WO2016030507A1

WO2016030507A1 - Detection of lactobacillus parabuchneri

Info

Publication number: WO2016030507A1
Application number: PCT/EP2015/069745
Authority: WO
Inventors: Rémy BRUGMMANN; Daniel Wüthrich; Hélène BERTHOUD; Stefan IRMLER; Hans Graber
Original assignee: Universität Bern; Agroscope
Priority date: 2014-08-29
Filing date: 2015-08-28
Publication date: 2016-03-03

Abstract

The present invention relates to a nucleic acid oligomer, wherein the nucleic acid oligomer consists of or comprises a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a contiguous nucleic acid sequence of the same length that is comprised within SEQ ID NO 01 or comprised within a nucleic acid sequence complementary to SEQ ID NO 01. The present invention further relates to the use of the nucleic oligomer for detecting Lactobacillus parabuchneri.

Description

Detection of Lactobacillus parabuchneri Description

The present invention relates to methods and means for detecting Lactobacillus

parabuchneri, particularly in foodstuff, more particular in milk or milk products.

Members of the genus Lactobacillus are important microorganisms for the production of foodstuff. They are used for the production of diary products, but selected members of the genus may also act as pests in the production of beverages such as beer or wine.

The suitability for food production of the species belonging to the genus Lactobacillus depends on their metabolism, which varies among the various species of the genus. Some of the species produce undesirable products, which may spoil the foodstuff. Traditional microbiological methods can be used to isolate and detect the microorganisms. However, those methods have the drawback that they take several days to complete. Therefore, a rapid method for species-specifically detecting or quantifying a member of the genus

Lactobacillus is desirable.

Based on this background it is the objective of the present invention to provide methods and means for detecting Lactobacillus parabuchneri in foodstuff or beverage, particularly in milk or a milk product such as cheese. The objective is attained by the subject matter of the independent claims.

During the investigation of histamine producing Lactobacillus parabuchneri in milk and cheese it was surprisingly found that the members of the species share a common unique nucleic acid sequence, which can be used for specific detection of L. parabuchneri.

According to a first aspect of the invention, a nucleic acid oligomer is provided, wherein the nucleic acid oligomer consists of or comprises a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a contiguous nucleic acid sequence of the same length that is

a. comprised within a sequence selected from the group comprising SEQ ID NO 01 , SEQ ID NO 05, SEQ ID NO 06, SEQ ID NO 07, SEQ ID NO 08, SEQ ID NO 09, SEQ ID NO 10, SEQ ID NO 1 1 , SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21 and SEQ ID NO 22, or

b. comprised within a nucleic acid sequence complementary to SEQ ID NO 01 , SEQ ID NO 05, SEQ ID NO 06, SEQ ID NO 07, SEQ ID NO 08, SEQ ID NO 09, SEQ ID NO 10, SEQ ID NO 1 1 , SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21 or SEQ ID NO 22.

The oligomer of the present invention has a length that enables specificity of the sequence in comparison to prokaryotic and eukaryotic DNA comprised in foodstuff. In particular, the oligomer has a length of more than 8, 9, 10,1 1 , 12, 13, 14, 15, 16, 17 or 18 nucleotides.

The term "a nucleic acid sequence complementary to SEQ ID NO 01 " in the context of the present specification refers to a nucleic acid sequence that when aligned antiparallel to SEQ ID NO 01 (or any one of sequences of SEQ ID NO 05 to 22), particularly meaning when SEQ ID NO 01 (or any one of sequences of SEQ ID NO 05 to 22) being orientated from the 5' end to the 3' end and the nucleic acid sequence complementary to SEQ ID N01 (or any one of sequences of SEQ ID NO 05 to 22) being orientated from the 3' end to the 5' end, the nucleotide bases at each position are complementary, for example a guanine nucleoside moiety on the complementary sequence binds to a cytosine moiety on SEQ ID NO 01 . In other words, the nucleic acid sequence complementary to SEQ ID NO 01 can be regarded as the anti-sense strand to SEQ ID NO 01 , which is the sense strand.

In the context of the present specifications the terms sequence identity and percentage of sequence identity refer to the values determined by comparing two aligned sequences. Methods for alignment of sequences for comparison are well-known in the art. Alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981 ), by the global alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci. 85:2444 (1988) or by computerized implementations of these algorithms, including, but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Unless otherwise stated, sequence identity values provided herein refer to the value obtained using the BLAST suite of programs using default parameters (Altschul et al., J. Mol. Biol. 215:403-410 (1990)). Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology-Information

(http://blast.ncbi.nlm.nih.gov/). One example for comparison of amino acid sequences is the BLASTP algorithm that uses default settings such as: Expect threshold: 10; Word size: 3; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: Existence 1 1 , Extension 1 ; Compositional adjustments: Conditional compositional score matrix adjustment. One such example for comparison of nucleic acid sequences is the BLASTN algorithm that uses the default settings: Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match/Mismatch Scores: 1.-2; Gap costs: Linear

The nucleic acid sequence that is characterized by SEQ ID NO can uniquely be found in the members of the species Lactobacillus parabuchneri. Thus the nucleic acid oligomer of the invention can be used as a specific primer to amplify the unique nucleic acid sequence in, for example, a polymerase chain reaction (PCR) providing a valuable tool for detecting minimal amounts of Lactobacilli parabuchneri in any sample of interest. Likewise the nucleic acid oligomer of the invention can be used as specific probe for detecting the unique nucleic acid sequence or an amplificate thereof.

In certain embodiments, the nucleic acid oligomer of the invention consists of 8 to 50 nucleotides. In certain embodiments, the nucleic acid oligomer of the invention consists of 15 to 40 nucleotides. In certain embodiments, the nucleic acid oligomer of the invention consists of 18 to 30 nucleotides. In certain embodiments, the nucleic acid oligomer of the invention consists of 18 to 25 nucleotides.

In certain embodiments, the sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a contiguous nucleic acid sequence of the same length consists of 8 to 50 nucleotides. In certain embodiments, the sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a contiguous nucleic acid sequence of the same length consists of 15 to 40 nucleotides. In certain embodiments, the sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a contiguous nucleic acid sequence of the same length consists of 18 to 30 nucleotides. In certain embodiments, the sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a contiguous nucleic acid sequence of the same length consists of 18 to 25 nucleotides.

"Nucleotides" in the context of the present invention are nucleic acid or nucleic acid analogue building blocks, oligomers of which are capable of forming selective hybrids with DNA or RNA sequences or oligomers (specifically with SEQ ID NO 01 or a contiguous sequence thereof having a length of 20, 30, 40, 50 or more base pairs) on the basis of base pairing. The term nucleotides in this context includes the classic ribonucleotide building blocks adenosine, guanosine, uridine (and ribosylthymin), cytidine, the classic deoxyribonucleotides deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine. It further includes analogues of nucleic acids such as phosphotioates, 2O-methylphosphothioat.es, peptide nucleic acids (PNA; N-(2-aminoethyl)-glycine units linked by peptide linkage, with the nucleobase attached to the alpha-carbon of the glycine) or locked nucleic acids (LNA; 2Ό, 4'C methylene bridged RNA building blocks). The hybridizing sequence may be composed of any of the above nucleotides, or mixtures thereof.

In certain embodiments, the nucleic acid oligomer of the invention is a DNA oligomer. In certain embodiments, the nucleic acid oligomer of the invention comprises deoxynucleotides, phosphothioate deoxynucleotides, LNA and/or PNA nucleotides or mixtures thereof.

In certain embodiments, the nucleic acid oligomer consists of or comprises a nucleic acid sequence characterized by SEQ ID NO 02, SEQ ID NO 03 or SEQ ID NO 04. In certain embodiments, the nucleic acid oligomer comprises a detectable or quantifiable label. Such nucleic acid oligomers may be used as specific probes for example real-time PCR.

The term "detectable or quantifiable label" in the context of the present specification particularly refers to a compound or moiety that is covalently or non-covalently attached to the nucleic acid oligomer of the invention and that comprises a detectable or measureable quality such as absorbance, luminescence, radioactivity, redox potential or an enzymatic activity.

In certain embodiments, the detectable or quantifiable label is luminescent compound or moiety. In certain embodiments, the detectable or quantifiable label is selected from fluorescein (CAS number 2321 -07-5), carboxyfluorescein (CAS number 3301 -79-9)

Atto612Q, Cy5 (6-[3,3-dimethyl-2-[(1 E,3E,5E)-5-(1 ,3,3-trimethylindolin-2-ylidene)penta-1 ,3- dienyl]indol-1-ium-1-yl]hexanoic acid), LC640, LC610, LC640, LC670, LC690, LC705 or TAMRA (Carboxy-tetramethylrhodamine).

In certain embodiments, the nucleic acid oligomer of the invention comprises a first FRET (Fluorescence Resonance Energy Transfer) partner molecule at the 5' end of the oligomer and a second FRET partner at the 3' end of the oligomer, wherein the first and the second FRET partner are able to interact in such way that the luminescent signal of first FRET partner is changed with spatial approximation of the first FRET partner and the second FRET partner. Alternatively, the nucleic acid oligomer of the invention comprises a first FRET partner at the 3' end of the oligomer and a second FRET partner at the 5' end of the oligomer. Such labelled nucleic acid oligomers may be used as specific probes in a polymerase-chain-reaction, wherein the labelled nucleic acid oligomer as described above specifically binds to a nucleic acid sequence comprised with SEQ ID NO 01 or comprised within a nucleic acid sequence complementary to SEQ ID NO 01. Upon amplification of the nucleic acid sequence the labelled nucleic acid oligomer can be degraded by a nucleic acid polymerase having 5'-3' exonuclease activity resulting in a change of the spatial

approximation of the first and the second FRET partner.

In certain embodiments, the nucleic acid oligomer of the invention comprises an additional nucleic acid sequence at the 5' end of the oligomer comprising a first FRET partner and an additional nucleic acid sequence at the 3' end of the oligomer comprising a second FRET partner, wherein the additional nucleic acid sequence at the 5' end is complementary to the additional nucleic acid sequence at the 3' end, and wherein the first and the second FRET partner are able to interact in such way that the luminescent signal of first FRET partner is changed with spatial approximation of the first FRET partner and the second FRET partner. Alternatively, the nucleic acid oligomer of the invention comprises an additional nucleic acid sequence at the 3' end of the oligomer comprising a first FRET partner and an additional nucleic acid at the 5' end of the oligomer comprising a second FRET partner, wherein the additional nucleic acid sequence at the 5' end is complementary to the 3' end, and wherein the first and the second FRET partner are able to interact in such way that the luminescent signal of first FRET partner is changed with spatial approximation of the first FRET partner and the second FRET partner.

Such nucleic acid oligomers may be used as a molecular beacon (see US07385043 B1 , US 5925517 B1 and US6,461 ,817 B1 , all of which are incorporated herein by reference), wherein the oligomers form a hairpin loop, wherein the hairpin loop comprises a stem-loop region comprising the paired additional nucleic acid sequences at the 5' end and the 3' end and a loop region comprising the nucleic acid oligomer of the invention. In presence of nucleic acid characterized by SEQ ID NO 01 or a nucleic acid complementary to SEQ ID NO 01 the loop region hybridize with nucleic acid resulting in a spatial separation of the first FRET partner from the second FRET partner accompanied by a change of the luminescent signal of the first and/or the second FRET partner.

A first FRET partner in the context of the present specification refers to a molecule that is able to get excited by electromagnetic radiation, to emit electromagnetic radiation after excitation or to transfer energy after excitation to another molecule for example through nonradiative dipole-dipole coupling. Such first FRET partner may also be signified as donor chromophore.

A second FRET partner in the context of the present specification refers to a molecule that is able to quench the luminescence of the first FRET-Partner by energy transfer from the first FRET partner in the electronically excited state to the second FRET partner, whereby the efficiency of the energy transfer and thus the quenching rate is distance dependent. Such second FRET partner may also be signified as acceptor chromophore.

In certain embodiments, the first FRET partner is fluorescein (CAS No. 2321 -07-5) or carboxyfluorescein (CAS number 3301-79-9).

In certain embodiments, the second FRET partner is selected from Atto612Q, Cy5 (6-[3,3- dimethyl-2-[(1 E,3E,5E)-5-(1 ,3,3-trimethylindolin-2-ylidene)penta-1 ,3-dienyl]indol-1-ium-1- yl]hexanoic acid), LC640, LC610, LC640, LC670, LC690, LC705 or TAMRA (Carboxy- tetramethylrhodamine).

In certain embodiments, the second FRET partner is not luminescent. In certain

embodiments, the second FRET partner is a dark quencher, particularly a black hole quencher such as BHQ-0 (Biosearch Technologies, USA), BHQ-1 (Biosearch Technologies, USA), BHQ-2 (Biosearch Technologies, USA), BHQ-3 (Biosearch Technologies, USA) or BHQ-10 (2-[(E)-[4-[(4-hydroxy-4-oxo-butyl)-methyl-amino]phenyl]azo]-5-[(E)-(4- oxoniosulfonylphenyl)azo]benzenesulfonate), Dabysyl (dimethylaminoazobenzenesulfonic acid), a Qxl quencher such as QXL 490 (AnaSpec, Inc., USA), QXL 570 (AnaSpec, Inc., USA), QXL 610 (AnaSpec, Inc., USA), QXL 670 (AnaSpec Inc., USA) or QXL 680 (AnaSpec, Inc., USA), Iowa black FQ (Integrated DNA Technologies, Inc., USA), Iowa black RQ

(Integrated DNA Technologies, Inc., USA), IRDye QC-1 (LI-COR Biosciences GmbH, Germany) or Eclipse Dark Quencher (Eurogentec Deutschland GmbH, Germany)

According to a further aspect of the invention a combination of nucleic acid oligomers is provided. The combination of nucleic acid oligomers comprises

a first nucleic acid oligomer consisting of or comprising a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a first contiguous nucleic acid sequence of the same length that is comprised within SEQ ID NO 01 , and a second nucleic acid oligomer consisting of or comprising a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a second contiguous nucleic acid sequence of the same length that is comprised within a nucleic acid sequence complementary to SEQ ID NO 01.

Such combination of nucleic acid oligomers may be used as a set of specific primers for amplifying both a nucleic acid having a sequence comprised within SEQ ID NO 01 and a nucleic acid having a sequence complementary to SEQ ID NO 01 . Alternatively, such combination may be used as a set of probes for specifically detecting both nucleic acids described above.

In certain embodiments, the first contiguous nucleic acid sequence is located upstream of a nucleic acid sequence that is complementary to the second contiguous nucleic acid sequence.

The term "upstream" in the context of the present specification particularly means that with regard to the nucleic acid sequence that is complementary to the second contiguous nucleic acid sequence the first contiguous nucleic acid sequence is located toward the 5' end of SEQ ID NO 01.

In certain embodiments, the first contiguous nucleic acid sequence is located 50 to 500 nucleotides upstream of the nucleic acid sequence that is complementary to the second contiguous nucleic acid sequence with regard to the 5' end of both first and second contiguous nucleic acid sequence. Using such a combination of nucleic acid oligomers as primers in a polymerase chain reaction will result in the formation of a double-stranded amplificate of 50 to 500 base pairs length. In certain alternative embodiments, a combination is provided, wherein the combination of nucleic acid oligomers comprises

a first nucleic acid oligomer consisting of or comprising a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a first contiguous nucleic acid sequence of the same length that is comprised within a sequence selected from the group comprising SEQ ID NO 01 , SEQ ID NO 05, SEQ ID NO 06, SEQ ID NO 07, SEQ ID NO 08, SEQ ID NO 09, SEQ ID NO 10, SEQ ID NO 1 1 , SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21 and SEQ ID NO 22 and further comprising a first FRET partner, and

a second nucleic acid oligomer consisting of or comprising a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a second contiguous nucleic acid sequence of the same length that is comprised within a nucleic acid sequence complementary to SEQ ID NO 01 (or any one of sequences of SEQ ID NO 05 to 22) and further comprising a second FRET partner,

wherein the first contiguous nucleic acid sequence is located 1 to 20 nucleotides upstream of the nucleic acid sequence that is complementary to the second contiguous nucleic acid sequence with regard to the 3' end of both first and second contiguous nucleic acid sequence. In certain embodiments, the first contiguous nucleic acid sequence is

complementary to the second nucleic acid sequence.

Such nucleic acid oligomers according to the above alternative embodiments may be used as probes for detecting a double-stranded nucleic acid having a sequence comprised within SEQ ID NO 01 .

According to an alternative to the above aspect, a combination of nucleic acid oligomers is provided, wherein the combination comprises

a first nucleic acid oligomer consisting of or comprising a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a first contiguous nucleic acid sequence of the same length that is comprised within SEQ ID NO 01 and further comprising a first FRET partner, and

- a second nucleic acid oligomer consisting of or comprising a sequence at least 80%,

85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a second contiguous nucleic acid sequence of the same length that is comprised with SEQ ID NO 01 further comprising a second FRET partner,

wherein the 3' end of first contiguous nucleic acid sequence is located 1 to 20 nucleotides upstream of the 5' end of the second contiguous nucleic acid sequence. According to another alternative to the above aspect, a combination of nucleic acid oligomers is provided, wherein the combination comprises

a first nucleic acid oligomer consisting of or comprising a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a first contiguous nucleic acid sequence of the same length that is comprised within a nucleic acid sequence complementary to SEQ ID NO 01 and further comprising a first FRET partner, and a second nucleic acid oligomer consisting of or comprising a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a second contiguous nucleic acid sequence of the same length that is comprised within a nucleic acid sequence complementary to SEQ ID NO 01 and further comprising a second FRET partner,

wherein the 3' end of first contiguous nucleic acid sequence is located 1 to 20 nucleotides upstream of the 5' end of the second contiguous nucleic acid sequence.

In certain alternative embodiments, the first nucleic acid oligomer comprises the second FRET partner, and the second nucleic acid oligomer comprises the first FRET partner.

In certain embodiments, each of the first nucleic acid oligomer, the second nucleic acid oligomer, the first contiguous nucleic acid sequence and the second contiguous nucleic acid sequence according to the above aspect or alternatives independently of each other consist of 8 to 50 nucleotides. It is understood that in this context, the first contiguous nucleic acid sequence cannot be longer than the corresponding first nucleic acid oligomer, and the second contiguous nucleic acid sequence cannot be longer than the corresponding second nucleic acid oligomer. In certain embodiments, each of the first nucleic acid oligomer, the second nucleic acid oligomer, the first contiguous nucleic acid sequence and the second contiguous nucleic acid sequence independently of each other consist of 15 to 40 nucleotides. In certain embodiments, each of the first nucleic acid oligomer, the second nucleic acid oligomer, the first contiguous nucleic acid sequence and the second contiguous nucleic acid sequence independently of each other consists of 18 to 30 nucleotides. In certain embodiments, each of the first nucleic acid oligomer, the second nucleic acid oligomer, the first contiguous nucleic acid sequence and the second contiguous nucleic acid sequence independently of each other consists of 18 to 25 nucleotides.

According to yet another aspect of the invention, the use of a nucleic acid oligomer or a combination according to the invention for detecting and/or quantifying Lactobacillus parabuchneri is provided. In certain embodiments, Lactobacillus parabuchneri is detected and/or quantified in a sample obtained from a foodstuff or a beverage. In certain

embodiments, Lactobacillus parabuchneri is detected and/or quantified in a sample obtained from a beer, milk or a milk product. In certain embodiments, Lactobacillus parabuchneri is detected and/or quantified in a cheese sample.

According to a further aspect of the invention a method for detecting and/or quantifying Lactobacillus parabuchneri is provided. The method comprises:

- providing a sample,

contacting the sample with a nucleic acid oligomer or a combination according to the invention,

performing an amplification reaction with the sample in presence of nucleic acid polymerase, wherein in presence of Lactobacillus parabuchneri in the sample a nucleic acid sequence of Lactobacillus parabuchneri is amplified yielding an amplificate, and

detecting the presence of the amplificate and/or quantifying the amplificate.

The term "amplification reaction" in the context of the present specification particularly refers to a reaction, wherein the nucleic acid sequence of Lactobacillus is duplicated at least one time, particularly at least 10 times, and more particular at least 30 times.

The term "nucleic acid sequence of Lactobacillus parabuchneri in the context of the present specification particularly refers to a nucleic acid sequence that is characterized by SEQ ID NO 01 (or any one of sequences of SEQ ID NO 05 to 22) or that is complementary to SEQ ID NO 01 (or any one of sequences of SEQ ID NO 05 to 22).

In certain embodiments, the sample is obtained from a foodstuff or a beverage. In certain embodiments, the sample is obtained from milk or a milk product. In certain embodiments, the sample is obtained from cheese.

In certain embodiments, the amplification reaction comprises the steps of

a denaturing step, wherein said sample is heated up to an appropriate temperature causing the melting of any double stranded nucleic acid comprised within the sample, particularly the melting of the nucleic acid sequence of Lactobacillus parabuchneri if Lactobacillus parabuchneri is present in the sample,

an annealing step, wherein in presence of Lactobacillus parabuchneri in the sample the nucleic acid oligomer of the invention or the first and/or the second nucleic acid oligomer of the combination of the invention specifically binds to the nucleic acid sequence of Lactobacillus parabuchneri, and wherein particularly the annealing step is performed at an appropriate temperature depending on the length and composition of the nucleic acid oligomer or the first and/or second nucleic acid oligomer of the combination, and a synthesis step, wherein the annealed nucleic acid oligomer or the annealed first and/or second nucleic acid oligomer are elongated by the nucleic acid polymerase by adding nucleotides to the nucleic acid oligomer that are complementary to the nucleic acid sequence of Lactobacillus parabuchneri forming the amplificate of the nucleic acid sequence of Lactobacillus parabuchneri.

In certain embodiments, the amplificate is detected and/or quantified with a dye that particularly preferentially binds to double-stranded DNA. In certain embodiments, the amplificate is detected and/or quantified with an intercalating dye. Examples for such an intercalating dye include without being restricted to ethidium bromide (CAS number 1236-45- 8) and SYBR Green I (CAS number 163795-75-3).

In certain alternative embodiments, the amplificate is detected and/or quantified with a nucleic acid oligomer according to the invention comprising a detectable and/or quantifiable label or with a combination according to the invention, wherein the first nucleic acid oligomer or the second nucleic acid oligomer comprises a detectable and/or quantifiable label.

Particularly, such labelled nucleic acid acts as a specific probe for detection or quantification of the amplificate.

In certain embodiments, the nucleic acid oligomer comprising a detectable and/or quantifiable label consists of or comprises SEQ ID NO 04. In certain embodiments, the nucleic acid oligomer comprising a detectable and/or quantifiable label consists of or comprises SEQ ID NO 04, wherein the label is fluorescein.

In certain embodiments of any of the methodological aspects of the invention described herein, the method further comprises a step of designating said food product for discard if said method detects the presence of Lactobacillus parabuchneri or if the quantity of

Lactobacillus parabuchneri is above a predefined threshold, and /or designating said food product as marketeable or "passed" if said method fails to detect the presence of

Lactobacillus parabuchneri or if the quantity of Lactobacillus parabuchneri is below a predefined threshold. The method of the invention may include a step of labelling the foodstuff from which the sample was taken for further inspection, or labelling the foodstuff for being discarded or otherwise disposed of or for preventing the foodstuff to be sold or included in preparations for sale or human consumption.

In certain embodiments of any of the methodological aspects of the invention described herein, the method further comprises a step of acting upon the detection of the presence of any one of the sequences used in confirming the presence of L. parabuchneri in foodstuff. Particularly, the method may comprise a step of discarding the foodstuff, or a step of disposing of the foodstuff, or a step of preventing the foodstuff to be sold or included in preparations for sale or human consumption. According to yet another aspect of the invention, a kit for detecting and/or quantifying Lactobacillus parabuchneri is provided, particularly in a foodstuff or a beverage, more particular in milk or a milk product, even more particular in cheese. The kit comprises a nucleic acid oligomer according to the invention or a combination according to the invention. Such kit may be used for performing the method of the invention described above.

In certain embodiments, the kit comprises a first combination according to the invention and a nucleic acid oligomer according to the invention comprising a detectable and/or quantifiable label, or

a second combination according to the invention, wherein at least one of said first and said second nucleic acid oligomer comprises a detectable and/or quantifiable label.

In certain embodiments, the kit comprises a first combination according to the invention and a nucleic acid oligomer according to the invention comprising a first FRET partner and a second FRET partner.

In certain embodiments, the kit comprises a first combination according to the invention and a second combination according to the invention comprising

a first nucleic acid oligomer consisting of or comprising a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a first contiguous nucleic acid sequence of the same length that is comprised within SEQ ID NO 01 (or any one of sequences of SEQ ID NO 05 to 22) and further comprising a first FRET partner, and

a second nucleic acid oligomer consisting of or comprising a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a second contiguous nucleic acid sequence of the same length that is comprised within a nucleic acid sequence complementary to SEQ ID NO 01 (or any one of sequences of SEQ ID NO

05 to 22) and further comprising a second FRET partner,

wherein the first contiguous nucleic acid sequence is located 1 to 20 nucleotides upstream of the nucleic acid sequence that is complementary to the second contiguous nucleic acid sequence with regard to the 3' end of both contiguous nucleic acid sequences.

In certain alternative embodiments, the kit comprises a first combination according to the invention and a second combination comprising

a second nucleic acid oligomer consisting of or comprising a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a second contiguous nucleic acid sequence of the same length that is comprised within SEQ ID NO 01 (or any one of sequences of SEQ ID NO 05 to 22) and further comprising a second FRET partner,

wherein the 3' end of the first contiguous nucleic acid sequence is located 1 to 20

nucleotides upstream of the 5' end of the second contiguous nucleic acid sequence.

In certain embodiments, the kit comprises a nucleic acid oligomer characterized by SEQ ID NO 02, a nucleic acid oligomer characterized by SEQ ID NO 03, and optionally a nucleic acid oligomer characterized by SEQ ID NO 04 comprising a detectable and/or quantifiable label. Wherever alternatives for single separable features such as, for example, a certain label or a certain FRET partner are laid out herein as "embodiments", it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein.

The invention is further characterized, without limitations, by the following examples, from which further features, advantages and embodiments can be derived. The examples are meant to illustrate but not limit the invention.

Description of the figures

Fig. 1 shows a scheme of the genome of Lactobacillus parabuchneri FAM21731 .

Fig. 2 shows an alignment of the histidine carboxylase cluster (HDC) of Lactobacillus parabuchneri.

Fig. 3 shows the detection of Lactobacillus parabuchneri in milk; the number CFU per ml is plotted against the number of copies of the target gene per ml of inoculated milk samples, as determined by qPCR. Data were obtained from three independent experiments.

Examples

Various histamine producing bacilli were isolated from cheese, which were identified as L. parabuchneri (unpublished data). When one of this isolates, namely L. parabuchneri FAM21731 , was used as adjunct in the manufacture of semi-hard cheese the concentration of histamine increased during cheese ripening. Interestingly, also L. parabuchneri

FAM21835 was isolated, a strain which was not able to produce histamine.

The genome of L. parabuchneri FAM21731 was sequenced using next-generation sequencing technology. The draft genome sequence was compared to public available genome sequences of L. buchneri and other related species. Thereby, a gene sequence was identified which is unique for L. parabuchneri. Based on this sequence a real-time PCR method was developed to analyze various strains of L. parabuchneri. Furthermore, the method was used to detect and enumerate L. parabuchneri in cheese and milk samples. The results show that the PCR system is specific for the detection and quantification of L.

parabuchneri in dairy products. The use of this method is of interest since it can be employed to locate potential sources of /., parabuchneri contamination on farms and in dairy plants.

The genome L. parabuchneri FAM21731 was sequenced using Pacbio and illumina technology. By combining the reads of both sequencers, a chromosome of 2.6 Mb was obtained. Annotation was performed using Prokka (rapid prokaryotic genome annotation). An open reading frame designated as Lpbuc_01040 (17682 bp) did not show homology to genes present in the non-redundant Genbank database (Nov. 2013) (grey circle)

De novo assembly of whole genome

The Lactobacillus parabuchneri strain FAM21731 was sequenced using PacBio technology. The De novo assembly of the PacBio reads using HGAP resulted in three contigs. The three contigs were found to be circular and it was possible to close the three contigs using manual correction. It resulted in a chromosome (2,600,578 bp) and two plasmids (58,093 bp; 67,905 bp). Remapping of the PacBio reads using quiver (smrtanalysis-2.2.0) resulted in no found false called bases or Indels.

The Annotation of the genome using Prokka (1 .6) resulted in 2,644 predicted genes of which 2,566 were determined as protein coding sequences.

Additionally 4 strains extracted out of different cheeses (table 1 ) were sequenced using Ion torrent technology. The assembly of the Ion torrent reads was performed using newbler (2.7). The assembly sizes were between 2,483,239 bp and 2,752,406 bp. Table 1 :

Histamine production

The genome of /., parabuchneri FAM21731 encodes a histidine decarboxylase (hdc), which was also found in other L. parabuchneri strains. The amino acid sequence of HDC was compared with the HDC from various other lactic acid bacteria (Fig. 2). The grey scaled blocks illustrate similarity and represent the length of 10 amino acid residues. Interestingly, two different types of hdc are found in lactic acid bacteria.

By comparing this hdc gene and the neighboring genes with already published genome revealed that a cluster of four genes is highly conserved (HDC cluster). The 4 proteins are a histidyl-tRNA synthetase, a hypothetical protein, a histidine decarboxylase and a histidine histamine anti-porter. Homologs of those proteins were found in several other Lactobacillus species.

By studying the HDC gene cluster of other species 4 different kinds of gene composition and arrangements were found. The cluster found in L. parabuchneri shows the same gene order as the ones in Tetragenococcus halophilus, Tetragenococcus muriaticus, Lactobacillus hilgardii, Lactobacillus sakei, Lactobacillus saerimneri, Lactobacillus vaginalis, Lactobacillus fermentum, Lactobacillus reuteri. Focusing on the phylogeny of the HDC protein, it can also be observed that the protein is closely related to those species. Comparing these results to the phylogeny of the core genome of the LactbacHli it can be seen that the HDC proteins are closer related, especially of Lactobacillus sakei, than the core genome. Furthermore, the HDC cluster is located on a plasmid in Lactobacillus hilgardii, Tetragenococcus halophilus and Tetragenococcus muriaticus. This leads to the conclusion that the HDC cluster was transferred via horizontal gene transfer among the species that are carrying the same cluster. Thus in contrast to the unique sequence Lpbuc_01040 (SEQ ID NO 01 ) the gene sequences of the hdc cluster are not appropriate targets for detecting L. parabuchneri.

Thin-layer chromatography analysis showed that twelve of the thirteen strains, which have been isolated from cheese and sequenced (data not shown), are producing histamine (table 2). Only the strain FAM21838 does not show any production of histamine.

Table 2:

Methods

PacBio Sequencing

5 pg of high molecular weight DNA from Lactobacillus parabuchneri were sheared in a

Covaris g-TUBE (Covaris, Woburn, MA, USA) to obtain 20 kb fragments. After shearing the DNA size distribution was checked on a FragmentAnalyzer (Advanced Analytical

Technologies, Ames, IA, USA). A SMRTbell library was produced with the PacBio DNA Template Prep Kit 2.0 (Pacific Biosciences, Menlo Park, CA, USA) according to the manufacturer's recommendations. The library was sequenced on two SMRT cells with P4/C2 chemistry on a PacBio RSI I system at 120 min movie length. Sequencing yielded 1 18Ό00 post filter reads corresponding to 629 Mb with a mean read length of 5,326 bases.

Assembly of PacBio reads

The resulting reads were assembled using the HGAP (Hierarchical Genome Assembly Process) standard procedure. To close the cyclic DNA of the chromosome and the plasmid, the Assembly was performed twice. The resulting scaffolds of both assemblies were alignment and the ends of the scaffold were replaced by the contiguous sequence of the other assembly. The assembly resulted in one chromosome (2,600,578 bp) and two plasmids (58,093 bp and 67,905 bp).

Annotation of the Genome

Prokka (version 1 .6) was used to perform the annotation of the genome. Further functional annotation of the CDSs was performed by using blast2go-pipe (version 2.5, (NCBI non- redundant database, March 2014)) and interproscan (version 5.3-46.0). Genomic islands were detected using pai-ida (version 1 .1 ).

Assembly of Ion-torrent data

To assemble the ion-torrent reads of the Lactobacillus parabuchneri strains, newbler (version 2.7) was used.

Detection of histamine production

To determine the formation of histamine, strains were grown in MRS broth supplemented with 0.3% (w/v) L-histidine for 3 d at 30 °C. Afterwards, an aliquot of the culture supernatant was diluted 100-fold in methanol. The diluted culture supernatant (1 μΙ_) was applied bandwise onto HPTLC cellulose plates (Merck, Germany). Histamine (500 ng) was used as reference. The plate was developed with 1 -propanol-25% ammonia (4 + 1 , v/v). After drying, the plates were immersed into Pauly's reagent, which is a detection reagent for imidazole derivatives

DNA extraction from media

Bacteria grown in media were collected by centrifugation at 4000 ^χ g for 10 min at 25 °C. DNA was extracted from the pellet with robot extraction (BioRobot EZ1 , EZ1 DNA Tissue kit; Qiagen, Switzerland).

DNA extraction from milk

DNA was extracted from milk as follows. Briefly, 4 ml. of milk were added to 4 ml. solution containing 0.95% (w/v) Triton X-100, 4 x 10⁸ cfu Lactobacillus casei FAM 18121 and 0.24% trypsin. The mixture was incubated for 15 min at 55 °C followed by cooling on ice. After the addition 4 ml. of n-pentane and vigorous shaking, the mixture was centrifuged at 4000 ^χ g for 45 min at 25 °C. The supernatant was discarded. The pellet that contained bacteria was treated with 100 μΙ_ of lysozyme (50 mg mL-1 ) and 50 μΙ_ of mutanolysin (2000 U ml.^"1) for 1 h at 37 °C followed by the addition of 10 pL of proteinase K (>600 mAU mL-1 ) and incubation for 1 h at 56 °C. Finally, DNA was extracted from suspension with robot extraction (BioRobot EZ1 , EZ1 DNA Tissue kit; Qiagen, Switzerland).

DNA extraction from cheese

Cheese (10 g) was added to 90 mL peptone water (10 g L-1 peptone from casein, 5 g L-1 sodium chloride, 20 g L-1 trisodium citrate dihydrate, pH 7) in a Stomacher filter bag, heated for 10 min at 40 °C and then homogenized for 3 min (Masticator, IUL Instruments GmbH, Germany). Afterwards, 50 pL of 10% (w/v) SDS were added to 10 mL of homogenizate which was then centrifuged at 4000 ^χ g for 30 min at 25 °C. The supernatant was discarded. The pellet which contained bacteria was resuspended in 1 mL of TES (100 mM Tris-HCI, 10 mM EDTA, 25% (w/v) sucrose, pH 8.0) containing 5 mg of lysozyme and incubated for 1 h at 37 °C. Afterwards, the suspension was centrifuged. DNA was extracted with robot extraction (BioRobot EZ1 , EZ1 DNA Tissue kit; Qiagen, Switzerland)

Real-time PCR

A qPCR method was developed based on the sequence of Lpbuc_01040 (SEQ ID NO 01 ). The assay was tested with DNA extracted from different lactobacillus species (e.g. L. casei, L. plantarum, L. hilgardii, L. buchneri). Only DNA extracted from strains belonging to L.

parabuchneri gave a positive signal (data not shown). Raw milk was then inoculated with various concentrations of L. parabuchneri. DNA extracted from the milk was used for qPCR analysis. The results of the qPCR are shown in figure 3.

Real-time PCR was carried out using the Rotor-Gene RG 3000-A or 6000 machine (Corbett Research, Australia). To quantify the target gene the PCR reaction mixture (12 pL) contained 1x qPCR MasterMix No ROX (Eurogentec, Switzerland), 300 nM of primer_forward (5'- TGGAATTAACGGCTTGCTCTTAC-3', SEQ ID NO 02), 300 nM of primer_reverse (5'- CAACCC AATTAG G CCAAG G A-3' , SEQ ID NO 03), 100 nM of probe (FAM-5'- CCGGTTCTGCTCATTGGCGCA-3'-BHQ-1 , SEQ ID NO 04) and 2 pL of DNA. Water served as negative control (no template control). The cycling conditions were as follows: 2 min at 50 °C, 10 min at 95 °C followed by 40 cycles consisting of 15 s at 95 °C and 60 s at 60 °C.

To quantitate the amount of the target gene in a test sample, a standard curve was used. Therefore, serial dilutions of the plasmid pGEM-T easy which contains a partial sequence of the target gene were run together with the test samples. A standard curve was constructed by plotting the CT value obtained during amplification of each dilution against the log of the dilution factor (or log of the copy number). The target quantity of the test samples was then determined by interpolation. All standard dilutions and test samples were assayed in triplicate.

Over 400 L. parabuchneri isolates obtained from milk and cheese samples have been detected with the qPCR method so far (data not shown), of which two strain do not contain the hdc-cluster.

Claims

1. A nucleic acid oligomer, particularly of a sequence length of more than eight, ten, twelve or fifteen nucleotides, wherein said nucleic acid oligomer consists of or comprises a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a contiguous nucleic acid sequence of the same length that is

a. comprised within SEQ ID NO 01 or

b. comprised within a nucleic acid sequence complementary to SEQ ID NO 01 .

2. The nucleic acid oligomer according to claim 1 , wherein said nucleic acid oligomer consists of 8 to 50 nucleotides, particularly 15 to 40 nucleotides, more particular 18 to 30 nucleotides, even more particular 18 to 25 nucleotides.

3. The nucleic acid oligomer according to claim 1 or 2, wherein said sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to said contiguous nucleic acid sequence of the same length has a length of 8 to 50 nucleotides, particularly 15 to 40 nucleotides, more particular 18 to 30 nucleotides, even more particular 18 to 25 nucleotides.

4. The nucleic acid oligomer according to any one of claims 1 or 2, wherein said nucleic acid oligomer is a DNA oligomer.

5. The nucleic acid oligomer according to any one of the preceding claims, wherein said nucleic acid oligomer consists of or comprises a nucleic acid sequence characterized by SEQ ID NO 02, SEQ ID NO 03 or SEQ ID NO 04.

6. The nucleic acid oligomer according to any one of the preceding claims, further comprising a detectable and/or quantifiable label.

7. A combination of nucleic acid oligomers comprising:

a. a first nucleic acid oligomer consisting of or comprising a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a first contiguous nucleic acid sequence of the same length that is comprised within SEQ ID NO 01 , and

b. a second nucleic acid oligomer consisting of or comprising a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a second contiguous nucleic acid sequence of the same length that is comprised within a nucleic acid sequence complementary to SEQ ID NO 01 .

8. The combination of nucleic acid oligomers according to claim 7, wherein within SEQ

ID NO 01 said first contiguous nucleic acid sequence is located upstream of a nucleic acid sequence that is complementary to said second contiguous nucleic acid sequence.

9. The combination of nucleic acid oligomers according to any one of claims 7 or 8, wherein each of said first nucleic acid oligomer, said second nucleic acid oligomer, said first contiguous nucleic acid sequence and said second contiguous nucleic acid sequence independently of each other consists of 8 to 50 nucleotides, particularly 15 to 40 nucleotides, more particular 18 to 30 nucleotides, even more particular 18 to 25 nucleotides.

10. Use of a nucleic acid oligomer according to any one of the preceding claims or of a combination of nucleic acid oligomers according to any one of the preceding claims for detecting and/or quantifying Lactobacillus parabuchneri, particularly in a sample obtained from a foodstuff or a beverage, more particular in a sample obtained from beer, milk or a milk product, even more particular in a cheese sample.

1 1 . A method for detecting and/or quantifying Lactobacillus parabuchneri, wherein the method comprises:

providing a sample, particularly a sample obtained from a foodstuff or a beverage, more particular a sample obtained from beer, milk or a milk product, even more particular a cheese sample,

contacting said sample with a nucleic acid oligomer according to any one of claims 1 to 6 or a combination of nucleic acid oligomers according to any one of claims 7 to 9,

performing an amplification reaction with said sample in presence of a nucleic acid polymerase, under conditions allowing for the amplification of a nucleic acid sequence of Lactobacillus parabuchneri if Lactobacillus parabuchneri is present in said sample, yielding an amplificate, and

detecting the presence or absence of said amplificate and/or quantifying said amplificate, optionally relating the presence, absence or quantification of said amplificate to the presence, absence or a quantity of Lactobacillus parabuchneri.

12. The method according to claim 1 1 , wherein said amplificate is detected or quantified with

a nucleic acid oligomer according to any one of claim 1 to 6 comprising a detectable or quantifiable label, or with a combination according to any one of claims 7 to 9, wherein said first nucleic acid oligomer or said second nucleic acid oligomer comprises a detectable and/or quantifiable label.

13. The method according to claim 12, wherein said nucleic acid oligomer comprising a detectable or quantifiable label consists of or comprises SEQ ID NO 04.

14. A method for quality control on samples of a food product, comprising the steps of a. Taking a sample of the food product

b. Performing a method for detecting or quantifying Lactobacillus parabuchneri according to any one of claims 1 1 , 12 or 13,

c. Designating said food product for discard if said method detects the presence of Lactobacillus parabuchneri or if the quantity of Lactobacillus parabuchneri is above a predefined threshold, and /or designating said food product as marketeable or "passed" if said method fails to detect the presence of Lactobacillus parabuchneri or if the quantity of Lactobacillus parabuchneri is below a predefined threshold;

d. optionally, discarding the food product if said food product is designated for discard in step c.

15. A kit for detecting and/or quantifying Lactobacillus parabuchneri, comprising a nucleic acid oligomer according to any one of claims 1 to 6 or a combination of nucleic acid oligomers according to any one of claims 7 to 9, and optionally a DNA polymerase and / or deoxynucleotides.

16. The kit for detecting and/or quantifying Lactobacillus parabuchneri according to claim 15, comprising a first combination of nucleic acid oligomers according to any one of claims 7 to 9, and

a nucleic acid oligomer according to any one of claims 1 to 6 comprising a detectable and/or quantifiable label, or

a second combination according to any one of claims 7 to 9, wherein at least one of said first and said second nucleic acid oligomer comprises a detectable and/or quantifiable label.

17. The kit for detecting and/or quantifying Lactobacillus parabuchneri according to any one of claims 15 or 16, comprising a nucleic acid oligomer characterized by SEQ ID NO 02, a nucleic acid oligomer characterized by SEQ ID NO 03, and optionally a nucleic acid oligomer characterized by SEQ ID NO 04 comprising a detectable and/or quantifiable label.