Lactobacillus specific probes

Provided herein are sequence primers and probe for the detection of Lactobacillus species as well as diagnostic kit thereof.

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Description
TECHNICAL FIELD OF THE INVENTION

The present invention relates to probes and primers for quantification of Lactobacillus species.

BACKGROUND OF THE INVENTION

Generally, the intestinal flora of human milk-fed infants is primarily composed of the genus Bifidobacterium and Lactobacillus. The genera Bifidobacterium and Lactobacillus are considered to be important in maintaining a well-balanced intestinal microbiota and it has been postulated that they have several health-promoting effects.

The approach up to now was to promote Lactobacillus in general, i.e. on the genus level. The genus Lactobacillus consists of many different species, which differ in metabolism, metabolites, enzyme activities, oligo- and polysaccharide utilisation, cell wall composition, and interaction with the host's immune system. It therefore can be expected that not every species of the genus Lactobacillus has the same functional effect on the infant. Examples of different Lactobacillus species are L. rhamnosus, L. casei, L. paracasei, L. reuteri, L. fermentum, L. plantarum, L. acidophilus, and L. delbrueckii. For example, species producing large amounts of D-lactate, such as L. delbrueckii, may cause acidosis in young infants. These results indicate that the stimulation of the genus Lactobacillus per se in the babies' colon may not be sufficient. It is therefore the aim to achieve a Lactobacillus flora in formula-fed infants that is reminiscent to the Lactobacillus flora of human milk fed babies on a species level.

So far, quantification of Lactobacillus on species level in faecal samples or intestinal biopsies has not been adequately performed. Plating, followed by colony determination and conventional PCR techniques only result in qualitative or semi-quantitative data. Fluorescence in situ hybridisation is not suitable, because of the low concentration of Lactobacillus species and the available FISH probes are not sufficiently specific.

Therefore, there is a need for a method to quantify Lactobacillus species distribution in the gastrointestinal tract and/or faeces of infants.

EP0674650 relates to nucleic acid sequences which preferentially bind to the rRNA or rDNA of microorganisms which cause the spoilage of beer. The beer spoilage microorganisms are predominantly of the genera Lactobacillus and Pediococcus. The nucleic acids may be used as probes in assays to detect the presence of these microorganisms. Kits containing two or more probes are also described.

SUMMARY OF THE INVENTION

In order to quantitatively determine the Lactobacillus species distribution in faeces real-time PCR (5′nuclease assay) has to be used, for which species specific probes and primers are provided.

A method of species-specifically detecting and quantitatively assaying species of the genus Lactobacillus found in humans, particularly human infants, as well as a diagnostic kit for the detection and quantification of Lactobacillus species is provided.

DETAILED DESCRIPTION OF THE INVENTION

Probe Development and Diagnostic Kit

A method of species-specifically detecting and quantitatively assaying species of the genus Lactobacillus found in human faeces and/or intestine, particularly human infants, as well as a diagnostic kit for the detection and quantification of Lactobacillus species is provided.

Also provided herein is a method for quantifying Lactobacillus species, especially those found in humans, i.e. L. acidophilus, L. casei, L. delbrueckii, L. fermentum. L. paracasei, L. plantarum, L. reuteri and L rhamnosus using species specific oligonucleotide primes and probes.

These primers and probes can be used to identify species of Lactobacillus and Lactobacillus-like species via FISH, PCR, DGGE, TGGE, dot blot hybridisation and real time PCR methods. All these techniques have in common that it involves a hybridisation step with nucleotides. Preferably real time PCR is used to quantify Lactobacillus species.

Each of the sequences described below may have additional bases bonded to the 5′- or 3′-terminal thereof as long as it functions as a probe.

These oligonucleotides can be prepared by conventional means for chemical synthesis, for example by an automated DNA synthesiser. DNA fragments containing the above-mentioned sequences can be prepared by enzymatic cleavage of genes from the corresponding Lactobacillus species.

For the purpose of the present invention, the development of primers and probes specific to the Lactobacillus species for use in the 5′nuclease assay was as follows:

Duplex 5′ nuclease assays were developed for L. acidophilus, L. casei, L. delbrueckii, L. fermentum, L. paracasei, L. plantarum, L. reuteri and L rhamnosus as a proportion of the total cells of the genus Lactobacillus. We developed the 5′ nuclease assays on the intergenic spacer of 16S-23S rDNA instead of the 16S rDNA gene, which is normally used for the phylogenetic analyses and specific detection of bacteria. The choice for the intergenic spacer greatly depended on the fact that contamination and sensitivity issues were described for Real-Time PCR when 16S rDNA was used. Furthermore, a large similarity between the 16S rDNA sequences of the different Lactobacillus species was shown (Leblond-Bourget et. al. 1996), which made it almost impossible to develop primer and probe sets specific for the different Lactobacillus species. Surprisingly, these problems could be avoided by using the intergenic spacer region.

For the development of primers and probes the different sequences of the 16S-23S intergenic spacer region of the different Lactobacillus species (L. acidophilus [AB102855 (3), AF182726 (5), U32971 (7)]a, L. amylovorus [AF182732 (5)], L. animalis [AY526616, AY526614], L. brevis [AB102858 (3), AF405353 (2)], L. bulgaricus [Z75475], L. casei [AB102854 (3), AF405352 (2), AF182729 (5), AF121200 (6)], L. collinoides [AB117957, AB117955], L. crispatus [AF182719 (5), AF074857 (6)], L. curvatus [AF074858 (6), U97135 (1), U97129 (1)], L. delbrueckii [AB102856 (3), AB035485 (5), AB035484 (5), U32969 (7), U32968 (7), U32967 (7)], L. farciminis [AF500491 (4), AF500490 (4)], L. fermentum [AF182720 (5)], L. frumenti [AJ616011], L. gasseri [AB102860 (3), AF182721 (5), AF074859 (6)], L. graminis [U97136 (1), U97130 (1)], L. hamsteri, L. helveticus [AF182728 (5)], L. jensenii [AB035486 (5), U32970 (7)], L. johnsonii [AF074860 (6)], L. mindensis [AJ616016], L. panis [AJ616012], L. paracasei [AB035487 (5), AF182724 (5), U32964 (7)], L. paralimentarius [AJ616014], L. paraplantarum [U97138 (1), U97132 (1)], L. pentosus [U97141 (1), U97140 (1), U97134 (1)], L. plantarum [AB102857 (3), AF405354 (2), AF182722 (5), U97139 (1), U97133 (1)], L. sakei [U97137 (1), U97131 (1)], L. salivarius [AB102859 (3), AB03488 (5), AF182725 (5)], L. sharpeae [AF074861 (6)], L. reuteri [AF182723 (5)], L. rhamnosus [AF182730 (5), AF121201 (6), U32966 (7)], L. ruminis [AF080103], L. vaginalis [AF182731], L. zeae [AF074862]a) were retrieved from Genbank, EMBL and DDBJ databases. All retrieved sequences were aligned using DNASIS for Windows V2.5 (Hitachi Software Engineering Co., Ltd., Wembley, UK). (a=accession codes, 1: Berthier et al. 1998. FEMS Microbiol Left 161:97-106. 2: Dobson et al. 2002. Int J Syst Evol Microbiol 52:2003-10. 3: Massi et al. 2004. J Appi Microbiol 96:777-86. 4: Rachman et al. 2003. J Appi Microbiol 95:1207-16. 5: Song et al. 2000. FEMS Microbiol Left 187:167-73. 6: Tannock et al. 1999 Appl Environ Microbiol 65:4264-7. 7: Tilsala-Timisjarvi, A., and T. Alatossava. 1997. Int J Food Microbiol 35:49-56.)

The overall conserved regions of the sequences were used to design primers and probes for the Lactobacillus genus. To increase specificity and sensitivity, TaqMan Minor groove Binding (MGB) probes were used. Conserved regions in the sequences of the different kind of subspecies, which showed little homology with other species were used to design primers and probes for respectively L. acidophilus, L. casei, L. delbrueckii, L. fermentum, L. paracasei, L. plantaurum, L. rhamnosus and L. reuteri.

The primers and TaqMan MGB probes were designed with help of Primer Express 1.5a (Applied Biosystems, Nieuwerkerk a/d IJssel, N L). We applied the following criteria: The probe and primers should have a GC content of 30 to 80% and runs of more than 3 identically nucleotides (especially for guanidine (G)) should be avoided. The melting temperature (Tm) of the probe should be between 68° C. and 70° C., whereas the primers should have a melting temperature 10° C. below the melting temperature of the probe. Furthermore, no G on the 5′ end of the probe should be present and the strand with more cytosine (C) than G was selected. The last 5 nucleotides at the 3′ end of the primers should have no more than two G and/or C bases. Finally, the amplicon length should be less than 150 base pairs. The designed primers and TaqMan MGB probes are shown in table 1 and were tested on specificity using the Basic Local Alignment Search Tool (BLAST).

The probe designed for the detection of the genus Lactobacillus consists of an oligonucleotide with the 5′ reporter dye VIC™ (Applied Biosystems, NL) and the 3′ quencher NFQ-MGB™ (Applied Biosystems, NL) and the probes for the different Lactobacillus species of oligonucleotides with the 5′ reporter dye 6-carboxy-fluorescein (FAM™) and the 3′ quencher NFQ-MGB™ (Applied Biosystems, NL). For determination of the total bacterial load a broad-range (universal probe and primer set is used, which is described by Nadkarni, et al, 2002, Microbiology 148:257-266. The universal probe consists of oligonucleotides with the 5′ reporter dye 6-carboxy-fluorescein (FAM™) and the 3′ quencher dye 6-carboxy-tetramethyl-rhodamine (TAMRA™). The designed probes are shown in table 1.

TABLE 1 Designed primers and probes for use in the 5′ nuclease assays. % Primers & Tm G BLAST ID Amplicon SEQ Target Probes Sequence (5′ → 3′) (° C.) C number length ID No L. acido- F_acid_IS GAAAGAGCCCAAAC 59 43 1089017502- 85 1 philus CAAGTGATT 26171- 2029659558 40 R_acid_IS CTTCCCAGATAATT 59 37 1089017571- 2 CAACTATCGCTTA 27139- 5254509477 P_acid_IS TACCACTTTGCA 70 45 2 3 GTCCTACA 1089017717- 29310- 1542960554 15 L. casei F_case_IS CTATAAGTAAGCTTT 59 36 1037022798- 132 4 GATCCGGAGATTT 023495-2136 R_case_IS CTTCCTGCGGGTAC 59 55 1037022917- 5 TGAGATGT 024843- 29627 P_case_IS ACAAGCTATGAATT 70 38 1037022752- 6 CACT TGC 023005- 20772 L. del- F_delb_IS CACTTGTACGTTGA 58 30 1089018504- 94 7 brueckii AAACTGAATATCTTA 4206- Aa 6452981190 6 R_delb_IS CGAACTCTCTCGGT 58 55 1089018475- 8 CGCTTT 6841- 1666577681 51 P_delb_IS CCGAGAATCATTGA 68 44 1089018437- 9 GATC 6309- 1639882274 98 L. fer- F_ferm_IS AACCGAGAACACCG 58 50 1036676682- 88 10 mentum CGTTAT 09669-23287 R_ferm_IS ACTTAACCTTACTGA 58 38 1036676709- 11 TCGTAGATCAGTCA 010209-2351 P_ferm_IS TAATCGCATACTCA 68 32 1036676736- 12 ACTAA 010547- 20717 L. para- F_paca_IS ACATCAGTGTATTG 60 38 1038306417- 80 13 casei CTTGTCAGTGAATA 016220- C 23561 R_paca_IS CCTGCGGGTACTGA 60 55 103806445- 14 GATG TTTC 016796-3050 P_paca_IS TGCCGCCGGCCAG 70 85 1038306524- 15 018375-2626 L. F_plan_IS TGGATCACCTCCTT 58 42 1038305707- 144 16 planta- TCTAAGGAAT 03107-18756 rum R_plan_IS TGTTCTCGGTTTCAT 58 26 1038305742- 17 TATGAAAAAATAa 04177-12861 P_plan_IS ACATTCTTCGAAACT 68 32 1038305778- 18 TTGT 04682-12880 L. F_reut_IS ACCGAGAACACCGC 59 48 1089025339- 93 19 reuteri GTTATTT 29395- 1292800472 16 R_reut_IS CATAACTTAACCTAA 59 28 1089025385- 20 ACAATCAAAGAT 30347- TGTCT 3755823275 4 P_reut_IS ATCGCTAACTCAATT 69 28 1089025413- 21 AAT 30287- 2611284585 4 L. F_rham_IS CGGCTGGATCACCT 59 58 1023708254- 97 22 rhamno- CCTTT 09591-2284 sus R_rham_IS GCTTGAGGGTAATC 59 52 1023708352- 23 CCCT CAA 010389- 16127 P_rham_IS CCTGCACACACGAA 69 55 1023708453- 24 A 011313-6655 Genus F_alliact_IS TGGATGCCTTGGCA 58 55 1024485925- 92 25 Lacto- CTAGGA 024664- bacillus 30598 R_alliact_IS AAATCTCCGGATCA 58 35 1024478788- 26 AAGCTTACTTAT 024701- 16287 P_alliact_IS TATTAGTTCCGTCCT 68 40 1024478009- 27 TCATC 017753- 28422 All F_eub TCCTACGGGAGGCA 59 Nadkarni et. 466 bpa bacteria GCAGT al. 2002 R_eub GGACTACCAGGGTA 58 TCTAATCCTGTT P_eub CGTATTACCGCGGC 70 TGCTGGCAC
aIn these cases some adjustments (more than 3 consecutive nucleotides or an amplicon length greater then 150 bp) were made to the guidelines to find an appropriate primer and probe set.

Labelled preparations are prepared by labelling the oligonucleotide with a detectable marker by conventional means. Labelling markers which may be used include radioisotopes, fluorescent substances, enzymes, biotin and haptens. Hybridisation between the labelled preparation and a sample can be performed in accordance with known techniques, such as dot blot hybridisation and northern hybridisation. The hybrid which are formed can be confirmed through the detection of the labelled preparation by known means, for example, autoradiography using radioisotopes, enzyme-labelled antibody techniques using enzyme or biotin, and the like.

Further, of these oligonucleotides, the DNA fragments represented by SEQ ID selected from SEQ ID No 1, SEQ ID No 2, SEQ ID No 4, SEQ ID No 5, SEQ ID No 7, SEQ ID No 8, SEQ ID No 10, SEQ ID No 11, SEQ ID No 13, SEQ ID No 14, SEQ ID No 16, SEQ ID No 17, SEQ ID No 19, SEQ ID No 20, SEQ ID No 22, SEQ ID No 23, SEQ ID No 25, and SEQ ID No 26 respectively can be used as a primer in the PCR method for identification of species. More specifically, microbial cells to be identified are subjected to bacteriolysis, and any of the DNA fragments of SEQ ID selected from SEQ ID No 1, SEQ ID No 4, SEQ ID No 7, SEQ ID No 10, SEQ ID No 13, SEQ ID No 16, SEQ ID No 19, SEQ ID No 22, SEQ ID No 25, respectively and SEQ ID selected from SEQ ID No 2, SEQ ID No 5, SEQ ID No 8, SEQ ID No 11, SEQ ID No 14, SEQ ID No 17, SEQ ID No 20, SEQ ID No 23, SEQ ID No 26, respectively is added thereto as a primer, followed by treatment with a DNA polymerase. If DNA amplification is observed using electrophoresis, etc., this means that the cells possess a gene portion which corresponds to the DNA fragment used, i.e. the cells are identified to be of the same species as the origin of the DNA fragment primer.

Accordingly, there are provided oligonucleotides comprising SEQ ID selected from SEQ ID No 1, SEQ ID No 2, SEQ ID No 4, SEQ ID No 5, SEQ ID No 7, SEQ ID No 8, SEQ ID No 10, SEQ ID No 11, SEQ ID No 13, SEQ ID No 14, SEQ ID No 16, SEQ ID No 17, SEQ ID No 19, SEQ ID No 20, SEQ ID No 22, SEQ ID No 23, SEQ ID No 25, SEQ ID No 26, and sequences complementary thereto.

Also provided herein are oligonucleotide probe for detection of a nucleic acid target sequence which is characteristic of the species of the genus Lactobacillus, said probe being selected from:

1)-a labelled oligonucleotide which specifically hybridises to Lactobacillus acidophilus DNA represented by SEQ ID No 3 or a sequence complementary thereto;

2)-a labelled oligonucleotide which specifically hybridises to Lactobacillus casei DNA represented by SEQ ID No 6 or a sequence complementary thereto;

3)-a labelled oligonucleotide which specifically hybridises to Lactobacillus delbrueckii DNA represented by SEQ ID No 9 or a sequence complementary thereto;

4)-a labelled oligonucleotide which specifically hybridises to Lactobacillus fermentum DNA represented by SEQ ID No 12 or a sequence complementary thereto;

5)-a labelled oligonucleotide which specifically hybridises to Lactobacillus paracasei DNA represented by SEQ ID No 15 or a sequence complementary thereto;

6)-a labelled oligonucleotide which specifically hybridises to Lactobacillus plantarum DNA represented by SEQ ID No 18 or a sequence complementary thereto;

7)-a labelled oligonucleotide which specifically hybridises to Lactobacillus reuteri DNA represented by SEQ ID No 21 or a sequence complementary thereto;

8)-a labelled oligonucleotide which specifically hybridises to Lactobacillus rhamnosus DNA represented by SEQ ID No 24 or a sequence complementary thereto;

9)-a labelled oligonucleotide which specifically hybridises to Lactobacillus DNA represented by SEQ ID No 27 or a sequence complementary thereto.

Further provided herein is a method of species-specifically detecting species of the genus Lactobacillus found in human, particularly human infants, comprising the steps of:

(A) contacting a sample with an oligonucleotide probe in a hybridising solution, wherein said probe is selected from the group consisting of:

1) a labelled oligonucleotide which specifically hybridises to Lactobacillus acidophilus DNA represented by SEQ ID No 3 or a sequence complementary thereto;

2) a labelled oligonucleotide which specifically hybridises to Lactobacillus casei DNA represented by SEQ ID No 6 or a sequence complementary thereto;

3) a labelled oligonucleotide which specifically hybridises to Lactobacillus delbrueckii DNA represented by SEQ ID No 9 or a sequence complementary thereto;

4) a labelled oligonucleotide which specifically hybridises to Lactobacillus fermentum DNA represented by SEQ ID No 12 or a sequence complementary thereto;

5) a labelled oligonucleotide which specifically hybridises to Lactobacillus paracasei DNA represented by SEQ ID No 15 or a sequence complementary thereto;

6) a labelled oligonucleotide which specifically hybridises to Lactobacillus plantarum DNA represented by SEQ ID No 18 or a sequence complementary thereto;

7) a labelled oligonucleotide which specifically hybridises to Lactobacillus reuteri DNA represented by SEQ ID No 21 or a sequence complementary thereto;

8) a labelled oligonucleotide which specifically hybridises to Lactobacillus rhamnosus DNA represented by SEQ ID No 24 or a sequence complementary thereto;

9) a labelled oligonucleotide which specifically hybridises to all Lactobacillus DNA represented by SEQ ID No 27 or a sequence complementary thereto, and

(B) determining whether said probe hybridises to nucleic acids in said sample so as to detect whether said species of said genus is present in said sample.

The present invention also encompasses a method of species-specifically detecting species of the genus Lactobacillus found in human, particularly human infants, comprising the steps of:

a) performing a nucleic acid sequence amplification procedure using a primer set comprising the oligonucleotide primer of SEQ ID No selected from SEQ ID No 1, SEQ ID No 4, SEQ ID No 7, SEQ ID No 10, SEQ ID No 13, SEQ ID No 16, SEQ ID No 19, SEQ ID No 22, SEQ ID No 25, and respectively with the oligonucletide primer of SEQ ID selected from SEQ ID No 2, SEQ ID No 5, SEQ ID No 8, SEQ ID No 11, SEQ ID No 14, SEQ ID No 17, SEQ ID No 20, SEQ ID No 23, SEQ ID No 26; and

b) determining whether the oligonucleotide probe above mentioned hybridises to the nucleic acid target sequence, preferably the oligonucleotide probe is of SEQ ID No selected from SEQ ID No 3, SEQ ID No 6, SEQ ID No 9, SEQ ID No 12, SEQ ID No 15, SEQ ID No 18, SEQ ID No 21, SEQ ID No 24, SEQ ID No 27.

The present method is beneficial for the manufacture of a diagnostic kit. Accordingly, a diagnostic kit is herein provided for the detection in a sample of Lactobacillus species selected from L. acidophilus, L. casei, L. delbrueckii, L. fermentum, L. paracasei, L. plantarum, L. reuteri and L. rhamnosus, by means of hybridisation analysis, comprising at least a DNA probe as mentioned above as well as one or more further means required for hybridisation analysis, such as denaturation liquid, a hybridisation liquid, a washing liquid, a solid carrier, a hybridisation vessel and label detecting means. Also herein provided is a diagnostic kit for the detection in a sample of the above-mentioned Lactobacillus species by means of PCR analysis, comprising a set of DNA primers as mentioned above as well as one or more further means required for PCR analysis, such as a polymerase, a polymerisation liquid, an oil overlay, a reaction vessel and means for detecting the amplified DNA. In one embodiment the diagnostic kit comprises at least a DNA probe as described above and a set of primers as described above and optionally further means as mentioned above.

The present probes and methods are particularly suitable for quantifying Lactobacillus on a species level in the intestine and/or faeces. The probe and primers with SEQ ID 25, 26, and 27 are not related to specific Lactobacillus species but to the genus Lactobacillus. SEQ ID 25, 26, 27 are used to quantify the genus Lactobacillus and to relate the quantity of specific Lactobacillus species to all lactobacilli. Thus it may be said that SEQ ID NOs 1-24 are used for detecting and identifying a species and that SEQ ID NOs 25-27 are used for quantifying this identified species. Preferably Lactobacillus species are quantified in infant faeces, since lactobacilli play an important role in the infants' health. The results obtained from such an analysis can be used for selecting suitable Lactobacillus species as a probiotic supplement or probiotic food or component in a (helath) nutritional composition and/or selecting non-digestible saccharides selectively stimulating Lactobacillus species to be incorporated into a (health) nutritional composition. These probiotics and/or prebiotics are preferably to be administered to the subject low in specific Lactobacillus species in order to rebalance the intestinal flora on a Lactobacillus species level and prevent or treat disorders associated with this imbalance. Preferably, the disorder is selected from the group consisting of allergy, atopic disease, eczema, diarrhoea, intestinal inflammation and infections. Likewise, the present probes and methods are preferably applied for selecting probiotics and/or non-digestible saccharides for administration to animals in order to promote animal health, more preferably the gastro-intestinal health of animals. Thus a method for preparing a nutritional composition is provided, said method comprising selecting suitable Lactobacillus species and/or selecting non-digestible saccharides selectively stimulating Lactobacillus species by applying the oligonucleotide probes according to the present invention and preferably the oligonucleotide primers according to the present invention, preferably in a method according to the present invention and incorporating said selected Lactobacillus species and/or non-digestible saccharides into a (health) nutritional composition.

The present probes and methods are preferably used for quantification and species determination of systemic infections by species from the genus Lactobacillus.

The present probes and methods are preferably used in controlling and/or monitoring food fermentation by Lactobacillus species.

EXAMPLE

Validation and Optimisation of the Developed Probes and Primers for Lactobacilli

The bacterial strains used to validate the assays for the relative quantification of the different Lactobacillus species are listed in Table 2.

TABLE 2 Bacterial strains (origin) used in this study. Lactobacillus Strains Origina Other Strains Origina L. acidophilus ATCC 4356 Bacillus cereus ATCC 11778 ATCC 521 Bacteroides fragilis LMG 10263T DSM 20079T Brevibacterium casei ATCC 35513T L. amylovorus DSM 20552 Clostridium difficile ATCC 9689T L. bavaricus JCM 1128 Enterococcus feacalis DSM 20478T L. brevis LMG 18022 Escherichia coli ATCC 35218 L. bulgaricus ATCC 11842T Listeria ATCC 7644 monocytogenes L. casei ATCC 393T Pediococcus DSM 20284T acidilactici DSM 20011T Propionibacterium DSM 4901 avidum L. crispatus ATCC 33820T Pseudomonas DSM 1117 aeruginosa L. curvatus ATCC 51436 Saccharomyces DSM 2548 cerevisiae L. delbrueckii JCM 1106 Salmonella ATCC 14028 typhimurium JCM 1248T Staphylococcus aureus ATCC 29213 L. fermentum DSM 20052T Bifidobacterium ATCC 15703T adolescentis L. gasseri LMG 11496T Bifidobacterium DSM 20098T angulatum Bifidobacterium DSM 20456T bifidum L. helveticus CNRZ 32 Bifidobacterium ATCC 25527T animalis L. johnsonii ATCC 33200T Bifidobacterium DSM 20093T gallicum L. kefir LMG 11496 Bifidobacterium ATCC 27534T dentium L. kefirgranum DSM 10550T Bifidobacterium breve ATCC 15700T L. paracasei ATCC 11582 Bifidobacterium ATCC 27539T catenulatum ATCC 27216 Bifidobacterium LMG 8811T infantis L. pentosus JCM 8334 Bifidobacterium ATCC 15707T JCM 8338 longum L. plantarum DSM 20174T NCIMB 8826 L. reuteri LMG 9213T L. rhamnosus ATCC 53103 ATCC 7469T L. sake DSM 6333 L. salivarius ATCC 11741T
aATCC: American Type Culture Collection; CNRZ: Centre National de Recerches Zootechniques, France; DSM: Deutsche Sammlung von Mikroorganismen und Zellkulturen, Germany; JCM: Japan Collection of Microorganisms, Japan; LMG: Laboratory for Microbiology, University of Gent, Belgium; NCIMB: National Collections of Industrial and Marine Bacteria, UK;

TType strain

All strains of lactobacilli, bifidobacteria, propionibacteria, enterococci, pediococci and Saccharomyces were cultured overnight under suitable conditions stored at −20° C. DNA was extracted by thawing 5 ml of frozen overnight cultures by methods known in the art and stored at −20° C.

The specificity of each duplex 5′ nuclease assay was tested by performing a 25 μl amplification of the different strains. These 25 μl PCR reactions were performed using 2.5 μl DNA template, 12.5 μl TaqMan Universal Master Mix (Applied Biosystems), optimised concentrations of the primers and probes (see table 3), followed by running the TaqMan Universal Temperature Profile, on the ABI Prism 7700 (Applied Biosystems, Nieuwerkerk a/d IJssel, N L). All of the 5′ nuclease assays were specific for the Lactobacillus species for which they were developed and the 5′nuclease assay for determination of the genus Lactobacillus detected all Lactobacillus species tested, but no other bacteria like Propionibacterium, Enterococcus or Pediococcus species.

The optimised concentrations of the primers and probes are given in table 3. The CV values for reproducibility and repeatability for the different kind of 5′ nuclease assays were determined and can be found in table 4.

TABLE 3 Optimised final primer and probe concentrations used in the different duplex 5′ nuclease assays Forward Reverse Probe Target 5′ nuclease assay Primer (nM) Primer (nM) (nM) L. acidophilus L. acidophilus 900 900 200 Genus Lactobacillus 900 900 200 L. casei L. casei 900 900 200 Genus Lactobacillus 300 300 50 L. delbrueckii L. delbrueckii 300 300 100 Genus Lactobacillus 900 900 200 L. fermentum L. fermentum 300 300 100 Genus Lactobacillus 300 300 100 L. paracasei L. paracasei 300 300 100 Genus Lactobacillus 300 300 100 L. plantarum L. plantarum 300 300 100 Genus Lactobacillus 300 300 100 L. reuteri L. reuteri 300 300 100 Genus Lactobacillus 900 900 200 L. rhamnosus L. rhamnosus 900 450 200 Genus Lactobacillus 150 100 100 Genus Genus Lactobacillus 600 600 100 Lactobacillus All bacteria 300 300 100

TABLE 4 The CV values for reproducibility and repeatability of the 5′ nuclease assays Target Reproducibilitya (CV) Repeatabilityb (CV) L. acidophilus 0.109 0.127 L. casei 0.058 0.063 L. delbrueckii 0.059 0.035 L. fermentum 0.067 0.048 L. paracasei 0.052 0.110 L. plantarum 0.134 0.135 L. reuteri 0.062 0.113 L. rhamnosus 0.047 0.053
areproducibility is determined by testing monocultures (100%) in ten fold and calculation of the CV (%) based on the gained results

brepeatability is determined by testing monocultures (100%) three times in four fold and calculation of the CV (%) based on results gained.

The developed and validated duplex 5′ nuclease assays show a good specificity, sensitivity, reproducibility and repeatability and can therefore be seen as a good tool for enumeration of the different Lactobacillus species in faeces or intestinal biopsies.

Claims

1. Oligonucleotides comprising SEQ ID selected from SEQ ID No 1, SEQ ID No 2, SEQ ID No 4, SEQ ID No 5, SEQ ID No 7, SEQ ID No 8, SEQ ID No 10, SEQ ID No 11, SEQ ID No 13, SEQ ID No 14, SEQ ID No 16, SEQ ID No 17, SEQ ID No 19, SEQ ID No 20, SEQ ID No 22, SEQ ID No 23, SEQ ID No 25, SEQ ID No 26, and sequences complementary thereto.

2. Oligonucleotide probe for detection of a nucleic acid target sequence which is characteristic of the species of the genus Lactobacillus, said probe being selected from:

1) a labelled oligonucleotide which specifically hybridises to L. acidophilus DNA represented by SEQ ID No 3 or a sequence complementary thereto;
2) a labelled oligonucleotide which specifically hybridises to L. casei DNA represented by SEQ ID No 6 or a sequence complementary thereto;
3) a labelled oligonucleotide which specifically hybridises to L. delbrueckii DNA represented by SEQ ID No 9 or a sequence complementary thereto;
4) a labelled oligonucleotide which specifically hybridises to L. fermentum DNA represented by SEQ ID No 12 or a sequence complementary thereto;
5) a labelled oligonucleotide which specifically hybridises to L. paracasei DNA represented by SEQ ID No 15 or a sequence complementary thereto;
6) a labelled oligonucleotide which specifically hybridises to L. plantarum DNA represented by SEQ ID No 18 or a sequence complementary thereto;
7) a labelled oligonucleotide which specifically hybridises to L. reuteri DNA represented by SEQ ID No 21 or a sequence complementary thereto;
8) a labelled oligonucleotide which specifically hybridises to L. rhamnosus DNA represented by SEQ ID No 24 or a sequence complementary thereto;
9) a labelled oligonucleotide which specifically hybridises to the genus Lactobacillus DNA represented by SEQ ID No 27 or a sequence complementary thereto.

3. A method of species-specifically detecting species of the genus Lactobacillus found in human, particularly human infants, comprising the steps of:

(A) contacting a sample with an oligonucleotide probe in a hybridising solution, wherein said probe is selected from the group consisting of: 1) a labelled oligonucleotide which specifically hybridises to L. acidophilus DNA represented by SEQ ID No 3 or a sequence complementary thereto; 2) a labelled oligonucleotide which specifically hybridises to L. casei DNA represented by SEQ ID No 6 or a sequence complementary thereto; 3) a labelled oligonucleotide which specifically hybridises to L. delbrueckii DNA represented by SEQ ID No 9 or a sequence complementary thereto; 4) a labelled oligonucleotide which specifically hybridises to L. fermentum DNA represented by SEQ ID No 12 or a sequence complementary thereto; 5) a labelled oligonucleotide which specifically hybridises to L. paracasei DNA represented by SEQ ID No 15 or a sequence complementary thereto; 6) a labelled oligonucleotide which specifically hybridises to L. plantarum DNA represented by SEQ ID No 18 or a sequence complementary thereto; 7) a labelled oligonucleotide which specifically hybridises to L. reuteri DNA represented by SEQ ID No 21 or a sequence complementary thereto; 8) a labelled oligonucleotide which specifically hybridises to L. rhamnosus DNA represented by SEQ ID No 24 or a sequence complementary thereto; 9) a labelled oligonucleotide which specifically hybridises to the genus Lactobacillus DNA represented by SEQ ID No 27 or a sequence complementary thereto; and
(B) determining whether said probe hybridises to nucleic acids in said sample so as to detect whether said species of said genus is present in said sample.

4. A method of species-specifically quantifying the detected species of the genus Lactobacillus found in humans, particularly in human faeces, according to claim 3, wherein said quantifying method is real time PCR.

5. A method of species-specifically detecting species of the genus Lactobacillus found in human, particularly human infants, comprising the steps of:

a) performing a nucleic acid sequence amplification procedure using a primer set comprising the oligonucleotide primer of claim 1, preferably the oligonucleotide primer of SEQ ID No selected from SEQ ID No 1, SEQ ID No 4, SEQ ID No 7, SEQ ID No 10, SEQ ID No 13, SEQ ID No 16, SEQ ID No 19, SEQ ID No 22, SEQ ID No 25, and respectively with the oligonucleotide primer of SEQ ID selected from SEQ ID No 2, SEQ ID No 5, SEQ ID No 8, SEQ ID No 11, SEQ ID No 14, SEQ ID No 17, SEQ ID No 20, SEQ ID No 23, SEQ ID No 26; and
b) determining whether an oligonucleotide probe of claim 2, preferably respectively the oligonucleotide probe of SEQ ID No selected from SEQ ID No 3, SEQ ID No 6, SEQ ID No 9, SEQ ID No 12, SEQ ID No 15, SEQ ID No 18, SEQ ID No 21, SEQ ID No 24, SEQ ID No 27, hybridises to a nucleic acid target sequence.

6. A diagnostic kit for the detection in a sample of Lactobacillus species selected from L. acidophilus, L. casei, L. delbrueckii, L. fermentum, L. paracasei. L. plantarum, L. reuteri, and L. rhamnosus, by means of hybridisation analysis, comprising at least a DNA probe according to claim 2 as well as one or more further means required for hybridisation analysis, such as denaturation liquid, a hybridisation liquid, a washing liquid, a solid carrier, a hybridisation vessel and label detecting means.

7. A diagnostic kit for the detection in a sample of Lactobacillus species selected from L. acodophilus, L. casei, L. delbrueckii, L. fermentum, L. paracasei, L. plantarum, L. reuteri, and L. rhamnosus by means of PCR analysis, comprising a set of DNA primers according to claim 1 as well as one or more further means required for PCR analysis, such as a polymerase, a polymerisation liquid, an oil overlay, a reaction vessel and means for detecting the amplified DNA.

8. Use of the oligonucleotide sequences and methods of any of the preceding claims for quantifying Lactobacillus species in faeces.

9. Use of the oligonucleotide sequences and methods of any of the preceding claims for selecting Lactobacillus species and/or non-digestible saccharides selectively stimulating the growth of Lactobacillus species, to be incorporated into a nutritional composition.

Patent History
Publication number: 20060194241
Type: Application
Filed: Feb 28, 2006
Publication Date: Aug 31, 2006
Inventors: Jan Knol (Wageningen), Monique Haarman (Wageningen)
Application Number: 11/364,963
Classifications
Current U.S. Class: 435/6.000; 536/24.100
International Classification: C12Q 1/68 (20060101); C07H 21/04 (20060101);