EP2430192A1 - Methode de detection d'adn procaryote extrait d'un echantillon de selles - Google Patents
Methode de detection d'adn procaryote extrait d'un echantillon de sellesInfo
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- EP2430192A1 EP2430192A1 EP10727071A EP10727071A EP2430192A1 EP 2430192 A1 EP2430192 A1 EP 2430192A1 EP 10727071 A EP10727071 A EP 10727071A EP 10727071 A EP10727071 A EP 10727071A EP 2430192 A1 EP2430192 A1 EP 2430192A1
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- Prior art keywords
- seq
- specific
- sequence
- dna
- sequences
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Definitions
- the present invention relates to a method for detecting and preferably quantifying DNA, preferably comprising prokaryotic DNA, extracted from a stool sample of an individual, in particular for the molecular determination of the composition of the intestinal flora in stool.
- the present invention also relates to a DNA detection and quantification kit that is useful for implementing a method for detecting and quantifying DNA according to the invention.
- microbiota here called gut microbiota
- gut microbiota the composition of the intestinal flora in humans because this flora is involved in the physiological processes of the digestion of beverages and foods, and its role is suspected or substantiated in different pathological processes digestive and extradigestive.
- the role of the gut microbiota in obesity is a hot topic [Turnbaugh PJ et al. - A core gut microbiome in obesity an lean twins. Nature 2009; 457: 480-4].
- the weight status of an individual is the result of the number of calories ingested (role of the diet), the number of calories extracted during digestion (role of the gut microbiota), the number of calories stored (role of adipose tissue) and the number of calories expended (role of physical exertion).
- a first study showed that the obese individuals had a bias of the ratio of the bacteria of the phylum Firmicutes compared to the bacteria of the phylum Bacteroidetes (ratio Firmicutes / Bacteroidetes or F / B) compared to the control individuals due mainly to the decrease of the Bacteroidetes in the subjects obese [Ley RE et al. - Human gut microbes associated with obesity. Nature 2006; 444: 1022-3].
- Bacteroides thetaiotaomicron results in increased caloric conversion in xenobiotic mice [Samuel BS et al. -A humanized gnotobiotic mouse model of host-archaeal-bacterial mutualism.
- pathologies in which the intestinal microbiota is known or suspected to play a role include the genesis of colonic cancers [Pique JM et al. - Methane production and colon cancer. Gastroentorology 1984; 87: 601-5] and other chronic inflammatory digestive conditions [Peled Y. et al. - Factors affecting methane production in humans. Gastrointestinal diseases and alterations of flora colony - Dig. Dis. Sci. 1987; 32: 267-71; Waever GA et al. - Incidence of methanogenic bacteria in sigmoidoscopy population: an association of methanogenic bacteria in diverticulosis. Gut 1986; 27: 698-704].
- the SSU rDNA gene of M. smithii is detected and quantified in a single sample and is also not specific for M. smithii but is found in any other methanogenic archae.
- the DNA extraction protocol used does not involve chemical lysis or lysis by heat.
- the inventors have discovered that the extraction methods used in the state of the art to extract the DNA from the stool were not sufficiently effective to lead to reliable results in terms of quantification, in particular when seeks to quantify thick-walled Archae-type prokaryotes.
- the inventors have developed a method for quantifying certain species of microorganisms and certain phyla of microorganisms, from a stool sample, routinely applicable in research laboratories and medical and veterinary diagnostic laboratories, and comprising a marker internal quality.
- the inventors developed a method for the molecular quantification of bacteria of the phylum Firmicutes, specifically bacteria of the genus Lactobacillus, and bacteria of the phylum Bacteroidetes.
- the choice of phyla firmicutes and bacteroidetes has been explained above.
- the choice of the genus Lactobacillus, which is a genus of the phylum Firmicutes, is justified by the fact that the growth promoters and in particular Lactobacillus were associated in chickens with a very important weight gain especially in those which were supplemented in Lactobacillus during childhood [Khan M et al. Growth-promoting effects of single-dose intragastrically administered probiotics in chickens.
- the inventors have developed a technique for determining and quantifying each of the species / phyla of interest by quantitative PCR using Archae Methanobrevibacter smithii as a control of the quality of prokaryotic DNA extraction from the stool sample.
- Methanobrevibacter smithii a very difficult to extract microbe Archae, should be present in all human stool specimens because of its function in the detoxification of products derived from food processing in the intestine. by methane synthesis, and therefore its identification and quantification should constitute a positive control of the successful realization of the extraction and quantification of the sample.
- the inventors have therefore first developed a technique for extracting DNA from microorganisms from the stool, which enabled them to verify by the constant presence of Archae Methanobrevibacter smithii in human stools the quality of this extraction process. Then, they developed a method for quantifying microorganisms of interest, with a view to developing a routine protocol allowing, from a stool sample in humans or animals, to quantify the presence of microorganisms. of interest, including Methanobrevibacter smithii, Lactobacillus spp., Firmicutes and Bacteroidetes.
- the present invention thus provides a high performance protocol for the extraction of prokaryotic DNAs (Bacteria and Archaea) and for the relative quantification of the intestinal prokaryotic flora that is exhaustive, efficient, reproducible and easy to implement in a routine laboratory so as to facilitate the analysis. a large number of samples for both diagnostic and epidemiological purposes.
- the inventors have developed a technique for extracting DNA by alternating mechanical lysis and enzymatic lysis, which they have controlled by introducing into a salt the control of the known quantities of prokaryotes (Bacteria). , Archaea) whose extraction of DNA is difficult because of the presence of a particularly thick cell wall such as Methanobrevibacter smithii. Indeed, there can be no reliable quantification without optimal DNA extraction.
- the inventors have optimized all the steps of extraction of bacterial lysis until the choice of the extraction method is manual or automatic, taking into account the efficiency and reproducibility of the method. Finally, the inventors compared the extraction technique used with that used by the main authors published on this subject, as described in Example 1.
- the DNA extraction method as well as the real-time PCR quantification tools developed by the inventors, enabled them to confirm that Methanobrevibacter smithii was present in 100% of individuals, at very variable rates, in particular according to their weight status.
- the inventors have deduced the interest of systematically analyzing in salt the presence of Methanobrevibacter smithii, at least as a control of the quality of extraction of the stool DNA in the sample tested.
- Methanosphaera stad manae was present only in the stools of 38% of individuals. It is this additional data which also led the inventors to propose the detection of Methanobrevibacter smithii as a control of a good extraction of DNA.
- the present invention provides a method for the detection and preferably quantification of DNA including, where appropriate, prokaryotic DNA, extracted from a stool sample of an individual, characterized in that one controls the quality of DNA extraction checking whether a specific DNA of Methanobrevibacter smithii, preferably quantified at a range of at least 10 4 M organisms, is detected therein. smithii / ml of said stool sample.
- the number of DNA copies of at least one specific Methanobrevibacter smithii DNA sequence is quantitatively quantitated by quantitative PCR, selected from the following specific sequences:
- the number of copies of said specific Methanobrevibacter smithii specific DNA is quantified by real-time PCR comprising PCR type enzymatic co-amplification of a specific sequence of methanobrevibacter smithii contained in a single molecule. in it it DNA extracted from the sample of salt them, and on the other hand in a sample of the synthetic DNA fragments serving as a calibration standard of quantification of DNA, said specific seq uence of Methanobrevibacter smithii being chosen among the following sequences or their complementary sequences: A sequence of the 16S RNA gene amplifiable by the following primer sequences:
- Antisense primers SEQ. ID. N ° 7: 5'-
- Methanobrevibacter smithii derived from the 16S RNA ribosomal gene and the rpoB gene are quantified.
- the inventors have observed that detection of Metha nobrevibacter smithii in 100% of individuals requires detection of two genes, preferably the amount of M. smithii being quantified at a rate of at least 10 4 M organisms. smithii / ml in said salt sample.
- a quantification of 10 4 organisms / ml of stool sample corresponds to only the detection sensitivity below which no DNA is detected with the SEQ primers. ID. nos. 6 and 7 for the rpoB gene of M. smithii and primers SEQ ID. No. 2 and 3 for the 16S RNA gene of M. smithii.
- real-time PCR amplification and quantification reactions are carried out using specific hydrolysis probes of Methanobrevibacter smithii.
- the Real-Time PCR technique consists of a classical PCR using direct and reverse sequence primers, and includes amplified product detection based on measurement. fluorescence emission proportional to the amount of amplified genes with a so-called "hydrolysis" probe.
- said probe is labeled with a fluorescence emitter or 5 'fluorophore and a 3' fluorescence emission blocking agent.
- This blocking agent absorbs the fluorescence emitted when the fluorophore and the blocking agent are close. When the fluorophore and the blocking agent are separated, the fluorescence emission is no longer absorbed by the blocking agent.
- the Taq polymerase causes a hydrolysis of the probe and thus a release of nucleotides and fluorophore in solution. Fluorescence emission will therefore be proportional to the number of amplifiers.
- the principle of real-time PCR is based on the ability of Taq polymerase during the elongation step to hydrolyze a hybridized probe on the DNA to be copied, this hydrolysis allowing the fluorescence emission, which allows it to quantification.
- two different targets can be quantified by introducing into the reaction mixture two probes and one probe directed against a first target, and two other primers and probes directed against the other target. Both probes are labeled with different fluorophores.
- a large synthetic DNA fragment is used as the calibration standard for quantifying DNA, said large fragment of synthetic DNA comprising said specific sequences of each of said bacteria or species, respectively. prokaryotic whose concentrations are quantified.
- the presence of several molecular targets on the same nucleic fragment makes it possible to quantify different targets in the same sample and to homogenize them in a homogeneous manner, the quantization using the same standard of calibration of several molecular species. These allow comparison of the effectiveness of different PCR reactions between them and from one determination to another over time and avoids biases related to the positive control.
- DNA fragment is meant a DNA or oligonucleotide fragment whose sequences are written hereinafter in the 5 ' ⁇ 3' direction.
- an amplification and quantification reaction is carried out by PCR in real time, using specific hydrolysis probes respectively of each of said specific Methanobrevibacter smithii sequences, namely:
- SEQ. ID NO: 4 5'-CCGTCAGAATCGTTCCAGTCAG -3 '
- SEQ. ID NO: 8 5'-ATTTGGTAAGATTTGTCCGAATG -3 '
- a large synthetic DNA fragment serving as a DNA quantization calibration standard
- said large synthetic DNA fragment grouping specific sequence dies, preferably in the form of a plasmid, with a plurality of samples of large synthetic DNA fragment of different known concentrations.
- sequences of the amplified molecular targets have the following structure, with the ordered primer sequences and the framed probe sequences and in italic iq ue:
- the inventors have therefore developed a quantification tool that targets sequences that make it possible to quantify specifically and faithfully: (I) - Firmicutes (Fi), (2) - Bacteroidetes (Ba), (3) - Lactobacillus (La) in order to know by quantitative real-time PCR the relative composition of the intestinal microbiota of obese subjects, thin subjects and subjects with anorexia nervosa.
- Fi Firmicutes
- Ba Bacteroidetes
- La Lactobacillus
- specific sequence is understood to mean a sequence of the genome of said species Methanobrevibacter smithii, of said Lactobacillus gene or phylum Bacteroidetes or Firmicutes, which is not found in any other genome of microorganisms or living organisms.
- specific consensus sequence is understood to mean a sequence of the genome of said species Methanobrevibacter smithii, of said genus Lactobacillus or, respectively, of said phylum Bacteroidetes or phylum Firmicutes, which is found in all said strains of said species said genus or said phylum and not found in any other microorganism genome or living organism.
- the inventors have searched in silico for the 16S portion of the gene encoding the ribosomal RNAs for the mplification of almost all Bacteroidetes on the one hand and Firmicutes on the other hand eliminating all risks of cross-reactivity between the two groups.
- These different primers and probes systems being chosen in agreement with the experimental contingencies, the cross reactivities were tested in silico then experimentally using the DNAs extracted from stem bacteria.
- the primers and probes systems used to recognize Bacteroidetes do not recognize experimentally any DNA from Firmicutes strains and vice versa.
- the inventors chose the target on the tuf gene and a couple of primers and a probe were chosen according to the technical constraints of the real-time PCR. From the DNA of 20 strains of Lactobacillus not recognized by the pair of ace and the probe corresponding to the molecular target chosen, 20 tuff gene sequences not deposited in Genbanks were amplified and sequenced, consensus sequences Primers and probes were selected and tested on 96 DNA Lactobacillus strains. This system has probes and probes that allows the amplification of the maximum amount of Lactobacillus strains that is preserved.
- Lactobacillus sp selected have a greater sensitivity and recognize a greater number of bacteria than those described in the prior art.
- a quantitative amplification method is carried out by PCR using the following primers:
- SEQ. ID NO: 13 5'-TACATYCCAACHCCAGAACG-3 '
- SEQ. ID NO: 14 5 'AAGCAACAGTACCACGACCA -3'.
- SEQ. ID NO: 17 5'-GTCAGCTCGTGTCGTGA-3 '
- K is G or T and Y is C or T.
- SEQ. ID NO: 15 5'-AAGCCATTCTTRATGCCAGTTGAA -3 ', where R is A or G.
- N designates either I or one of A, T, C or G.
- variable nucleotides As defined above.
- Oligonucleotides of Seq. ID. No. 10, 13, 15, 18 and 19 are therefore implemented, in fact, in the form of equimolar mixtures of oligonucleotides of different sequences, the oligonucleotide dice of different sequences answering each SEQ sequence. ID. No. 10, 13, 15, 18 and 19 to the various possible definitions of sequences Nos. 10, 13, 15, 18 and 19 respectively, namely:
- the step of hybridization of the primers and elongation at 48 ° C. is carried out.
- a large synthetic DNA fragment is used as a quantification standard, which contains the specific sequences of the different molecular targets selected previously.
- the presence of multiple targets on the same fragment makes it possible to quantify different targets in the same sample and co-quantify them homogeneously using the same calibration range to detect different molecular targets in the measurement. where the constraints of the choice of primers and the probe allow it.
- This range of calibration maintained over time is the guarantor of the stability of the quantification system.
- This calibrator is diluted from 10 7 to 1 of each of the molecular targets to 5 .mu.l of DNA sample.
- said concentrations Fi (bacterium of phyla Firmicutes), Ba (bacteria of phyla Bacteroidetes) or La (bacterium Lactobacillus sp) are determined by enzymatic amplification of PCR type in real time and quantification of the DNA of said DNA fragments of seq. specific bacteria of the phylum Firmicutes and Bacteroidetes and the genus Lactobacillus.
- said bacterial concentrations are determined by enzymatic co-amplification of the real-time PCR type of said DNA fragments of specific seq uences of the phylum Firmicutes and Bacteroidetes and of the genus Lactobacil, respectively, contained in a par t in said DNA extracted from the sample, and on the other hand in synthetic DNA samples each comprising said specific sequences of said standard bacteria.
- calibration of quantification of DNA, said DNA fragments of specific sequences respectively of the phylum Firmicutes and Bacteroidetes and of the genus Lactobacillus having a size of 70 to 150 nucleotides, preferably 90 to 120 nucleotides.
- the detection and the quantification of said amplified fragments are carried out using probes labeled with sequences distinct from and amplified by the amplification primers, for each of said DNA fragments of specific sequences respectively of the phylum Firmicutes and Bacteroidetes and of the genus Lactobacillus, the markers of the different probes are markers that are different from each other, in particular the known fluorescent markers of the VIC and FAM type.
- consensus sequences specific for said bacteria are derived from:
- Bacteroidetes the fragment of positions 537 to 721 of the 16S ribosomal RNA gene of Bacteroides fragilis whose accession number in the RDP-II ribosomal library is S000000037.
- Lactobacillus bacteria for the bacteria of the genus Lactobacillus bacteria, a sequence of 90 bases, common to the bacteria Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, Lactobacillus ineri and Lactobacillus acidophilus within the tuf gene coding for the elongation factor at positions 253 to 343 of the GenBank reference gene AY 5621 91.1.
- "Probe” is understood here to mean an oligonucleotide, more preferably 20 to 30 nucleotides, which hybridises specifically with said specific sequence and thus makes it possible to detect and quantify it specifically by measuring the increase in bound fluorescence of the PCR reaction.
- the probe makes it possible to detect the amplified specific DNA and to quantify it by comparing the intensity of the signal with that of the standard of quantification.
- primer is meant herein an oligonucleotide of preferably 15 to 25 nucleotides which specifically hybridizes with one of the two ends of the sequence that the DNA polymerase will amplify in the PCR reaction.
- a method of determining the status of the intestinal flora of an individual advantageously comprises the quantification of the two phyla Bacteroides and Firmicutes, on the one hand, and, on the other hand, bacteria of the genus Lactobacillus and of the species Methanobrevibacter smithii.
- a large synthetic DNA fragment is used as the calibration standard for quantification of DNA, said large synthetic DNA fragment grouping said specific sequences respectively of each of said prokaryotic bacteria.
- the presence of several molecular targets on the same nucleic fragment makes it possible to quantify different targets in the same sample and co-quantify them in a homogeneous manner, the quantification using the same calibration range of several molecular species, allowing the comparison of the effectiveness of different PCR reactions between them and from one determination to another over time and avoids biases related to the positive control.
- a large synthetic DNA fragment is used as a DNA quantification calibration standard, said large synthetic DNA fragment containing such specific sequences as Methanobrevibacter smithii, respectively. , of the genus Lactobacillus, of phylum Bacteroidetes, and preferably of Phylu m Firmicutes, more preferably inserted into a plasmid.
- a restriction enzyme cleavage site is advantageously introduced into at least one of the molecular targets. This site is missing on the natural seq uence. Therefore, by enzymatic analysis and analysis of the amplified fragment on the agarose gel, or by using a real-time PCR probe that specifically recognizes the restriction site, it is possible to detect the possible presence of the contaminating plasmid. .
- a PCR type enzymatic amplification reaction of the DNA of at least one of said specific sequence of at least one of said agents in the DNA extracted from said samples according to the invention is carried out. to test and in the DNA of the standard calibration sample, using at least one set of primers capable of amplifying both at least said authentic specific sequence and said specific sequence modi fi ed ;
- the digested fragment is from the amplification of the molecular target inserted into the control plasmid, it contains the restriction site, and will be smaller in size than the undigested fragment.
- a real-time PCR reaction is carried out with forward and reverse primers of one of the molecular targets, and a specific probe of said exogenous sequence containing the restriction site.
- amplification and q ua ntification reactions are carried out by using sets of primers and hydrolysis probes specific for each of the different bacteria known as the specific sequences of each of the bacteria to be tested. and where appropriate a specific sequence of human DNA in the test sample, such as a specific sequence of albumin hu maine, and said specific sequence comprises a probe sequence flanked by suitable sequences to serve as a primer in a PCR type assay reaction of specific sequences.
- a plurality of PCR enzymatic amplification reactions, simultaneous or not, of each of said specific sequences of said bacteria are used with the same large standard synthetic DNA fragment, using a plurality of simultaneous PCR amplification reactions.
- no more than different sets of primers specific for each of the different said specific sequences of each of said bacteria, the sequences of the different primers not intersecting between the different said bacteria and said primers can be implemented in an enzymatic amplification reaction carried out according to the same protocol and, in particular, at the same hybridization temperature.
- a large first synthetic double-stranded DNA fragment of determined sequence comprising: a sequence in a determined order of a plurality of n second contiguous synthetic small DNA fragments, consisting essentially of dimerization of a plurality of n oligonucleotides by enzymatic amplification using a heat-resistant polymerase enzyme comprising:
- a first PCR nucleic acid amplification reaction step in the presence of a said polymerase enzyme, a series of n oligonucleotides of defined sequences, without exogenous primers, comprising a series of cycles under temperature conditions; allowing the hybridization of said oligonucleotides, followed by an elongation of the complex obtained, intended to put said oligonucleotides in sequence, in sequence, the sequences of said oligonucleotides corresponding successively and alternately to the sense and antisense sequences of the so-called second synthetic fragments , and each said oligonucleotide containing in its 5 'and 3' regions sequences complementary to those of the following and previous oligonucleotides, if any, and
- a second amplification step using primers specific for the 5 'and 3' ends of said direct strand of said large first synthetic fragment to be prepared, making it possible to produce identical copies of said large first fragment.
- This technique is therefore based on the use and control of an artifact of PCR, which consists in the hybridization of the primers with each other (dimerization of the primers). This phenomenon is observed in the case where the PCR conditions, in particular temperature, are poorly adapted, and the primers contain partially complementary sequences.
- the construction technique thus consists, from the target sequences, of choosing the sequences of the oligonucleotides, with alternating oligonucleotides of direct (“sense") or inverse (also called “reverse” or “antisense”) sequences.
- alternating oligonucleotides of direct (“sense") or inverse (also called “reverse” or “antisense") sequences In order to make it possible to put end to end of these oligonucleotides, care is taken to introduce in 3 'of the sequence of an oligonucleotide a nucleotide sequence complementary to the first nucleotides of the following oligonucleotide.
- oligonucleotides will be hybridized by their complementary parts, and thanks to the polymerase activity, for example Taq polymerase, a synthesis of 5 'at 3' is carried out in order to obtain double-stranded fragments.
- the final (assembled) fragment is synthesized by PCR using a pair of forward and reverse primers corresponding to the end sequences of the desired first large double stranded synthetic DNA fragment.
- said large synthetic DNA fragments are advantageously inserted into a plasmid.
- This technique of generic construction of a synthetic nucleotide fragment allows the contiguity of several molecular targets of interest. It is a simple method to implement, fast and reliable, and does not require heavy equipment and expensive.
- the present invention also relates to a diagnostic kit useful for the implementation of a diagnostic method and followed by the report Firmicutes / Bacteroidetes in the stool samples according to the invention, characterized in that it comprises:
- quantification of said specific prokaryotic DNAs of the bacterium is carried out.
- Bacteroidetes and Phylu m Firmicutes respectively, to ensure the diagnosis and / or monitoring of the weight status of an individual.
- the quantification method according to the invention makes it possible to correlate the monitoring of the weight status of a person with monitoring of bacteria BA (Bacteroidetes), FI (Firmicutes), LA (Lactobacillus) and M bacteria. (Methanobrevibacter smithii), in particular as follows:
- a decrease in BA (Bacteroidetes) and an increase in LA (Lactobacillus) represents a risk of weight gain, provided that LA (Lactobacillus) is greater than 10 6 .
- a decrease in Methanobrevibacter smithii may be indicative of a favorable evolution of this pathology, spontaneously or under the effect of therapeutic or non-medicinal therapeutic actions for obese patients in the process of Pharmaceutical treatment, an increase in BA (Bacteroidetes) and a decrease in LA (Lactobacillus) may be indicative of good treatment progress.
- specific supplements or treatments with regard to the bacteria concerned may also contribute to the therapeutics of the subjects concerned by seeking to specifically reduce the level of M. (Methanobrevibacter smithii) in anorexics, and increase the level of BA (Bacteroidetes). and lowering the level of LA (Lactobacillus) in obese patients or people on antibacterial therapy along the way.
- M. Metalobrevibacter smithii
- BA Bacilletes
- the present invention also provides a detection kit comprising reagents for carrying out PCR amplification reaction and at least one set of oligonucleotide primers and preferably in addition at least one probe oligonucleotide.
- a detection kit comprising reagents for carrying out PCR amplification reaction and at least one set of oligonucleotide primers and preferably in addition at least one probe oligonucleotide.
- said kit comprises at least one of two pairs of oligonucleotide primers and, preferably at least one of the oligonucleotide hydrolysis probes, capable of amplifying at least one of said dithes. sequences of Methanobrevibacter smithii derived from the gene
- the pair of oligonucleotides has sequences of SEQ sequences. ID # 2 and SEQ. ID No. 3 and, preferably, the oligonucleotide hydrolysis probe of sequence SEQ. ID No 4, and
- kits according to the invention comprises the pairs of oligonucleotide primers with preferably the oligonucleotides probes of hydrolysis, su ivants:
- the pair of oligonucleotides has sequences of SEQ sequences. ID No. 9 and SEQ. No. 10, preferably with a hydrolysis probe oligonucleotide of sequence SEQ. ID No. 11 for the amplification of a specific consensus sequence of the phylu m Bacteroidetes and,
- kits further includes:
- a kit comprises extraction reagents comprising at least one abrasive powdery product, preferably glass powder and a chemical and / or enzymatic lysis reagent.
- the present invention also provides a method characterized in that to perform the extraction of the prokaryotic DNA from said stool sample, the steps are performed in which:
- a homogeneous suspension of said stool sample is made in a buffer solution, preferably at a dilution of 50 to 150 g / l, and
- a mechanical lysis is carried out by mixing and stirring the slurry of step 1 with an abrasive spraying product, preferably preferably acid-washed glass powder, and preferably a heating step is carried out at 100 ° C. for 10 minutes, and
- step 3 chemical and / or enzymatic lysis is performed by mixing and agitate the suspension obtained in step 2) with chemical or enzymatic lysis buffer and then
- step 2) repeating step 2) with the mixture obtained in step 3), and
- step 3) is preferably repeated with the mixture obtained in step 4).
- the DNA is finally separated either by known methods such as by attachment to a column containing the silicate or washing to remove said DNA from the column or by washing and centrifugation. to recover said DNA fragments.
- the realization of the first mechanical lysis step makes it possible to optimize and promote the work of chemical or enzymatic lysing agents in step 2) by promoting their penetration into the proca ryote microorganisms allowing partial degradation.
- the thick walls of the thick-walled prokaryotic micro-organisms, and the complete degradation of the adjacent layers is obtained only by at least a second mechanical lysis after said first chemical or enzymatic lysis.
- step 2) heating at 100 ° C. makes it possible to finalize the degradation of the components of the walls and membranes of the microorganisms.
- FIG. 1 shows on the ordinate the percentage of sequences
- 16S ribosomal RNA and abscissa the results for phylu m Firmicutes (FI) bacteria, then bacteria phyl um Bacteroidetes (BA).
- the sensitivity rates are represented by the first column in fine lines, namely 88.94% for bacteria of the phylum Firmicutes and 89.89% for the bacteria of the phylum Bacteroidetes.
- the specificity rates are represented by the second column in g ras, namely 0.83% for the bacteria of phylum Firmicutes and 0.01% for the bacteria of the phylum Bacteroidetes.
- FIG. 2 represents the quantification of the bacteria of the phylum Bacteroidetes in salt collected from three groups of obese (O), control (L) and anorexic (A) individuals.
- the cross indicates the average; the horizontal line the median and the cu be represents the distribution of the values in the standard deviation.
- FIG. 3 represents the distribution of q uantities greater than 10 6 of the number of bacteria of the genus Lactobacillus detected and quantified by the method according to the invention in the stools of anorexic (A), obese (O) and control ( L).
- the numbers on the y-axis represent the number of individuals.
- II represents the Lactobacillus copy number greater than or equal to 10 6
- II represents the Lactobacillus copy number of less than 10 6 .
- Fig ure 4 represents the quantification of Archea Methanobrevibacter smithii according to the method of the invention in the stools of anorexiq ues (A), obese (O) and controls (L).
- Fig. 4 shows the standard deviation (height of the vertical line) and the average (cube area), the numbers in the ordinate being the average number of copies of Methanobrevibacter smithii.
- EXAMPLE No. 1 DNA extraction protocol according to the invention and comparison with the reference protocol published in the literature, for the detection of Archae Methanobrevibacter smithii in salt.
- Archae Methanobrevibacter smithii is a methanogen, that is to say an arche able to transform the products of digestion methane CH4 foods.
- Methanosphaera stadtmanae is also a methagenogenic Archae detected in salt. The inventors have argued that, given the importance of the biochemical methanogenesis reaction for intestinal physiology, Archae Methanobrevibacter smithii and not Methanosphaera stadtmanae was present and detectable in stool in all individuals.
- Methanobrevibacter smithii was effectively detected in almost all individuals by combining detection of the 16S RNA ribosomal 1 gene with that of the rpoB gene whereas Methanosphaera stadtmanae was detected only in 40% of individuals using the same system.
- the inventors have imagined having to lyse the wall of Archae in order to release the Archaic DNA in order to make it accessible to molecular detection based on the PCR technique. For this purpose, the inventors have put in place by successive trial and error the following protocol. Initially, the inventors mechanically lysed the samples by agitation in the presence of glass powder, followed by chemical lysis, and then extracted the DNA.
- a protocol that is the subject of the invention is therefore the following:
- 2- 250 .mu.l of suspension are introduced into a tube of 1.5 ml containing 0.3 g (equivalent to a volume of about 10 .mu.l, ie less than 4% of the final volume) of glass powder of which the particles measure less than 106 ⁇ m and are washed with acid to remove the gaseous, nucleic acids and DNAses and RNAses which may be derived from the processes for the manufacture of glass beads (reference G4649, Sigma, Saint Quentin Fallavier, France) and acted in the BIO Fast Prep 101 (Qbiogene, Strasbou rg, France) at maximum speed (6.5) for 90 seconds;
- the preparation is then heated at 100 ° C for 10 minutes and then brought back to the temperature a mbiante. Then, a standard NucleoSpin® Tissue Kit Kit protocol (Macherey Nagel, Hoerdt, France) and used as follows. 180 ⁇ l of lysis buffer T1 and 25 ⁇ l of proteinase K at 20 mg / ml are added. The mixture is incubated at 56 ° C overnight;
- a known DNA lysis buffer is chemically lyzed, that is to say 200 ⁇ l of buffer B3 (mixture in equal proportions of the buffers B1 [Guanid ine hydrochloride] and buffer B2 of the composition available from the supplier) are added and the mixture is incubated for 10 minutes at 70 ° C., 200 ⁇ l of ethanol are added, melted, applied on a silica column, centrifuged for 1 min at maximum speed, washed with 500 ⁇ l of buffer BW, centrifuged for 1 minute at a speed. maximum, washed with 600 ⁇ l of melon B5 centrifuged 1 minute at maximum speed;
- the inventors compared the protocol that is the subject of the invention with the reference protocol for the extraction of prokaryotic DNA from the stools.
- the inventors collected 50 stool samples collected from 50 individuals who submitted stool for exploration of diarrhea.
- These 50 salt samples benefited from a parallel extraction of DNA by the protocol object of the invention and by using the QIAAM extraction kit P Stool DNA mini kit (Qiagen, Courta beef, France) described in the literature [Eckburg PB. et al . Diversity of the intestinal microbial hu man flora. Science 2005 Vol ume 308 page 1635-1638].
- the inventors compared the number of copies of the 16S RNA gene and the rpoB gene per ml of salt using both extraction protocols.
- the numerical data were analyzed using Kruskal-Wall's non-para metric test in the EPIIN FO version 3.4. 1 (center on ten controlling and prevention, Atlanta, GA). P values were used to show significant differences between the two methods and were calculated using the non-parametric Kruskal-wallis method for 2 groups. A value of P less than 0.05 was considered significant.
- the 16S ribosomal RNA gene was detected according to the same method of detection and quantification with the primers SEQ. ID. No. 6 and 7 and the probe SEQ. ID. No. 8 described in Example 2, in 50/50 (100%) of individuals and the rpoB gene was detected in 49/50 (99%) of individuals with SEQ primers. ID. No. 2 and 3 and the probe SEQ. ID. # 4.
- the extraction protocol according to the invention has made it possible to detect between 2 and 3 logarithms of DNA copy plots that the Qiagen commercialized protocol with a P value. less than or equal to 0.00001 for the two genes analyzed.
- the negative controls remained strictly negative.
- the inventors For the quantification of M. smithii, the inventors used the SEQ leader sequences. ID. No. 2 and 3 and SEQ probe sequence. ID. No. 4 for the amplification of the 16S gene of ribosomal I 1 RNA of M. smithii, and the SEQ leader sequences. ID. No. 6 and 7 and SEQ probe sequence. ID. No. 8 for the amplification of M. smithii's rpoB. The inventors have established the following PCR quantitation calibration curves for the detection of M. smithii by amplification of the 16S rRNA gene and the rpoB gene, from known amounts of M. smithii obtained by culture of M. smithii on agar:
- the ribosomal database project (RDP-II): introducing myRDP space. Nucleic Acids Res 2007, 35: D169-D172] of 16S ribosomal RNA in the ribosomal RDP-II database.
- the phylum Bacteroidetes is represented by more than 34,000 sequences (only phyl um Protebacteria is more important than that of Bacteroidetes). Identifying a system of 3 fragments of sequences (primers and probe) targeting almost all species of phyla Firmicutes and Bacteroidetes respectively was a real challenge, especially since the system had to be specific; that is to say that a minimum of species outside the phylum considered should be detectable.
- the difficulty was that the small variability of the 16S ribosomal RNA gene nucleotide sequence (many conserved regions) was therefore identified by PCR primers and a probe.
- each of the sequences is very sensitive to the clade Firmicutes but not necessarily very specific.
- the meeting of the 3 sequences primers and probes SEQ. ID. No. 17, No. 18 and No. 19 provides high specificity and sensitivity for use in quantitative real-time PCR.
- the three primer sequences and SEQ probes. ID. No. 9, No. 10 and No. 11 for Bacteroidetes bacteria also confer high specificity and sensitivity for use in real-time quantitative PCR.
- the real-time PCR program included for the detection of Bacteroidetes: 95 ° C 15 min, then 45 cycles of 95 ° C 30s, 48 ° C 45s, 72 ° C 1 min), and, for the detection of Firmicutes and Lactobacillus and Methanobrevibacter smithii: 95 ° C 15min, then 45 cycles of 95 ° C 30s, 60 ° C 1min.
- the concentrations of the quantities of probes and primers have been adapted for each of the systems in order to maintain their effectiveness,
- the results indicate that the Bacteroidetes detection system has a sensitivity of 89.89%, and detects 30,237 of the 33,639 16S ribosomal RNA sequences of phylu m Bacteroidetes bacteria in the RDP-II database (Fig.
- the detection system of the Firmicutes has a sensitivity of 88.94% since it detects 83,576 of the 93,969 sequences of the 16S RNA ribosomal RNA gene from the phylogenetic Firmicutes of the RDP-II database.
- the previously described methods provide a high degree of specificity (99%) and sensitivity ( ⁇ 50 copies) for use in real-time PCR.
- Table 1 The results of Table 1 are expressed in terms of CT, ie the number of amplification cycles necessary for the amplification to start, this CT value in relation to the concentration of the nucleic product to be used, namely that more CT is low, the concentration is high.
- a calibration plasmid comprising a large hybrid synthetic DNA fragment containing the SEQ sequences was constructed. No. 1, No. 2, No. 12, No. 16 and No. 20, in the form of a double-stranded DNA fragment constructed by amplification reaction, as described in FR-2,882,063. .
- a calibration plasmid range of 7 was created , one copy per well, for each amplification reaction the ten points of the plasmid range were tested.
- the measurement of the F / B ratio is currently performed only by metagenomic, DNA chip and sequencing methods of large libraries of 16S ribosomal RNA gene clones, which are in the field of research but are not applicable. in diagnostic routine.
- the inventors have therefore thought to use the invention to reliably determine the ratio F / B by a real-time PCR technique, applicable routinely.
- Methanobrevibacter smithii allowed the inventors to observe that the number of Methanobrevibacter smithii (Figure 4) is higher in obese than in controls with an obese / control ratio of 1.72, but this is not statistically significant.
- the rate of Methanobrevibacter smithii is therefore interesting for a person suspected of anorexia.
- the obese have a digestive flora poor in Bacteroidetes and rich in Lactobacillus.
- the flora of anorexic is similar to controls for Firmicutes, Bacteroidetes and Lactobacillus but contains more Methanobrevibacter smithii.
- the method for detecting and quantifying bacterial components of the microbiota used and proposed by the inventors enables the specific, reliable, rapid and repetitive detection and quantification of these four groups of microorganisms in a large number of stool samples. This method can be very easily used in routine practice in laboratories.
- Table 1 List of microbial DNAs used to determine the sensitivity / specificity of the PCRs in real time for the detection of microorganisms belonging to the groups Firmicutes and Bacteroidetes, in the stool.
- A DNA extracted from the 108 strains tested by real-time PCR by the Bacteroidetes system.
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FR0953207A FR2945545B1 (fr) | 2009-05-14 | 2009-05-14 | Methode de detection d'adn procaryote extrait d'un echantillon de selles |
PCT/FR2010/050824 WO2010130914A1 (fr) | 2009-05-14 | 2010-04-30 | Methode de detection d'adn procaryote extrait d'un echantillon de selles |
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