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Lactobacillus specific probesRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic AcidLactobacillus specific probes description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060194241, Lactobacillus specific probes. Brief Patent Description - Full Patent Description - Patent Application Claims
TECHNICAL FIELD OF THE INVENTION [0001] The present invention relates to probes and primers for quantification of Lactobacillus species. BACKGROUND OF THE INVENTION [0002] 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. [0003] 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. [0004] 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. [0005] Therefore, there is a need for a method to quantify Lactobacillus species distribution in the gastrointestinal tract and/or faeces of infants. [0006] 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 [0007] 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. [0008] 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 [0009] Probe Development and Diagnostic Kit [0010] 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. [0011] 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. [0012] 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. [0013] 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. [0014] 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. [0015] 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: [0016] 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. [0017] 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)].sup.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). (.sup.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.) [0018] 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. [0019] 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.degree. C. and 70.degree. C., whereas the primers should have a melting temperature 10.degree. 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). [0020] The probe designed for the detection of the genus Lactobacillus consists of an oligonucleotide with the 5' reporter dye VIC.TM. (Applied Biosystems, NL) and the 3' quencher NFQ-MGB.TM. (Applied Biosystems, NL) and the probes for the different Lactobacillus species of oligonucleotides with the 5' reporter dye 6-carboxy-fluorescein (FAM.TM.) and the 3' quencher NFQ-MGB.TM. (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.TM.) and the 3' quencher dye 6-carboxy-tetramethyl-rhodamine (TAMRA.TM.). The designed probes are shown in table 1. TABLE-US-00001 TABLE 1 Designed primers and probes for use in the 5' nuclease assays. % Primers & Tm G BLAST ID Amplicon SEQ Target Probes Sequence (5' .fwdarw. 3') (.degree. 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- A.sup.a 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 TATGAAAAAATA.sup.a 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 bp.sup.a bacteria GCAGT al. 2002 R_eub GGACTACCAGGGTA 58 TCTAATCCTGTT P_eub CGTATTACCGCGGC 70 TGCTGGCAC .sup.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. Continue reading about Lactobacillus specific probes... 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