containing the single-
stranded nucleic acid Cl also preferably contains
reagents which aid in the hybridization such as eOy.
SSC, formamide or blocking reagents for nucleic acids
that are not to be detected. Additiorlal pipetting ~teps
for the addition o~ the hybridization solution can be
omitted by this means.
Single~stranded nucleic acid probes C1 can for example
be produced by chemical nucleic acid synthe~is according
to DE-A-39 16 871 or also arcording to EP-B-0 184 056.
Probe C contains on~ or several (immobilizable) groups I
capable of immobilization per nucleic acid strand.
Tha groups I capable o~ immobiliæation are for example
chemical groups which can be bound covalently to a solid
pha~e for example by means of a chemical or
photoreaction, or groups or parts of molecules which can
be bound or recognized by another molecule or part o~ a
molecule via group-specific interactions. Such groups
ar~ therefore e.g. haptens, antigens and antibodies,
nucleotide sequences, receptors, regulation sequences,
glycoproteins such as lectins, or even the binding
partners of binding proteins such as biotin or
iminobiotin. Vitamins and haptens are preferred, biotin,
fluorescein or steroids such as digoxigenin or digoxin
are particularly preferred. It is important for tha
inve~tion that in each hybrid D the immobilizable group
of the probe differs from the detectable group of the
nucleic acid B.
The mixture that contains the nucleic acid hybrid B when
the nucleic acid to be detected was present in the
- 20 ~ 2~7~
sample is subsequently contacted with a solid phase
which can specifically bind the hybrid D via the
immobilizable groups of the nucleic acid probe C.
The type of solid phase depends on the groups I capable
of immobilization. It preferably has an immobilizing
group R which can Pntsr into a binding intaraction with
I. If the immobilizable group is for example a hapten,
then a solid phase can be used which has antibodies
against this hapt~n on its surface. If the immobilizable
group is a vitamin, such as e.g. biotin, then the solid
pha~e can contain binding proteins such as avidin or
streptavidin in an immobilized form. Particularly
preferred groups I and R are biotin and streptavidin~
Immobilization via a group on the mod.ified nucleic acid
is particularly advantageous since this can be carried
out under milder conditions than for example
hybridization reaction~.
For the immobilization of the nucleic acids which are
formed, the reaction mixtura is preferably dispensed
into a vessel after formation of the nucleic acid
hybrids D, the sur~ace of this vessel being able to
react with the immobilizable group. The hybridization
reaction with the probe preferably takes place at the
same time as the immobilization. The vessel can for
example be a cuvette, a tube or a microtitr2 plate. It
is, however, also possible to use a solid phase in the
form of a porous material such as a membrane, a tissue
or a pad on which the reaction mixture is applied. It is
also possible to use so-called beads or latex particles.
The solid phase should have at least as many binding
sites for the immobilizable group of the probe as
nucleic acid hybrids D and thus nucleic acids B present.
2~2~78
- 21 -
The production o~ a preferred solid phase is described
in EP-A-0 344 578 which is referred to in its entirety.
After an incubation period during which the
immobilization reaction takes place, the liquid phase is
removed from the vessel, the porous material or the
pelleted beads~ The solid phase can subsequently be
washed with a suitable buf~er since the binding of the
hybrids D to the solid phase is very e~ficient. In this
connection the method according to the pr~sent invention
allows the use of particularly ~ew washing steps s:ince,
in contrast to the detectable probes used in the state
of the art, the probes which are dif~icult to separate
do not necessarily have to be completely remov~d, or
leads to comparatively low background signals.
The amount of modifiad nucleic acids bound to the soli~
phase can in principle be determined in a known manner,
whereby the steps which have to be carried out depend on
the type of the detectable group. In the case o~
directly detectable groups, for example fluorescent
labels, the amount of label is determined
fluorometrically. If the detectable group is a hapten,
then the modified nucleic acid is preferably reacted
with a labelled antibody against the hapten as described
analogously in EP-A-0 324 474~ The label can also be an
enzyme label such as B-galactosidase, alkaline
phosphatase or peroxidase. In the case of an enzyme
label, the amount of nucleic acid is measured by means
of the usually photometric, chemiluminometric or
fluorometric monitoring of a reaction of the enzyme with
a chromogenic, chemoluminogenic or fluorogenic
substrate. It is, however, also possible to monitor the
reaction electrochemically if a redox enzyme is used as
the label or to monitor a change in p~ by means of a pH
2~2~78
- 22 -
electrode. The mèasurement signal is a measure of the
amount of target nucleic acid which was originally
present and thus of bacteria to be detected. In initial
experiments it was found that even 1-5 genome
e~uivalents/reaction can be detected.
The detection of the nucleic acid can be carried out
qualitatively as well as quantitatively. In the case of
a quantitative analysis, it has proven to be expedient
to carry out a comparitive experiment with a sample of
known nucleic acid content. It is possible to establish
a calibration curve and this is recommended.
In an embodiment of the method using PCR,
oligonucleotides are added to the sample as primers P1
and P2. In this case P1 is complementary to a part of
the nucleic acid single strand A which represents both
the target and the template nucleic acid. P~ ~s
homologous to a part of ~ which is at a distance from
this. The mixture i5 now treated as described in
EP-A-0 201 184, whereby however, for example a
digoxigenin~labelled or fluorescein-labelled
deoxymononucleotide triphosphate is also used in
addition to the unmodified deoxymononucleotide
triphosphates. 20-30 amplification cycles are preferably
carried out. Afterwards single-stranded biotin-labelled
probe C i5 added. The mixture is incubated at a
temperature between 50 and 95C, preferably between 80
and 95C, particularly preferably between 85 and 95C
for ca. 1 to 15 min. An advantage of the thermal
denaturation at this stage, if desired after s~veral
amplification steps, is that it is desirable to add as
few reagents as possible. In the case of a chemical
de~aturation it may in extreme cases be necessary to add
very high concentrations of such reagents, for example
2 ~
- 23 ~
in order to avoid buffering effects also caused by the
reagents necessary for the lysis and their
neutralization. This can also be an advantage in khe
subsequent wall binding of the hybrids. The mixture i~
transferred to a streptavidin-coated vassel, preferably
after cooling the reaction mixture to ca. 37C, and
incubated again. The solution is remo~ed and the vessel
is washed. A conjugate of antibodies again~t digoxigenin
and an enzyme i5 added and it is incubated again. After
removing the solution and washing the vessel, it i5
reacted with a ~hromogenic substrate for the enzyme and
the formation of colour is observQd. Previously
detectably labelled probes have always been used in
prior art methodsr A complete separation of the non-
hybridized probes was necessary for the accuracy oiE the
measured result but was relatively laborousO
The method according to the present invention
circumvents this disadvantage in that instead of
hybridizing labelled probes and determining them, the
presence oE incorporated labelled mononucleotides is
measured and is used as a measure of the presence or the
amount of nucleic acids to be detected. The separation
of non-incorporated labelled mononusleotides can be
achieved simply and completely with the present method
since they are neither bound to the nucleic acids nor to
the surface. On the other hand, an excess of
immobilizable probe C does not interfere with the
determination since the immobilizable groups of probe C
are not used as a label and thus do not contribute to
the measured result. In particular it is easier to
calculate the binding capacity of the solid phase than
with incorporation of immobilizable NTPs.
2 ~ 7 ~
24 -
The method according to the present invention is
therefore very sensitive and selective, in addition it
can be carried out in a very short time.
The method according to the present invention is
suitable for the detection of bacterial species and also
for taxonomic groups of bacteria. The method can be used
particularly well for the determination of pathogenic
microorganisms, such as e.g. of Salmonella species,
Listeria monocytogenes, Campylobacter species, Vibrio
parahaemolyticus, Vibrio cholerae, E. coli,
Staphylococcus aureus, Clostridium perfringens,
ClostridiuM botulinum, Bacillus species, Yersinia
enterocolytica. The test i5 preferably possible in foods
such as milk, milk products such as cheese, kefir,
yoghourt, meat, fish~ seafood, vegetables, lettuce,
rice, cereals, eggs, poultry, spices, herbs and dried
foods. Ribonucleic acids as well as deoxyribonucleic
acids can be detected. Ribonucleic acids are preferred
as the target nucleic acid such as rRNA; parti~ularly
preferably 16 S or 23 S-rRNA.
In the method according to the present invention it is
possible to select different specificities for the
specific initiator and the nucleic acid probe. As a
result it is possible to a hieve a double specification.
Figure 1 shows a diagram oE the course of an embodiment
using a primer elongation (use of only one primer P1).
Figure 2 shows a diagram of the course of a preferred
embodiment using the PCR principle with two primers
whereby the nucleic acids B formed first are again used
as template nucleic acid.
- 25 ~ 2~
Figure 3 show~ a calibration curve which was obtained
according to example 1.
Figure 4 shows the nucleotide sequencle of the pximers
used in example 1 and of the probe (DNA; linear; single-
stranded; 20, 19 or 20 bp).
2 ~
- 26
Abbreviations:
A template nucleic acid
Al opposite strand o~ A
P1 primer complementary to A
E enzyme/enzyme complex
E1 DNA polymerase or reverse transcriptase
L detectable ~roup (label)
B product of reactlon a)
C nucleic acid probe
I immobilizable group
D nucleic acid hybrid oE B and C
R immobilizing group
F solid phase
S1 sequence complementary to A
Sl' anokher sequence complemenkary to A
S2 promote.r
S2' promoter
The invention is elucidated in more detail by the
following example:
o ~ ~)
- 27 -
Example
The bacteria are lysed with 1 % Triton X 100 using a
5 min incubation at 95C. 10 ~l of the lysis preparation
are used in a polymerase chain reaction. The primers used
bind to positions ~9-708 and 2223-2241 in the 23 S rRNA
gene of Listeria monosytogenes (Figure 4~, i.e. a
seguence of 1552 nucleotides is ampli~iecl. 5 fg to 5 ng
of chromo~omal DNA is used in the dilution series used
in this case.
30 PCR cycles ~15 sec 94C; 30 s~c 60C; 90 sec 72C)
are carried out in a volume o~ 100 ~l containiny 200 mM
of each primer; 50 mM KCl; 10 mM Tris~HCl, pH 8.5;
1.5 mM MgCl2; 100 ~g/ml gelatin; 200 ~M dATP; 200 ~M
dGTP; 150 ~M dTTP; 50 ~M digoxigenin~ 2'-deoxyuridine-
5'-dUTP (Dig-[ll] dUTP, Boehringer Mannhelm); 2.5 IJ
Thermus aquaticus tTaq) DNA polymerase. 20 ~l o~ the
amplification preparation and 40 n~ biotin-labelled
sample DNA, Figure 4, which binds to position 1191 1210
in the above~mentioned gene are denatured in a total
volume o~ 40 ~l for 10 min at 954C. Subsequently, 160 ~l
hybridizati~n solution (52.5 mM sodium phosphate~ pH
6.8; 6.25 x SSC [1 x SSC - 0.15 M NaCl; 0.015 M sodium
citrate] and 62.5 ~ formamide) is added and it is
pipetted into a streptavidin-coated microtitra plate.
The hybridization/wall binding is carried out for thrse
hours at 37C while shaking gently. After removing the
hybridization solution and washing three times with
0.9 % NaCl, 200 mU/ml <digoxigenin>-horseradish
peroxidase conjugate is added and incubated for 30 min
at 37C in 10 mM Tri~ HCl, pH 7.5; 0.9 % NaCl; 1 % BSA;
0.5 % Pluronic T68. After washing three times
(conditions see above) it is incubated with 0.1 % 2,2-
2 ~ PJ ~
- 28 -
azino di~3-ethylhenzothiazole]-(ABTS~ for 30 min at 37C
and the absorbance is measured at 405 nm.
The absorbanc2s ~or this reaction as well as ~or the
control reaction with biolabelled sample are shown in
Figure 3.
With the aid of this curve it is now also possible to
determine the nucleic acid concentrations present in
samples of unknown bacterial concsntration and thus the
amount o~ bacteria.
- 29 -
The patent specificatlons referred to herein
are more fully identified hereinafter:
European Patent Specification 0,200,362 filed
27.03.86, Published 10.12.86, Kary B. Muills
et al, assigned to Cetus Corporation.
European Patent Speci~ication 0,201,184 ~iled
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WO 89/11546 file 2~.05.89, Published
30011.89, Gunnar Paulsen et al.
European Patent Specification 0,324,474 filed
12.01.89, Published 19.07.89, Hans J. Hoitke
et al, assigned to Boehringer Mannheim GmbH.
European Patent Specification 0,357,011 filed
30.08.89, Published 07.03.90, Thomas G.
Laffler et al, assigned to Abbott
Laboratories.
European Patent Specification 0,297,379 filed
20.06.88, Published 04.01.89, Nanibhushan
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European Patent Specification 0,348,529 filed
23.12.88, Published 03.01.90.
WO 89/09281 filed 22.03.89, Published
05.10~89, Frits Wielaard, assigned to Akzo
N.V.
WO 84/02727 filed 09.01.84, Published
19.07.8~, David E. Kohne assigned to Gen-
Probe Partners.
German Offenlegungsschrift 39 29 030 (Laid
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07.03.91, Christoph Kessler et al, assigned
to Boehringer Mannheim GmbH.
European Patent Specification 0,237,362 filed
13.03.87, Published 16.09.87, Henry A. Erlich
et al, assigned to Cetus Corporation.
European Patent Specification 0,300,796 filed
21.07.88, Published 25.01.89, Martin Becker
et al, assigned to Syntex (U.S.A.) Inc.
European Patent Specification 0,370,694 filed
16.11.89, Published 30.05.90, Brent A.
Burdick et al, assigned to Cetus Corporation.
2 ~ 7 ~
- 30 -
European Patent Specification 0,379,369 filed
18.01.90, Published 25.07.90, Samuel Rose et
al, assigned to Syntex (U.S.~.) Inc.
European Patent Specification 0,272,098 Eiled
15.12.87, Published 22.06.88, George J.
Murakawa et al, assigned to City of Hope
National Medical Center.
WO 88/10315 filed 17.06.88, Published
29.12.88, Thomas R. Gingeras et al, assigned
to Siska Diagnostics, Inc.
European Patent Specification 0,310,229 filed
01.08.88, Published 05.04.89, Lawrence J.
Burg et al, assigned to The Board of Trustees
of the Leland Stanford Junior University.
European Patent Specification 0,356,021 filed
27.07.89, Published 28.02.90, Ale~ander F.
Markham et al, assigned to Imperial Chemical
Industries PLC.
WO 89/12696 filed 16.06.89, Published
28.12.89, Rodney ~. Richards et al, assigned
to Amgen Inc.
German Offenlegungsschrift 37 26 934 (Laid
Open Specifica-tion) filed 13.08.87, Published
23.02.89, Bernd Reckmann et al, assigned to
Merck Patent BmgH
WO 90/01069 filed l9.Q7.89, Published
08.02.90, David Segev, assigned to Segev
Diagnostics, Inc.
WO 87/06270 filed 15.04.87, Published
22.10.87, Barbara Chu et al, assigned to
The Salk Institute ~or Biological Studies and
The Trustees of Columbia University in the
City of New York.
WO 89/09835 filed 07.04.89, Published
19.10.89, Leslie E. Orgel. assigned to The
Salk Institute for Biological Studies.
European Patent Specification 0,373,960 filed
15.12.89, Published 20.06.90, Thomas R.
Gingeras et al, assigned to Siska
Disgnostics, Inc.
European Patent Specification 0,361,983 filed
02.10.89, Published 04.04.90, James E.
Ste~ano, assigned to Gene-Trak Systems.
~2~7~
- 31 -
European Patent Specification 0,329,822 filed
26.08.88, Published 30.08.89, Cheryl Davey et
al, assigned to Cangene Corporation.
German Offenlegungsschrift 39 16 871 (LaicL
Open Specification) filed 24.05.89, Published
29.11.90, Hartmut Seliger et al, assigned to
Boehringer Mannheim GmbH.
European Patent Specification 0,184,056 filed
16.11.85, Published 11.06 86, Nanibhushan
Dattagupta et al, assigned to Molecular
Diagnostics , Inc.
