US20120190028A1

US20120190028A1 - Nucleic acids and methods for the detection of enterobacter sakazakii (cronobacter spp.)

Info

Publication number: US20120190028A1
Application number: US13/389,366
Authority: US
Inventors: Cordt Grönewald; Kornelia Berghof
Original assignee: Biotecon Diagnostics GmbH
Current assignee: Biotecon Diagnostics GmbH
Priority date: 2009-08-07
Filing date: 2010-08-04
Publication date: 2012-07-26
Also published as: EP2287333B1; WO2011015356A1; EP2287333A1; US20150080258A1; US11479826B2

Abstract

Provided are detection means and method specific for the genus Cronobacter (E. sakazakii) which are sensitive by using a target molecule which is multiple existent in Cronobacter cells.

Description

BACKGROUND OF THE INVENTION

Enterobacter sakazakii is a pathogen of the Enterobacteriaceae family which is able to cause different diseases like necrotising enteritis, meningitis or septicemia and infects particularly prematured babies and infants with low immunity.
In comparison to other infections the official number of published E. sakazakii outbreaks is rather low, but the progressive course of E. sakazakii-induced diseases is often dramatic which is pointed out by a death rate of 40-80%.
Among several environmental origins of infection, milk-powder based baby food could be identified as a major source several times.
The shown coherence (FAO/WHO 2004, 2006) between the appearance of E. sakazakii in infant formulae and the coincidental occurrence of diseases has resulted in the current European regulation Nr. 2073/2005 which is defining microbiological criteria for foodstuffs. Therefore, E. sakazakii should not be detected in 300 g (n=30, c=0, m=negativ/10 g) dried infant formulae and dietary foods with special medical relevance which is to be designed for infants under the age of 6 months.
The primary edition of this regulation has resulted in an inspection requirement for E. sakazakii if the microbiological criterion for Enterobacteriaceae (negative results for 10 samples; starting material: 10 g) was not complied. However, an expertise of the european board for food safety has shown no general occurrence of Enterobacteriaceae and E. sakazakii at the same time. As a consequence, detections for Enterobacteriaceae and E. sakazakii must be done separately according European regulation Nr. 1441/2007. An exception is made if the individual company is able to document an explicit relationship between the appearance of Enterobacteriaceae and E. sakazakii.
As E. sakazakii strains are characterized by a diverse pheno- and genotypical heterogeneity, a taxonomic reclassification of E. sakazakii was accomplished. From this follows that the new introduced genus of Cronobacter contains the species C. sakazakii, C. malonaticus, C. muytjensii, C. turicensis and C. dublinensis as well as the three subspecies C. dublinensis subsp. dublinensis, C. dublinensis subsp. lausannensis and C. dublinensis subsp. lactaridi. In addition, two more strains are currently named as C. genomospecies 1 although they could not be allocated to these species.
As clinical isolates could be found in all different species of Cronobacter, a potential danger of the whole genera could be assumed. Therefore, the regulations developed for E. sakazakii could be transfered on Cronobacter spp. (Iversen et al., 2007, BMC Evol Biol. 7: 64).

Technology Up-to-Date

In the regulation 2073/2005 the international prestandard ISO/TS 22964 is mentioned as cultural method for the standard detection of E. sakazakii (Cronobacter spp.). This procedure includes an isolation based on chromogenic selection media after pre-enrichment in buffered peptone water and a selective enrichment in modified LST-Bouillon. Candidates will be checked if a characteristic yellow colouration can be recognized after inoculation on TSA-Agar. The yellow colonies must be confirmed by biochemical tests. This method is time- and laborintensive and can produce false-negative results. For example, some Cronobacter strains are not forming yellow colonies on TSA-Agar. (Besse et al., 2006, J. AOAC Int. 89: 1309-1316; Iversen et al., 2004, J. Clin. Microbiol. 42: 5368-5370).
Iversen et al. (2007, J. Clin. Microbiol. 45: 3814-3816) has shown that none of three tested chromogenic Cronobacter agars enable the detection of all Cronobacter strains. In the same study only 70 and 90% of the Cronobacter strains could be identified as E. sakazakii by biochemical tests API20E and ID32E. Another disadvantage of this cultural detection method is reasoned in a false-positive identification of some Enterobacteriaceae by using chromogenic or TSA-Agar and the API20E test kit (Iversen et al., 2007, J. Clin. Microbiol. 45: 3814-3816).
In comparison to these often variable phenotype-dependent cultural methods, molecular biological detection methods which are based on the hybridization of DNA-probes turned out to be more specific generally. These molecular biological detection methods are very sensitive and allow a fast detection of the microorganisms.
Especially methods which are combining an enrichment of nucleic acids and the detection by DNA-probes have gained more importance whereas the polymerase-chain-reaction (PCR) could be stated as very well-known example.
Several PCR-systems for the detection of E. sakazakii/Cronobacter spp. are already described. Here, the gene region rpsU/dnaG (Seo & Brackett, 2005, J Food Prot. 68: 59-63) and the genes gluA (Lehner et al., 2006, BMC Microbiol. 6: 15), ompA (Nair & Venkitanarayanan, 2006, Appl. Environ. Microbiol. 72: 2539-46) and zpx (Kothary et al., 2007, Appl. Environ. Microbiol. 73: 4142-51) served as DNA target sequences as well as parts of the ribosomal operon 16S rDNA (Lehner et al., 2004, BMC Microbiol. 4: 43; Malorny & Wagner, 2005, J. Food Prot. 68: 1623-1627; Hassan et al., 2007, Int. J. Food Microbiol. 116: 214-20) and the DNA-sequence between the tRNA-glu and 23S rDNA gene (Derzelle & Dilasser, 2006, BMC Microbiol. 6: 100).
In most cases only a limited number of Cronobacter strains were tested by these PCR-systems which are not covering the genetic diversity of the whole genus. In addition, the detection of members of the newly-described Enterobacter-species E. helveticus and E. pulveris were not tested although the organisms are closely related to the genus Cronobacter.
Cawthorn et al. (2008, Int. J. Food Microbiol. 127: 129-38) determined a specificity of max. 92% as they have compared 6 different PCR-systems in total which were described in the literature.
Iversen et al. (2007, J. Clin. Microbiol. 45: 3814-3816) found out a specificity of each 100% for 2 PCR-systems which were based on the rpsU/dnaG gen region. But in opposite to the ribosomal operons both gene regions only exist as single copy versions in the Cronobacter genom. (McClelland et al., 2008, GenBank accession number NC009778). As in total seven copies of the ribosomal operon could be found in the genome, a PCR-system which is built up on this genome region guarantees a significant higher sensitivity limit. Generally, a maximum of sensitivity is a very important feature for the detection of Cronobacter cells in contaminated infant formulae as the concentration of cells is quite low normally. (FAO/WHO 2006)
The regulation (EU) Nr. 1441/2007 rules that milk-powder based infant formulae must be analyzed for Enterobacteriaceae and E. sakazakii in parallel if a clear relationship between the occurrence of Enterobacteriaceae and E. sakazakii could not be found out. Cultural methods allow just a separate detection of the two parameters. In case of reference standards for E. sakazakii and Enterobacteriaceae only an enrichment in buffered peptone water is identically. The selective enrichment steps, isolation and identification have to be done separately. At the moment no biochemical or molecular biological method for the simultaneous detection of both parameters is described.
In comparison to cultural techniques, a drawback of molecular biological DNA-based methods like PCR is the non-selective detection of DNA from living and dead cells. By use of PCR a positive analysis result is often possible if only dead cells are in the sample, due to the high stability of DNA.
There have been a lot of methods developed in the past to circumvent this disadvantage of a PCR-based detection of microorganisms. Due to the less stability, one approach is the use of RNA instead of DNA as the target molecule. Detection will be accomplished by reverse transcription following PCR of the generated cDNA. But ribosomal RNA is not suitable as a viability marker, because of its high stability. Messenger RNA is in general much more unstable, but the detection of mRNA is sophisticated because of the omnipresent RNase and so only limited suitable for routine testing.
Recently were alternative methods published to selective amplify DNA of viable microorganisms. This selectivity will be achieved by compounds that can only penetrate cells with compromised cell walls and cell membranes. These compounds are phenanthridinium derivates with a light-sensitive azide group that can be covalently linked to DNA by photoactivation whereby amplification will be inhibited with high efficiency (Rudi et al., 2005, Appl. Environ. Microbiol. 71: 1018-1024; Nocker et al., 2006, J Microbiol Methods 67: 310-320).

DETAILED DESCRIPTION OF THE INVENTION

The object of this invention is to provide a detection method which is 100% specific for the genus Cronobacter (E. sakazakii) and at the same time very sensitive by using a target molecule which is multiple existent in Cronobacter cells. The complete coverage of the genus Cronobacter is very important, because all species are considered to be hazardous pathogens. On the other hand a false-positive detection of other bacteria like the closely related, non-pathogenic species E. helveticus or E. pulveris, which often mimic a presence of Cronobacter, has to be avoided. Such false-positive results are able to cause high economic losses. In most cases, the number of Cronobacter cells in contaminated samples is extremely low. Therefore a detection method has to be as sensitive as possible.
The inventors have surprisingly found out, that oligonucleotides or combinations thereof targeting the multi copy existing genomic region from the 3′-end of the 23S-till the 5′-end of the 5S-rDNA gene including the internal transcribed spacer region (ITS 2) enable such a specific and sensitive detection of Cronobacter spp.
They also discovered two oligonucleotides targeting this region that allow a specific detection of a subgroup of C. turicensis. FIG. 1 shows the DNA region as well as the hybridization positions of the oligonucleotides of this invention.

SEQ ID NO: 1:
TTA ACC TTA CAA CGC CAA AGA AGT CTG GCG TGT TGA GAG ACA ATT CAG

CTT GTG ACG GAT ARA CGT TCA TGG CGG AAG CGG TGR ACG RAC AGA

ATT TGC CTG GCG GCT GTA GCG CGG TGG TCC CA

SEQ ID NO: 1 comprises the 23S-ITS2-5S region of Cronobacter spp. (except for one subgroup of Cronobacter turicensis).

	SEQ ID NO: 2:
	ACC TTA CAA CGC CAA AGA AGT C

	SEQ ID NO: 3:
	TGT AGC GCG GTG GTC C

	SEQ ID NO: 4:
	AAG CGG TGA ACG GAC AGA ATT TGC CTG GC

	SEQ ID NO: 5:
	AAG CGG TGG ACG GAC AGA ATT TGC CTG GC

	SEQ ID NO: 6:
	AAG CGG TGA ACG AAC AGA ATT TGC CTG GC

	SEQ ID NO: 7:
	AAG CGG TGG ACG AAC AGA ATT TGC CTG GC

	SEQ ID NO: 8:
	TTC AGC TTG TGA CGG ATA AAC GTT CAT GGC G

	SEQ ID NO: 9:
	TTC AGC TTG TGA CGG ATA GAC GTT CAT GGC G

	SEQ ID NO: 10:
	GAA GTC TGG CGT GTT GAG AGA CAA T

	SEQ ID NO: 11:
	AAA CGT TCA TGG CGG AAG CGG TGA ACG AAC A

	SEQ ID NO: 12:
	AGA CGT TCA TGG CGG AAG CGG TGA ACG AAC A

	SEQ ID NO: 13:
	AGA CGT TCA TGG CGG AAG CGG TGG ACG AAC A

	SEQ ID NO: 14:
	AGA CGT TCA TGG CGG AAG CGG TGG ACG GAC A

	SEQ ID NO: 15:
	AAA CGT TCA TGG CGG AAG CGG TGG ACG AAC A

	SEQ ID NO: 16:
	AAA CGT TCA TGG CGG AAG CGG TGA ACG GAC A

	SEQ ID NO: 17:
	AAA CGT TCA TGG CGG AAG CGG TGG ACG GAC A

	SEQ ID NO: 18:
	AGA CGT TCA TGG CGG AAG CGG TGA ACG GAC A

SEQ ID NO: 2-18 are derived from SEQ ID NO: 1.

SEQ ID NO: 19:
TTA ACC TTA CAA CGC CAA AGA AGT CTG GCG TGT TGA GAG ACT ATT CAG

CTT GTG ACG GAT AAG ACC TGT GGC CGT GAG GCG GCA GGT GAC AGA

ATT TGC CTG GCG GCT GTA GCG CGG TGG TCC CA

SEQ ID NO: 19 comprises the 23S-ITS2-5S region of E. sakazakii LMG 2790 (C. turicensis). The strain LMG 2790 was reclassified to C. turicensis by the inventors after sequencing of a part of the 16S rDNA gene and sequence comparison to GENBANK database.

	SEQ ID NO: 20:
	GAG GCG GCA GGT GAC AGA ATT TG

	SEQ ID NO: 21:
	ACG GAT AAG ACC TGT GGC CG

SEQ ID NO: 20-21 are derived from SEQ ID NO: 19.

	SEQ ID NO: 22:
	CGC CAT TGT GCG AGG ATG GTG C

SEQ ID NO 22 is an oligonucleotide with an artificial sequence complementary to an amplification control nucleic acid.

DEFINITIONS

Fragments of Oligonucleotides

Fragments of oligonucleotides arise due to deletion of one or more nucleotides on the 5′ and/or 3′ end of an oligonucleotide.

Identical DNA Sequences/Percentage of Identity

For the determination of the identity (in the sense of complete matching, corresponding to 100% identity) of DNA or RNA sequences, partial sequences of a larger polynucleotide are considered. These partial sequences comprise ten nucleotides and are then identical when all 10 modules are identical for two comparative sequences. The nucleotides thymidine and uridine are identical. As partial sequences, all possible fragments of a larger polynucleotide can be considered.
As an example two polynucleotides are considered which comprise 20 nucleotides and which differ in the 5th module. In a sequence comparison six 10-way nucleotides are found which are identical and five which are not identical, because they differ in one module.
In addition, the identity can be gradually determined, whereby the unit is stated in percent. For the determination of the degree of identity partial sequences are also considered, which comprise as a minimum the length of the actually used sequence, e.g. as primer, or 20 nucleotides.
As an example, polynucleotide A with a length of 100 nucleotides and B with a length of 200 nucleotides are compared. A primer with a length of 14 nucleotides is derived from polynucleotide B. For the determination of the degree of identity, polynucleotide A is compared with the primer over its complete length. If the sequence of the primer occurs in polynucleotide A, whereby it however deviates in one module, then there is a fragment with a degree of identity of 13:14.fwdarw.92.3%.
In the second example the polynucleotides A and B previously mentioned are compared in their entirety. In this case all the possible comparative windows of a length of 20 nucleotides are applied and the degree of identity determined for them. If then nucleotides nos. 50-69 of polynucleotide A and B are identical with the exception of nucleotide no. 55, then a degree of identity of 19:20.fwdarw.95% arises for these fragments.

Multiplex PCR

A multiplex PCR is a Polymerase Chain Reaction or DNA or RNA amplification reaction in which more than two primers are used which are not regarded as a forwards-backwards primer pair. With the presence of all nucleotide target molecules to be detected, this leads to the creation of at least two different amplicons. These amplicons should at least differ in the region in which the primers link, but they can also be allocated to completely different genes.

Nucleotides

Nucleotides are the modules of the DNA or RNA. The following abbreviations are used:

G=Guanosine, A=Adenosine, T=Thymidine, C=Cytidine, R=G or A, Y=C or T,

K=G or T, W=A or T, S=C or G, M=A or C, B=C, G or T, D=A, G or T, H=A, C or T,

V=A, C or G, N=A, C, G or T, I=Inosine.

Real-Time Detection

In relation to this invention, real-time detection is defined as the simultaneous running of two processes: the detection of the DNA or RNA and a process which leads to the provision of a detectable amount of DNA or RNA. With this process the release of genomic DNA/RNA from cells may, for example, be involved or the enrichment of DNA/RNA from a complex mixture or the amplification of polynucleotides, e.g. through a PCR. Detection is the perception of a signal which correlates to the presence and possibly the amount of the DNA/RNA. In the case of the PCR this type of signal may increase with the increasing amplification of the target DNA. Real-time detection can be carried out also in a miniaturised form, e.g. on a chip. The signal can, for example, be produced through the fluorescent molecules of a probe, through radioactive molecules or through enzyme-coupled colour or fluorescence intensity.
The term real-time detection is synonymous to on-line detection.

Nucleic Acid

A “nucleic acid” is a DNA, RNA, PNA, or LNA which is obtained either through isolation from genomic DNA or from cDNA according to known standard methods and purified or generated artificially using known methods such as oligonucleotide synthesis or isolated as ribosomal RNA or mRNA from the organism or synthesised as PNA or LNA.
A “PNA” is a peptide nucleic acid in which instead of the phosphoric acid backbone of the DNA, 2-aminoethylglycin compounds occur.
A “LNA” is a locked nucleic acid in which the pentose moiety of DNA or RNA is modified to contain a 2′-O, 4′-C methylene linkage (1,2:5,6-di-O-isopropylene-alpha-D-allofuranose) to “lock” the nucleic acid in a certain conformational state.
According to the invention, in the nucleic acids, up to 20% of the nucleotides in 10 consecutive nucleotides, preferably however 1 nucleotide from a block of 10 consecutive nucleotides, may be replaced by nucleotides (e.g. inosin, etc.) which do not naturally occur in bacteria.
The nucleic acids may further contain modifications which allow the production of a signal that can be detected directly or indirectly. The expert is aware of the following modifications here:
(i) radioactive modifications, i.e. radioactive phosphorylation or radioactive marking with sulphur, hydrogen, carbon, nitrogen;
ii) colored groups (e.g. digoxygenin, etc.);
(iii) fluorescent groups (e.g. fluorescein, etc.);
(iv) chemoluminescent groups;
(v) groups for the immobilisation at a solid phase (e.g. biotin, streptag, antibodies, antigens, etc.); and/or
(vi) groups which allow an indirect or direct reaction with the help of antibodies, antigens, enzymes and/or substances with an affinity to enzymes or enzyme complexes;
or combinations of modifications according to two or more of the modifications listed under (i) to (vi). The term “modification” as used in this invention is understood to mean directly or indirectly detectable groups or groups for immobilisation at a solid phase which are attached to the nucleic acid. Metal atoms, radioactive, colored or fluorescent groups are directly detectable groups. Immunologically or enzymatically detectable groups are indirectly detectable groups, such as antigens and antibodies, haptenes or enzymes or parts of enzymes with an enzymatic effect. These indirect groups are detected in subsequent reactions. Preference is given to haptenes which are linked to an oligonucleotide and which are detected in a subsequent antibody reaction.

Primer

Primers are oligonucleotides which act as starter molecules during a PCR. Here, they hybridise on a target molecule, which may be, for example, DNA or RNA, and are lengthened by a polymerase. They can also however act as probes.

Probe

Probes are oligonucleotides or polynucleotides which hybridise on the target DNA or RNA molecules. They are used for the direct or indirect detection of these target DNA or RNA molecules. For this purpose, they can be coupled to fluorescent molecules or to molecules containing colouring agents. In addition they can be indirectly detected with an ELISA. In a special version they only produce a signal through FRET (Fluorescence Resonance Energy Transfer) when two probes hybridise adjacently in a defined manner. In this case a colouring agent on a probe is excited by a light beam and transfers its excitation energy to the colouring agent of the adjacent probe. This then emits light of a defined wavelength. In another special version signal generation relies on the 5′-3′ nuclease activity of Taq polymerase to cleave a dual-labelled probe during hybridization to the complementary target sequence. These dual-labelled probes consist of a fluorophore covalently attached to the 5′-end of the oligonucleotide probe and a quencher at the 3′-end. As long as the fluorophore and the quencher are in proximity, quenching inhibits any fluorescence signals. As the Taq polymerase extends the primer and synthesizes the nascent strand, the 5′ to 3′ exonuclease activity of the polymerase degrades the probe that has annealed to the template. Degradation of the probe releases the fluorophore from it and breaks the close proximity to the quencher, thus relieving the quenching effect and allowing fluorescence of the fluorophore.

Derivatives of the Oligonucleotides According to the Invention

Variations, Variants or derivatives of the oligonucleotides according to the invention are taken to mean sequences which differ in at least one nucleotide from the specific sequences according to SEQ ID numbers 1-22, for example, by at least one base interchange, an insertion, deletion or addition. These also include oligonucleotides which are at least 80% identical to one of the specific sequences according to SEQ ID numbers 1-22 and oligonucleotides with a comparable specificity of hybridisation. The latter signifies that the variation produces the same hybridisation pattern with a specified sample containing nucleic acid, such as the oligonucleotide with one of the specific sequences with one of the SEQ ID numbers 1-22.
Throughout the application, variants of any nucleic acid molecule of any of SEQ ID NO 1-22 are contemplated as alternatives to the respective oligonucleotide of SEQ ID NO 1-22. A variant of a nucleic acid molecule of any of SEQ ID NO 1-22 is

- a) a nucleic acid molecule which hybridizes under specific conditions with the respective nucleic acid molecule of any of SEQ ID NO 1-22;
- b) a nucleic acid molecule which is at least 70%, 80% or 90% identical with the nucleic acid molecule of a); or
- c) the complement of the nucleic acid molecule of SEQ ID NO 1-108 or of one of the variants a) to b).

Particular variants are also defined under “embodiments of the invention”.

Biochip

Biochip is taken to mean carriers for the high throughput of analyses as marketed, for example, by AFFYMETRIX. The chips enable the testing of numerous different nucleic acids on one carrier.

Hybridisation

The term “hybridisation” is understood as the double strand formation of two identical or similar nucleic acid fragments (DNA, RNA, PNA, LNA). Specific hybridisation is the term used if the hybridisation is carried out under strict conditions and gives a stable hybrid nucleic acid. In the terms of this invention, the feature “sequence which specifically hybridises with a sequence according to (i)” refers to a sequence which hybridises under strict conditions with the sequence according to (i).
The stringency of the hybridization conditions depends on various factors such as the base composition of the hybrid, the level and geometry of mispairing between the two nucleic acids, as well as reaction parameters like ionic strength and the reaction temperature. Appropriate conditions can be determined based on thermodynamic calculations (SantaLucia et. al. 1996), which have been incorporated into a variety of computer programs known to those skilled in the art. Examples of stringent conditions are set forth in the example section below.

Melting Curves and Melting Temperature

In relation to this invention, a melting curve analysis of a sample is defined as the following sequence of processes: the heating of the sample to a temperature high enough to denature the DNA in the sample; the cooling of the sample to a temperature below the annealing temperature of the target DNA; a slow heating of the sample with the simultaneous (real-time, online) detection of the dissociation (i.e., melting) of the DNA. Detection is the perception of a signal which correlates to the proportion of dissociated and not yet dissociated DNA. In the case of a melting curve after a PCR with hybridization probes or with a double strand binding dye, this type of signal may decrease with the increasing temperature. An inflexion point of the signal course is defined as melting temperature. The melting temperature of a sample is the point at which half the probes or the double strand binding dye have melted off the target DNA. For a better visualization, the first negative derivative of the signal course can be plotted against the temperature; the melting temperatures then appear as peaks of the curve. A melting curve can also be carried out in a miniaturised form, e.g. on a chip.
With a preferred embodiment the nucleic acid to be examined is passed to a PCR. This has the result that Cronobacter-specific amplicons are produced if nucleic acids of Cronobacter bacteria are present in the sample. Here in the simplest case, the PCR can be arranged as a simple linear PCR with only one oligonucleotide as primer, but preferably the PCR takes place however with so-called forwards and backwards primers for each genome section of the bacterial nucleic acid to be amplified.
With a further preferred embodiment various oligonucleotides and therefore various PCR runs are carried out in the form of a multiplex PCR. Here, different amplicons are created in the PCR in a single initiated reaction with the aid of the various oligonucleotides. Especially meaningful is the combination of oligonucleotides of this invention specific for the genus Cronobacter with oligonucleotides for the detection of the whole family Enterobacteriaceae because according to directive (EU) nr. 1441/2007 milk-based infant formula has to be analysed for Enterobacteriaceae and E. sakazakii in parallel unless a direct correlation could be found.
With a further preferred embodiment use is made of the so-called chip technology (biochips) in the detection method. Here, on one hand a large number of different analysis samples can be analysed on one chip in that the individual spots on the chip contain analysis material from different sources. On the other hand, the chip can carry a set of oligonucleotides, whereby each spot contains a specific oligonucleotide and this oligonucleotide pattern is brought into contact with analysis samples. In the case that the analysis material contains Cronobacter nucleic acid, it hybridises with the probes specific to the Cronobacter present on the chip and produces a corresponding signal pattern.
With a further preferred embodiment the detection method can include further steps, such as for example an amplification of the nucleic acid to be detected, whereby this preferably occurs using PCR and/or a southern hybridisation with Cronobacter-specific probes, whereby this hybridisation occurs without prior amplification or after amplification of the nucleic acid to be detected is concluded. Furthermore, the nucleic acid to be detected can be detected using the ligase chain reaction. Finally, the nucleic acid to be detected can be enriched by isothermal nucleic acid amplification.
With a further preferred embodiment, the amplification of the target nucleic acid can also take place using real-time detection.
With a further preferred embodiment the amplification of the nucleic acid to be detected and/or the detection of the contained amplicons occur on a biochip, whereby it is particularly preferable to carry out the amplification and detection on one chip.
According to the invention, as a means for carrying out the method described above, oligonucleotides are selected from a nucleic acid, comprising at least one sequence with one of the SEQ ID numbers 1-22 or variations thereof. The stated oligonucleotides can on one hand be used as primers within the scope of a PCR and on the other hand also as probes, for example within the scope of a southern blot hybridisation. Depending on the requirements of the desired detection, the specialist can form the suitable combination of oligonucleotides as primers or probes. Preferably, the stated oligonucleotides or combinations of them are used in the form of a kit for the detection of Cronobacter spp., whereby the kit also includes other reagents for the detection of bacteria or for conducting the detection reactions. In this respect, the reagents and enzymes required for the PCR and, where applicable, suitable carrier materials are also included, for example, such as it is desired with the chip technology.
The oligonucleotides or oligonucleotide combinations according to the invention are therefore a suitable means for the specific and reliable detection of Cronobacter spp. in any analysis samples.
With the invention of the polymerase chain reaction it is possible to amplify individual DNA polynucleotides and then to detect them with extremely high sensitivity. This technology opens up substantial new opportunities, but also exhibits new problems. For example, with the DNA amplification incorrect fragments can be easily amplified, leading to incorrect positive results in the analysis. In addition, it is very difficult to select the diagnostic DNA sequences characteristic to Cronobacter from the multitude of possibilities.
This invention consists of a method and oligonucleotides which enable a qualitative and quantitative detection of Cronobacter spp. The detection method consists all together of four steps: propagation of the bacteria, purification of the DNA/RNA, amplification of the polynucleotides and detection of them. In a special method the two last steps can also take place simultaneously.
The propagation of the bacteria occurs in that the matrix to be investigated, e.g. an infant formula sample is incubated with a currently available bacterial medium. Bacterial media are commercially available and can, for example, contain a proteolytically digested basic substance, such as peptone broth, and a buffer such as dipotassium hydrogen phosphate.
In the second step the polynucleotides are purified. To do this, the bacteria are normally first separated from the medium by centrifuging and/or filtration. A further washing stage may follow. Then the bacteria are broken down. This takes place by heating, by an alkaline or acidic environment or by reagents which destabilise the bacteria cell wall, such as deionising chemicals or lysozyme. The genomic DNA or the RNA can now be directly used in a PCR reaction or it is purified further. For this purification materials are suitable on the surface of which the polynucleotides bond, e.g. positively charged surfaces or silicate surfaces. This material can be mounted in columns and is commercially available.
To avoid an amplification of DNA from dead cells the cells could be treated with phenanthridinium derivates with a light-sensitive azide group like ethidium monoazide bromide (EMA) or propidium monoazide bromide (PMA) before the DNA isolation procedure starts. This treatment consists of two steps. In the first step cells and phenanthridinium derivates are incubated together in the dark to allow these compounds to penetrate cells with compromised cell membranes. Afterwards this mixture will be placed under a high power light bulb and the light-sensitive azide group react with the DNA. By this covalent bond an amplification of the DNA will be inhibited.
The PCR reaction and the detection of the amplicons have great importance in the detection of bacteria. As already explained, it is very difficult to find differences in DNA sequences between the genus Cronobacter and other bacteria, in particular closely related species like E. helveticus and E. pulveris.
To avoid false negative results, in a preferred embodiment of the invention, an amplification control nucleic acid is added during amplification. Accordingly, not only the microbial nucleic acid or a part thereof is amplified, but also the amplification control nucleic acid, which means that at least one amplification fragment in each case is created. The amplification control nucleic acid, e.g. a DNA fragment, is here an “internal standard molecule” which serves as an indicator for the effectiveness of the reaction (Ballagi-Pordany, Belak, 1996, Mol. Cell. Probes 10, 159-164). It is added in a defined quantity to the amplification reaction and amplified in parallel. The amplification control nucleic acid is preferably single or double strand and may be of unlimited length. Amplification control nucleic acids with a length of up to a thousand nucleotides have proved successful. In a preferred embodiment of this invention, the amplification control nucleic acid contains a sequence that is identical or complementary to the sequence of SEQ ID NO 22 or a part of it.
For good laboratory praxis in a diagnostic procedure it is recommended to monitor the whole analytical process by using external positive controls. In the case of the detection of Cronobacter spp. according to this invention the analysis of an additional positive control sample containing Cronobacter cells enables to check the complete process comprising microbial enrichment, DNA isolation and PCR. A drawback of the application of such a positive control is the possibility to get false-positive results due to cross-contamination by the positive control. False-positive results could be avoided by using of a positive control that can be differentiated from a true contamination by Cronobacter bacteria. A further embodiment of this invention is the discrimination of a rare subgroup from most common Cronobacter strains. This discrimination is possible via melting curve analysis using SEQ ID NO 4-7 and SEQ ID NO 8-9 or via direct detection of a subgroup of C. turicensis using SEQ ID NO 19-21.
Embodiments of the present invention are:

1. Nucleic acid molecule suitable as primer or probe in the detection of the genus Cronobacter, wherein
- i) the nucleic acid molecule
  - a) is a sequence comprising or consisting of a partial sequence of SEQ ID NO: 1, which partial sequence is highly conserved or conserved in all species of the genus Cronobacter; and
  - b) is between 10-50, preferably 15-40 and more preferably 20-35 nucleic acids long;
- ii) the nucleic acid molecule
  - a) is a sequence which hybridizes under specific or stringent conditions with the nucleic acid molecule of i); and
  - b) is between 10 and 50, preferably 15-40 and more preferably 20-35 nucleic acids long; or
- iii) the nucleic acid molecule
  - a) is at least 70, 75, 80, 85, 90 or 95% identical with the nucleic acid molecule, wherein identity may be determined over the stretch of at least 15, 20 or 25, preferably 15-50 nuclei acids, more preferably 15-40 and most preferably 20-35 nucleic acids; and
  - b) is between 10 and 50, preferably 15-40 and more preferably 20-35 nucleic acids long; or
- iv) the nucleic acid molecule is the complement of the nucleic acid molecule of any of i) to iii).

“Highly conserved” in embodiment 1 means suitable to hybridize under stringent conditions (59° C.-66° C.) with all members of the genus Cronobacter; at least with C. sakazakii, C. malonaticus, C. muytjensii, C. dublinensis, C. genomospecies 1 and preferably all or at least parts of the species C. turicensis; preferably not more than 4, 3, 2 or 1 mismatches in a stretch of 20 nucleotides occur with SEQ ID NO: 1; the highly conserved sequence may be a degenerate sequence.
“Conserved” in embodiment 1 means suitable to hybridize only under non-stringent conditions (50° C.-55° C.) with a subgroup of the species C. turicensis. “Conserved” sequences hybridize under stringent conditions (59° C.-66° C.) with the members of the genus Cronobacter C. sakazakii, C. malonaticus, C. muytjensii, C. dublinensis, C. genomospecies 1.

2. The nucleic acid molecule of embodiment 1, which is
- a) selected from SEQ ID NOs: 2, 3, which are primers, 4-7, which are degenerate probes; 8-9, which are degenerate probes, 10, which is a probe, or 11-18, which are degenerate probes; or
- b) a sequence which hybridizes under specific conditions with the nucleic acid molecule of a);
- c) at least 70, 75, 80, 85, 90 or 95% identical with the nucleic acid molecule of a), wherein identity may be determined over the stretch of at least 15, 20 or 25 preferably 15-50 nuclei acids, more preferably 15-40 and most preferably 20-35 nucleic acids; or
- d) the complement of the nucleic acid molecule of any of a) to c).
3. Nucleic acid molecule suitable as probe in the detection of a subgroup of Cronobacter, namely the species Cronobacter turicensis, wherein these probes may but do not have to detect all strains of Cronobacter turicensis and wherein
- i) the nucleic acid molecule
  - a) is a sequence comprising or consisting of a partial sequence of SEQ ID NO: 19, which partial sequence is highly conserved a subgroup of the species C. turicensis; and
  - b) is between 10 and 50, preferably 15-40, more preferably 20-35 nucleic acids long;
- ii) the nucleic acid molecule
  - a) is a sequence which hybridizes under specific conditions with the nucleic acid molecule of i); and
  - b) is between 10 and 50 preferably 15-40, more preferably 20-35 nucleic acids long; or
- iii) the nucleic acid molecule
  - a) is at least 70, 75, 80, 85, 90 or 95% identical with the nucleic acid molecule, wherein identity may be determined over the stretch of at least 15, 20 or 25 preferably 15-50 nuclei acids, more preferably 15-40 and most preferably 20-35 nucleic acids; and
  - b) is between 10 and 50, preferably 15-40, more preferably 20-35 nucleic acids long; or
- iv) the nucleic acid molecule is the complement of the nucleic acid molecule of any of i) to iii).

“Highly conserved” in embodiment 3 means suitable to hybridize under stringent conditions (59° C.-64° C.) with a subgroup of the species C. turicensis (for the subgroup of the species C. turicensis see e.g. example 3).

4. The nucleic acid molecule of embodiment 3, which is
- a) selected from SEQ ID NOs: 20-21, which are probes; or
- b) a sequence which hybridizes under specific conditions with the nucleic acid molecule of a);
- c) at least 70, 75, 80, 85, 90, or 95% identical with the nucleic acid molecule of a), wherein identity may be determined over the stretch of at least 15, 20 or 25 preferably 15-50 nuclei acids, more preferably 15-40 and most preferably 20-35 nucleic acids; or
- d) the complement of the nucleic acid molecule of any of a) to c).
5. Use of one or more nucleic acid molecules of any of embodiments 1 or 2 in the detection of the genus Cronobacter.

Preferably a pair of primer is employed, most preferably SEQ ID NOs: 2 and 3, optionally plus one or more probes, wherein most preferably the probes are selected from SEQ ID Nos 4-7 and/or 8-9. For all of these SEQ ID NOs respective variants are contemplated.
The nucleic acids used in embodiment 5 may be part of a kit suitable for the detection of the genus Cronobacter.

6. A combination of two or more nucleic acids of any of embodiments 1 to 2, optionally in combination with one or more nucleic acids of any of embodiments 3 to 4.

Suitable combinations include

- a) a pair of primers of embodiments 1 or 2;
- b) preferably a pair of primers of embodiments 1 or 2 plus 1 or 2 probes of embodiment 2;
- c) more preferably a pair of primers of embodiments 1 or 2 plus 1 or 2 probes of embodiment 2 plus 1 or 2 probes of embodiment 3 or 4; or
- d) more preferably a pair of primers of embodiment 1 or 2 plus probes of embodiments 3 or 4.

Optionally the combinations above may be provided in combination with two or more nucleic acids suitable as primer or probe in the detection of the genus Enterobacteriaceae. These Sequences may be those contemplated in European Patent application EP1254254—NUCLEIC ACID MOLECULES FOR DETECTING BACTERIA AND PHYLOGENETIC UNITS OF BACTERIA or variants of the respective sequences or SEQ ID NOs, which variants are defined as in this patent application.
The combinations of nucleic acids of embodiment 6 may be part of a kit.

7. Use of one or more nucleic acid molecules of any of embodiments 1 or 2 together with one or more nucleic acids of any of embodiments 3 to 4 in the detection of the genus Cronobacter and the discrimination of Cronobacter turicensis against other species.
8. A method for amplifying bacterial DNA of the taxonomic unit genus Cronobacter or for detecting Cronobacter in a sample, using primers, in which
- a) in a first amplification step the DNA of the genus Cronobacter is amplified with conserved primers of the present invention,
- b) optionally, in at least one further amplification step the DNA of the family Enterobacteriaceae is amplified with conserved primers;
- c) optionally, in a further detection step, the DNA fragments obtained by amplification step a) or b), which are specific for the genus Cronobacter or species within said genus or the family Enterobacteriaceae, respectively, are detected by means of probes,
  wherein the primers used in the first amplification step comprise a nucleic acid as defined in any of embodiments 1 or 2;
  wherein optionally the probes used in step c) comprise a nucleic acid as defined in any of embodiments 1 or 2 which are specific for the genus Cronobacter;
  wherein optionally the probes used in step c) comprise a nucleic acid as defined in any of embodiments 3 or 4 which are specific for the species within the genus Cronobacter; and
  wherein the primers and probes for the amplification and/or detection of the family Enterobacteriaceae are as set forth under embodiment 6.
9. The method of embodiment 8, wherein
- d) in a further detection step, the DNA fragments obtained by amplification step a) which are specific for the genus Cronobacter are detected by means of probes,
  wherein the probes used in step b) comprise a nucleic acid as defined in any of embodiments 1 or 2 which are specific for the genus Cronobacter.
10. The method of embodiment 8 or 9, wherein
- e) in a further detection step, the DNA fragments obtained by amplification step a) which are specific for the species Cronobacter turicensis are detected by means of probes,
  wherein the probes used in step c) comprise a nucleic acid as defined in any of embodiments 3 or 4.
11. The method of any of embodiments 8 to 10, wherein
- f) in at least one further amplification step the DNA of the taxonomic unit Enterobacteriaceae is amplified with conserved primers.
12. The method of embodiment 11, wherein
- g) in a further detection step, the DNA fragments obtained by amplification step f) which are specific for Enterobacteriaceae are detected by means of probes.
13. The method of any of embodiments 8 to 12, wherein the process involves a PCR amplification of the nucleic acid to be detected.
14. The method of any of embodiments 8 to 12, wherein the process involves a Southern Blot hybridization.
15. The method of any of embodiments 9 to 14, wherein one or more probes are modified or labelled so that it can generate a signal in analytical detection procedures which are known per se, with the modification selected from (i) radioactive groups, (ii) coloured groups, (iii) fluorescent groups, (iv) groups for immobilization of a solid phase, and (v) groups which allow a direct or indirect reaction, especially using antibodies, antigens, enzymes, and/or substances with affinity to enzymes or enzyme complexes.
16. The method of any of embodiments 8 to 15, including a step
of amplifying an amplification control nucleic acid, which is added during amplification step a).

A suitable probe for detecting the amplification control is SEQ ID NO:22 or a variant thereof.

17. The method of any of embodiment 9 to 13 or 15 to 16 further including a melting curve analysis, wherein the hybridization behavior of one or more particular probes is studied with time and is used to discriminate between different species of a genus.

Preferably a C. turicensis subgroup may be discriminated against other Cronobacter species.

18. The method of any of embodiments 8 to 13 or 15 to 17, wherein viable Cronobacter or other Enterobacteriaceae cells are discriminated against non-viable Cronobacter or other Enterobacteriaceae cells, respectively, wherein an intercalator, e.g. EMA or PMA, is employed to inactivate the DNA in non-viable cells.

FIGURES OF THE INVENTION

FIG. 1 shows the DNA region as well as the hybridization positions of the oligonucleotides of this invention.

FIG. 2 shows the Comparison of the 23S-ITS2-5S region of the type strain of C. sakazakii (DSM 4485) with sequences of the species E. helveticus, E. pulveris and E. turicensis.

FIG. 3 shows a melting curve analysis for differentiation of C. turicensis from other Cronobacter spp

FIG. 4 shows the simultaneous detection of Cronobacter spp. and Enterobacteriaceae by probe-based multiplex real-time PCR.

FIG. 5 shows the selective amplification of DNA from viable Cronobacter cells after treatment with EMA. Enrichment cultures with 10⁶/ml viable or dead C. sakazakii cells were prepared with or without treatment with ethidium monoazide bromide (EMA). The resulting DNA extracts were tested with the multiplex real-time PCR for Cronobacter and Enterobacteriaceae detection.

EXAMPLES

Example 1

Comparison of Three Primer Pairs Targeting the Ribosomal Operon Regarding Detection Sensitivity of DNA of Cronobacter spp.

Genomic DNA was isolated from pure cultures of the bacteria listed in Table 1 using known standard methods. Dilutions (DNA concentration 100 pg/μl and 1 pg/μl) of these preparations were then added to three different PCR mixes with the following compositions:
Mix A: with primer pair es-16s-for (CAA GTC GAA CGG TAA CAG GG)/es-16s-rev (GTC CCC CAC TTT GGT CCG) targeting the 16S rDNA gene published by Malorny & Wagner, 2005 (J. Food Prot. 68: 1623-1627).
Mix B: with primer pair ESFor (ATC TCA AAA MTG ACT GTA AAG TCA CGT T)/ESRevB (CCG AAR AAG TMT TCG KGC TGC GA) targeting the region between tRNA-glu- and 23S rDNA-gene published by Derzelle & Dilasser, 2006 (BMC Microbiol. 6: 100).
Mix C: with primer pair SEQ ID NO: 2/SEQ ID NO: 3 targeting the 23S-ITS2-5S region of this invention.


	Final Conc.	Final Conc.	Final Conc.
Component	Mix A	Mix B	Mix C

H₂O	—	—	—
PCR buffer	1 x conc.	1 x conc.	1 x conc.

MgCl₂	4.5	mM	3	mM	3.75	mM
dNTP-mix	200	μM	200	μM	200	μM

each

Primer es-16s-for	400	nM	—	—
Primer es-16s-rev	400	nM	—	—

Primer ESFor	—	500	nM	—
Primer ESRevB	—	500	nM	—

SEQ ID NO: 2	—	—	600	nM
SEQ ID NO: 3^a)	—	—	600	nM

Taq DNA Polymerase

0.06

U/μl

0.06

U/μl

0.06

U/μl

Sybr Green ®	0.25 x conc	0.25 x conc	0.25 x conc
Sample DNA	Var.	Var.	Var.

^a)Complement sequence was used

As described by Derzelle & Dilasser the “FastStart Taq Polymerase” (Roche Diagnostics) was used to prevent unspecific amplification. The DNA intercalating fluorescent dye Sybr Green® was added to estimate the PCR efficiency by measuring the CT-values on a real-time PCR instrument.
The PCR was carried out on a LightCycler® 480 instrument (Roche Diagnostics) according to the following protocols:


Protocol 1 for mix A and C

Initial denaturation	95° C.	10	min.
Amplification (45 cycles)	95° C.	15	s.
	66° C.	30	s.
	72° C.	5	s.
	84° C.	10	s. (fluorescence acquisition)

Protocol 2 for mix B and C

Initial denaturation	95° C.	10	min.
Amplification (45 cycles)	95° C.	15	s.
	60° C.	10	s.
	72° C.	25	s. (fluorescence acquisition)

TABLE 1

Detection sensitivity of primer pair SEQ ID NO 2 and
SEQ ID NO 3 (mix C) in comparison to two other
primer pairs targeting the ribosomal operon

CP-Value¹

		Mix A	Mix B	Mix C	Mix C
	DNA	Proto-	Proto-	Proto-	Proto-
Strain	amount	col	1	col 2	col 1	col 2

C. sakazakii DSM 4485	250	pg	15.5	22.1	15.9	16.3
C. sakazakii DSM 4485	2.5	pg	23.5	33.7	22.7	24.6
C. dublinensis subsp.	250	pg	29.4	21.8	16.7	16.9
dublinensis DSM 18705
C. dublinensis subsp.	2.5	pg	31.8	31.1	23.5	24.3
dublinensis DSM 18705
C. dublinensis subsp.	250	pg	18.8	23.1	15.7	16.1
lactaridi DSM 18707
C. dublinensis subsp.	2.5	pg	25.4	33.4	22.6	23.8
lactaridi DSM 18707
C. malonaticus DSM 18702	250	pg	16.3	24.0	16.5	17.8
C. malonaticus DSM 18702	2.5	pg	23.5	34.4	23.5	24.6
C. muytjensii ATCC 51329	250	pg	34.5	23.7	15.7	16.0
C. muytjensii ATCC 51329	2.5	pg	negative	33.8	22.5	22.9
C. turicensis DSM 18703	250	pg	20.8	32.0	15.6	16.2
C. turicensis DSM 18703	2.5	pg	25.3	35.2	22.5	22.7

¹CP-Value: The crossing point is calculated by the LightCycler Software as the cycle at which PCR amplification begins its detectable exponential phase

Example 2

Discrimination of C. turicensis from Other Cronobacter spp. by Melting Curve Analysis

Genomic DNA was isolated from pure cultures of the bacteria listed in FIG. 2 using known standard methods. The DNA of these preparations was then added to the PCR mix with the following composition (final volume 20 μl):


	Component	final Concentration

	H₂O	—
	PCR buffer	1 x conc.

	MgCl₂	3.75	mM
	dNTP-mix	200	μM each
	SEQ ID NO: 2	1	μM
	SEQ ID NO: 3^a)	300	nM
	SEQ ID NO: 4-7^b)	45	nM each
	SEQ ID NO: 8-9^c)	100	nM each
	Taq DNA Polymerase	0.06	U/μl
	Sample DNA	125	fg/μl

^a)Complement sequence
^b)3′-labeled with fluorescein, complement sequence
^c)5′-labeled with Cy5, complement sequence

FIG. 2 shows the Comparison of the 23S-ITS2-5S region of the type strain of C. sakazakii (DSM 4485) with sequences of the species E. helveticus, E. pulveris and E. turicensis.
The PCR was carried out on a LightCycler® 2.0 instrument (Roche Diagnostics) according to the following protocol:


Initial denaturation	95° C.	10	min.
Amplification (45 cycles)	95° C.	0	s.
	59° C.	30	s.
	72° C.	5	s.
Melting curve analysis	95° C.	0	s.

(1 cycle)	40° C.	45	s.
				{close oversize brace}	ramp rate 0, 1°/s
	75° C.	0	s.

Cooling	40° C.	30	s.

The melting curve analysis for differentiation of C. turicensis from other Cronobacter spp is shown in FIG. 3. Coupled to fluorescent molecules oligonucleotides of SEQ ID NO: 4-7 and SEQ ID NO: 8-9 which hybridise adjacently enable the differentiation of C. turicensis from other Cronobacter species. In addition it is possible to discriminate between different strains of C. turicensis.

Example 3

Specific Detection of a Subgroup of C. turicensis

Genomic DNA was isolated from pure cultures of the bacteria listed in Table 2 using known standard methods. The DNA of these preparations was then added to the PCR mix with the following composition (final volume 20 μl):


	Component	Final Concentration

	H₂O	—
	PCR buffer	1 x conc.

	MgCl₂	3.75	mM
	dNTP-mix	200	μM each
	SEQ ID NO: 2	600	nM
	SEQ ID NO: 3^a)	600	nM
	SEQ ID NO: 20^b)	180	nM
	SEQ ID NO: 21^c)	200	nM
	Taq DNA Polymerase	0.06	U/μl
	Sample DNA	125	fg/μl

^a)Complement sequence
^b)3′-labeled with fluorescein, complement sequence
^c)5′-labeled with Cy5, complement sequence

The PCR was carried out on a LightCycler® 2.0 instrument (Roche Diagnostics) according to the following protocol:


Initial denaturation	95° C.	10	min.
Amplification (45 cycles)	95° C.	0	s.
	59° C.	30	s. (fluorescence acquisition)
	72° C.	5	s.
Cooling	40° C.	30	s.

The results of the real-time PCR using the oligonucleotides SEQ ID NO: 20 and SEQ ID NO: 21 as probes are shown in table 2. Only the two C. turicensis strains LMG 2790 and E609 were detected.

TABLE 2

Specific PCR detection of a C. turicensis subgroup using
SEQ ID NO 2, 3, 20 and 21

Species	Strain	PCR result

C. turicensis	LMG 2790	+
C. turicensis	E609	+
C. turicensis	DSM 18703	−
C. turicensis	BCD 15786	−
C. dublinensis subsp. dublinensis	DSM 18705	−
C. dublinensis subsp. lactaridi	DSM 18707	−
C. malonaticus	DSM 18702	−
C. muytjensii	ATCC 51329	−
C. sakazakii	DSM	4485	−
C. genomospecies 1	NCTC 9529	−

Example 4

Detection of Cronobacter spp. by Probe-Based Real-Time PCR

Genomic DNA was isolated from pure cultures of the bacteria listed in Table 3 using known standard methods. The DNA of these preparations (concentration in the area of approx. 10 ng-100 pg) as well as the amplification control DNA were then added to the PCR mix with the following composition (final volume 20 μl):


	Component	final Concentration

	H₂O	—
	PCR buffer	1 x conc.

MgCl₂	3.75	mM
dNTP-mix	200	μM each
SEQ ID NO: 2	600	nM
SEQ ID NO: 3^a)	600	nM
SEQ ID NO: 4-7^b)	45	nM each
SEQ ID NO: 20^b)	180	nM
SEQ ID NO: 8-9^c)	100	nM each
SEQ ID NO: 21^c)	200	nM
SEQ ID NO: 22^d)	200	nM
Taq DNA Polymerase	0.06	U/μl
Control DNA	0.25	fg/μl

	Sample DNA	var.

^a) Complement sequence
^b)3′-labeled with fluorescein, complement sequence
^c)5′-labeled with Cy5, complement sequence
^d)5′-labeled with LC-Red ® 610

Amplification of Cronobacter spp. DNA was detected in channel 670/back 530. In cases of a negative result amplification of the control DNA should be visible in channel 610/back 530. The results of the probe-based real-time PCR are shown in table 3. All tested Cronobacter strains comprising all species and subspecies as well as C. genomospecies 1 were detected. In contrast, none of the tested bacterial strains not belonging to this genus was registered with this system.
The amplification control was detected in channel 610/back 530 for all samples with a negative result for Cronobacter DNA. The quality of these non-Cronobacter DNA samples was checked with consensus PCR system specific for all eubacteria targeting the 16S rDNA (Barry et al., 1990, Biotechnology 8: 233-236).

TABLE 3

Specific PCR detection of the genus Cronobacter using
SEQ ID NO 2, 3, 4-7, 8-9, 20 and 21

	Species	Strain¹	PCR result

	Cronobacter dublinensis subsp.	DSM 18705	+
	dublinensis
	Cronobacter dublinensis subsp.	DSM 18707	+
	lactaridi
	Cronobacter dublinensis subsp.	NCTC 9844	+
	Cronobacter malonaticus	DSM 18702	+
	Cronobacter muytjensii	ATCC 51329	+
	Cronobacter sakazakii	DSM 4485	+
	Cronobacter turicensis	DSM 18703	+
	Cronobacter turicensis	LMG 2790	+
	Cronobacter turicensis	E609	+
	Cronobacter turicensis	BCD 15786	+
	Cronobacter genomospecies 1	NCTC 9529	+
	Enterobacter sakazakii	BCD 15300	+
	Enterobacter sakazakii	BCD 15301	+
	Enterobacter sakazakii	BCD 15361	+
	Enterobacter sakazakii	BCD 15381	+
	Enterobacter sakazakii	BCD 15410	+
	Enterobacter sakazakii	BCD 15428	+
	Enterobacter sakazakii	BCD 15429	+
	Enterobacter sakazakii	BCD 15430	+
	Enterobacter sakazakii	BCD 15431	+
	Enterobacter sakazakii	BCD 15432	+
	Enterobacter sakazakii	BCD 15433	+
	Enterobacter sakazakii	BCD 15434	+
	Enterobacter sakazakii	BCD 15435	+
	Enterobacter sakazakii	BCD 15436	+
	Enterobacter sakazakii	BCD 15437	+
	Enterobacter sakazakii	BCD 15438	+
	Enterobacter sakazakii	BCD 15439	+
	Enterobacter sakazakii	BCD 15440	+
	Enterobacter sakazakii	BCD 15441	+
	Enterobacter sakazakii	BCD 15442	+
	Enterobacter sakazakii	BCD 15443	+
	Enterobacter sakazakii	BCD 15444	+
	Enterobacter sakazakii	BCD 15445	+
	Enterobacter sakazakii	BCD 15446	+
	Enterobacter sakazakii	BCD 15447	+
	Enterobacter sakazakii	BCD 15448	+
	Enterobacter sakazakii	BCD 15449	+
	Enterobacter sakazakii	BCD 15450	+
	Enterobacter sakazakii	BCD 15451	+
	Enterobacter sakazakii	BCD 15452	+
	Enterobacter sakazakii	BCD 15459	+
	Enterobacter sakazakii	BCD 15460	+
	Enterobacter sakazakii	BCD 15461	+
	Enterobacter sakazakii	BCD 15462	+
	Enterobacter sakazakii	BCD 15463	+
	Enterobacter sakazakii	BCD 15464	+
	Enterobacter sakazakii	BCD 15469	+
	Enterobacter sakazakii	BCD 15470	+
	Enterobacter sakazakii	BCD 15471	+
	Enterobacter sakazakii	BCD 15472	+
	Enterobacter sakazakii	BCD 15473	+
	Enterobacter sakazakii	BCD 15474	+
	Enterobacter sakazakii	BCD 15475	+
	Enterobacter sakazakii	BCD 15476	+
	Enterobacter sakazakii	BCD 15477	+
	Enterobacter sakazakii	BCD 15478	+
	Enterobacter sakazakii	BCD 15488	+
	Enterobacter sakazakii	BCD 15489	+
	Enterobacter sakazakii	BCD 15490	+
	Enterobacter sakazakii	BCD 15492	+
	Enterobacter sakazakii	BCD 15495	+
	Enterobacter sakazakii	BCD 15497	+
	Enterobacter sakazakii	BCD 15498	+
	Enterobacter sakazakii	BCD 15499	+
	Enterobacter sakazakii	BCD 15500	+
	Enterobacter sakazakii	BCD 15501	+
	Budvicia aquatica	DSM 5075	−
	Buttiauxella agrestis	DSM 4586	−
	Cedecea davisae	DSM 4568	−
	Citrobacter amalonaticus	DSM 4593	−
	Citrobacter freundii	BCD 4696	−
	Citrobacter koseri	DSM 4595	−
	Edwardsiella tarda	DSM 30052	−
	Enterobacter aerogenes	DSM 30053	−
	Enterobacter amnigenus	DSM 4486	−
	Enterobacter cancerogenus	CCM 2421	−
	Enterobacter cloacae	DSM 30054	−
	Enterobacter cowanii	CCM 7015	−
	Enterobacter gergoviae	BCD 674	−
	Enterobacter helveticus	DSM 18396	−
	Enterbacter hormaechei	DSM 12409	−
	Enterobacter pulveris	DSM 19144	−
	Enterobacter pyrinus	DSM 12410	−
	Enterobacter turicensis	DSM 18397	−
	Erwinia carotovora	DSM 30168	−
	Erwinia crysanthemi	DSM 4610	−
	Escherichia coli	DSM 30083	−
	Escherichia hermannii	DSM 4560	−
	Escherichia vulneris	DSM 4564	−
	Hafnia alvei	DSM 30163	−
	Klebsiella oxytoca	DSM 5175	−
	Klebsiella pneumoniae	DSM 30102	−
	Kluyvera ascorbata	DSM 4611	−
	Kluyvera cryocrescens	DSM 4588	−
	Leclercia adecarboxylata	DSM 5077	−
	Morganella (Proteus)morganii	DSM 30164	−
	Pantoea agglomerans	DSM 3493	−
	Pantoea ananatis	DSM 30070	−
	Pantoea dispersa	DSM 30073	−
	Proteus hauseri	DSM 30118	−
	Proteus mirabilis	DSM 788	−
	Providencia alcalifaciens	DSM 30120	−
	Providencia stuartii	DSM 4539	−
	Rahnella aquatilis	DSM 4594	−
	Raoultella (Klebsiella)planticola	DSM 4617	−
	Raoultella (Klebsiella)terrigena	DSM 2687	−
	Salmonella Abony	DSM 4224	−
	Salmonella bongori	BCD 14407	−
	Serratia ficaria	DSM 4569	−
	Serratia marcescens	DSM 1636	−
	Serratia proteamaculans	DSM 4487	−
	Serratia rubidaea	DSM 4480	−
	Shigella boydii	DSM 7532	−
	Yersinia aldovae	ATCC 35236	−
	Yersinia enterocolitica	DSM 4780	−
	Yersinia frederiksenii	ATCC 33641	−

For all negative tested strains was the amplification control positive.
¹Abbreviations stands for the following culture collections:
ATCC: American Type Culture Collection, Manassas, USA
BCD: BIOTECON Diagnostics culture collection, Potsdam, Germany
CCM: Czech Collection of Microorganisms, Brno, Czech Republic
DSM: German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany
LMG: BCCM/LMG Bacteria Collection, Gent, Belgium
NCTC: National Collection of Type Cultures, Salisbury, UK

Example 5

Simultaneous Detection of Cronobacter spp. and Enterobacteriaceae by Probe-Based Multiplex Real-Time PCR

Genomic DNA was isolated from pure cultures of the bacteria listed in FIG. 3 using known standard methods. The DNA of these preparations (concentration approx. 5-50 ng) as well as the amplification control DNA was then added to the multiplex PCR mix with the following composition (final volume 25 μl):


	Component	Final Concentration

	H₂O	—
	PCR buffer	1 x conc.

MgCl₂	3.75	mM
dNTP-mix	200	μM each
SEQ ID NO: 2	600	nM
SEQ ID NO: 3^a)	600	nM
SEQ ID NO: 8-9^b)	125	nM each
SEQ ID NO: 21^b)	250	nM
Enterobacteriaceae forward-primer	600	nM
Enterobacteriaceae reverse-primer	600	nM
Enterobacteriaceae probes 1-4^c)	150	nM each
Taq DNA Polymerase	0.06	U/μl
Control DNA	0.25	fg/μl

	Sample DNA	var.

^a) Complement sequence
^b)5′-labeled with FAM, 3′-labeled with a quencher, complement sequence
^c)5′-labeled with HEX, 3′-labeled with a quencher

The Enterobacteriaceae forward-primer is equivalent to SEQ ID NO 2 (TTC GGG TTG TCA TGC CAA TG), the Enterobacteriaceae reverse-primer to the complement sequence of SEQ ID NO 78 (ACC CGT GAG GCT TAA CCT TAC AAC ACC GAA), and the Enterobacteriaceae probes 1-4 are equivalent to SEQ ID NO 3 (CTG AAA GCA TCT AAG CGC GAA ACT TG), SEQ ID NO 4 (CTG AAA GCA TCT AAG CGG GAA ACT TG), SEQ ID NO 5 (CTG AAA GCA TCT AAG CAC GAA CTT G) and SEQ ID NO 6 (CTG AAA GCA TCT AAG CAC GAA ACT TG) of the invention WO 01/023606.
The PCR was carried out on a LightCycler® 480 instrument (Roche Diagnostics) according to the following protocols:


Initial denaturation	95° C.	5	min.
Amplification (40 cycles)	95° C.	10	s.
	65° C.-61° C.	70	s. (fluorescence acquisition)

	step down each cycle by 0.1° C.

The results of the probe-based multiplex real-time PCR are shown in FIG. 4, which shows the simultaneous detection of Cronobacter spp. and Enterobacteriaceae by probe-based multiplex real-time PCR. The specific detection of Cronobacter DNA occurred in the FAM-whereas the DNA amplification of the complete Enterobacteriaceae family could be detected in the HEX-channel.

Example 6

Selective Amplification of DNA from Viable Cronobacter Cells

An according to ISO/TS 22964 pre-enriched sample of infant formula was subcultivated in buffered peptone water (BPW). This subculture were inoculated to 10⁶dead C. sakazakii cells per ml. Inactivation was done by incubation of a C. sakazakii culture with 5×10⁸/ml for 10 min at 70° C. (inactivation checked by plating). As controls additional samples were inoculated with viable C. sakazakii cells.
For selective inactivation of DNA from dead cells 100 μl of each sample were mixed with 300 μl of a solution containing 20 μg/ml ethidium bromide monoazide (EMA). After incubation for 5 minutes in dark, the samples were placed on ice, exposed for 5 minutes to a 500 W halogen light bulb in approx. 15-20 cm distance. Afterwards the mixtures were centrifuged (5 min. at 10 000×g) and the supernatant discarded. DNA isolation was performed by resuspension of the cell pellet in 200 μl lysis solution containing Chelex x-100 and incubation at 95-100° C. for 10 minutes. As controls the DNA from 100 μl of all spiked samples was isolated without a treatment with EMA.
PCR mix and protocol were the same as in example 5. The results of this experiment are shown in FIG. 5.
As shown in FIG. 5 the addition of DNA from dead Cronobacter cells without a treatment with EMA to the Cronobacter specific PCR system results in clearly visible fluorescence curves, whereas in the treated samples no amplification is detectable. On viable cells the PCR detection sensitivity is only slightly reduced by this EMA-procedure. Therefore the PCR-based method of this invention could be combined with the EMA-technique and enables a sensitive detection of only viable Cronobacter cells.

Claims

1. A nucleic acid molecule suitable as a primer or probe in the detection of the genus Cronobacter, wherein

i) the nucleic acid molecule

a) is a sequence comprising or consisting of a partial sequence of SEQ ID NO: 1, which partial sequence is highly conserved or conserved in all species of the genus Cronobacter, and

b) is between 10 and 50 nucleic acids long;

ii) the nucleic acid molecule

a) is a sequence which hybridizes under specific conditions with the nucleic acid molecule of i) and

b) is between 10 and 50 nucleic acids long;

iii) the nucleic acid molecule

a) is at least 70% identical to the nucleic acid molecule of i) or ii); and

b) is between 10 and 50 nucleic acids long; or

iv) the nucleic acid molecule is the complement of the nucleic acid molecule of any of i) to iii).

2. The nucleic acid molecule of claim 1, which is

a) a nucleic acid molecule selected from any of SEQ ID NOs: 2-18; or

b) a nucleic acid molecule which hybridizes under specific conditions with the nucleic acid molecule of a);

c) a nucleic acid molecule which is at least 70% identical to the nucleic acid molecule of a); or

d) the complement of the nucleic acid molecule of any of a) to c).

3. A nucleic acid molecule suitable as a primer or probe in the detection of Cronobacter turicensis, wherein

i) the nucleic acid molecule

a) is a sequence comprising or consisting of a partial sequence of SEQ ID NO: 19, which partial sequence is highly conserved in a subgroup of the species Cronobacter turicensis; and

b) is between 10 and 50 nucleic acids long;

ii) the nucleic acid molecule

b) is between 10 and 50 nucleic acids long; or

iii) the nucleic acid molecule

a) is at least 70% identical to the nucleic acid molecule of i) or ii); and

b) is between 10 and 50 nucleic acids long; or

4. The nucleic acid molecule of claim 3, which is

a) selected from SEQ ID NOs: 20-21; or

b) a sequence which hybridizes under specific conditions with the nucleic acid molecule of a);

c) at least 70% identical to the nucleic acid molecule of a); or

d) the complement of the nucleic acid molecule of any of a) to c).

5. A method of using one or more nucleic acid molecules of claim 1 in the detection of the genus Cronobacter.

6. A combination of two or more of the nucleic acid molecules of claim 1 in combination with one or more nucleic acid molecules suitable as a primer or probe in the detection of Cronobacter turicensis, wherein

i) the nucleic acid molecule

b) is between 10 and 50 nucleic acids long;

ii) the nucleic acid molecule

b) is between 10 and 50 nucleic acids long; or

iii) the nucleic acid molecule

a) is at least 70% identical to the nucleic acid molecule of i) or ii); and

b) is between 10 and 50 nucleic acids long; or

7. A method of using of one or more nucleic acid molecules of claim 1 together with one or more nucleic acid molecules suitable as a primer or probe in the detection of Cronobacter turicensis, wherein

i) the nucleic acid molecule

b) is between 10 and 50 nucleic acids long;

ii) the nucleic acid molecule

b) is between 10 and 50 nucleic acids long; or

iii) the nucleic acid molecule

a) is at least 70% identical to the nucleic acid molecule of i) or ii); and

b) is between 10 and 50 nucleic acids long; or

iv) the nucleic acid molecule is the complement of the nucleic acid molecule of any of i) to iii),

in the detection of the genus Cronobacter and the discrimination of Cronobacter turicensis against other species.

8. A method for amplifying bacterial DNA of the taxonomic unit genus Cronobacter, using primers, in which

a) in a first amplification step the DNA of the genus Cronobacter is amplified with conserved primers, wherein the primers used in the first amplification step comprise a nucleic acid molecule of claim 1.

9. The method of claim 8, wherein

b) in a further detection step, the DNA fragments obtained by amplification step a) which are specific for the genus Cronobacter are detected by means of probes, wherein the probes used in step b) comprise a nucleic acid molecule, wherein

i) the nucleic acid molecule

a) is a sequence comprising or consisting of a partial sequence of SEQ ID NO: 1 which partial sequence is highly conserved or conserved in all species of the genus Cronobacter, and

b) is between 10 and 50 nucleic acids long;

ii) the nucleic acid molecule

b) is between 10 and 50 nucleic acids long;

iii) the nucleic acid molecule

a) is at least 70% identical to the nucleic acid molecule of i) or ii); and

b) is between 10 and 50 nucleic acids long; or

10. The method of claim 9, wherein

c) in a further detection step, the DNA fragments obtained by amplification step a) which are specific for the species Cronobacter turicensis are detected by means of probes,

wherein the probes used in step c) comprise a nucleic acid molecule, wherein

i) the nucleic acid molecule

b) is between 10 and 50 nucleic acids long;

ii) the nucleic acid molecule

b) is between 10 and 50 nucleic acids long; or

iii) the nucleic acid molecule

a) is at least 70% identical to the nucleic acid molecule of i) or ii); and

b) is between 10 and 50 nucleic acids long; or

11. The method of claim 10, wherein

d) in at least one further amplification step the DNA of the taxonomic unit Enterobacteriaceae is amplified with conserved primers.

12. The method of claim 11, wherein

e) in a further detection step, the DNA fragments obtained by amplification step d) which are specific for the genus Enterobacteriaceae are detected by means of probes.

13. The method of claim 12, including a step of amplifying an amplification control nucleic acid, which is added during amplification step a), wherein, the amplification control nucleic acid is SEQ ID NO:22.

14. The method of claim 13, further including a melting curve analysis, wherein C. turicensis is discriminated against other Cronobacter species.

15. The method of claim 14, wherein viable cells of the genus Cronobacter or of other Enterobacteriaceae are discriminated against respective non-viable Cronobacter or other non-viable Enterobacteriaceae cells.

16. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule is at least 95% identical to the nucleic acid molecule of i) or ii).

17. The nucleic acid molecule of claim 2, wherein the nucleic acid molecule is at least 95% identical to the nucleic acid molecule of a).

18. The nucleic acid molecule of claim 3, wherein the nucleic acid molecule is at least 95% identical to the nucleic acid molecule of i) or ii).

19. The nucleic acid molecule of claim 4, wherein the nucleic acid molecule is at least 95% identical to the nucleic acid molecule of a).