CN101509034A - Human body intestinal canal flora detection parting and quantitative reagent kit - Google Patents
Human body intestinal canal flora detection parting and quantitative reagent kit Download PDFInfo
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Abstract
The invention relates to a method for detecting, genotyping and quantifying human intestinal flora, consisting of universal primer PCR amplification and specific probe LDR. In the PCR amplification, the 16sr DNA fragment of selected bacteria genus is amplified by the designed universal primer synchronously; the specific LDR probe of each bacteria genus is utilized to achieve the goal of genotyping through ligation reaction; and LDR signal is detected by ABI Prism 377 type sequencer, and the detected fluorescence value is taken as the quantifying basis. Aiming at the universal primer of intestinal microflora, the detection method of the invention effectively amplifies all target intestinal microflora 16sr DNA sequence, thereby ensuring that each target sequence and the template of low copy therein can be detected, further ensuring the specificity of the specific probe of each bacteria genus, completely identifying each target sequence and realizing multiplex genotyping quantification.
Description
Technical field
The invention belongs to the microbial nucleic acids diagnostic field, particularly relate to a kind of human body intestinal canal flora detection parting and quantification kit.
Background technology
Bacterium in the enteron aisle plays an important role to the growing of human body, immunologic function, nutrition, drug metabolism, pathogenesis and health etc.The separation of bacterium in the enteron aisle, evaluation and quantitative for the function of studying flora in the enteron aisle and extremely important with the mutual relationship of body.Some classifying methods have been set up at present both at home and abroad at entero-bacte rRNA gene, but these method ubiquities unstable result, operation easier is big, flux is little, can't quantitatively wait weakness, have hindered research institution and clinical unit that intestinal microflora is carried out effective scientific research and detection.
Existing detection of intestinal bacteria method mainly is divided into two kinds, and the one, traditional microbial culture adds enumeration, and the one, the molecular biology quantitative analysis method.
Microbial culture adds a kind of traditional method of counting that enumeration is the entero-bacte qualitative, quantitative, mainly is to realize by special culture medium inoculation culture, bacterial strain counting according to the bacterium phenotypic characteristic.Yet mostly under the situation, often mixed various bacteria in the testing sample, must utilize the selectivity differential medium to carry out appraisal counting this moment, the oxygen tolerance that various bacteriums are different, nutritional requirement, the form of antibiotics sensitivity and bacterium colony and color characteristic have constituted the basis of differentiating. and the microbiotic that adds in the substratum also has in various degree restraining effect to bacterium itself simultaneously, and the specificity substratum is just special relatively, though up to the present there is not a kind of complete ideal selective medium yet. these reasons make traditional bacterial cultivation can count viable bacteria, but it is time-consuming, effort, the cultivation difficulty is big, it counts also out of true. and the discriminating of traditional bacterial cultivation is according to being based on bacterial morphological, the physiology and biochemistry feature, and these features only are the specific manifestations of bacterium under the specific culture condition
Opposite molecular biology quantitative analysis method is that the nucleotide sequence with bacterial genomes serves as to differentiate the basis, be not subject to the interference of external environment factor, do not rely on the substratum performance, not limited by incubation time, make quantitative analysis results more stable, more responsive, more save time, simultaneously repeatability is better. bacterial cell internal ribosome quantity reaches 10
4～10
5The branch that ribosome-RNA(rRNA) (rRNA) has special district and conserved regions again, therefore the finger printing of 5S rRNA, 16S rRNA and 23S rRNA and sequence are often used as the method for the comparative microbiology classification. and wherein the sequence of 16S rRNA reaches 1500～12000 Nucleotide, its sequence length is moderate, have the branch of conserved regions and variable region concurrently, so its sequence data is usually used in designing gene probe and PCR (polymerase chain reaction,PCR) amplimer.Because probe and primer have high susceptibility and specificity, make it carry out appraisal counting to bacterium on each levels such as genus, kind, subspecies even strain.The Protocols in Molecular Biology that is used for the entero-bacte quantitative analysis at present mainly contain dot blot (dot blot hybridization), fluorescence in situ hybridization (fluorescence in situ hybridization, FISH), fluorescence in situ hybridization is in conjunction with flow cytometry (FISH combined with flow cytometry) and polymerase chain reaction (PCR) technology.
Summary of the invention
Technical problem to be solved by this invention provides a kind of human body intestinal canal flora detection parting and quantification kit, detection method of the present invention have typing, quantitatively fast, the high and low cost of flux of speed a bit.
A kind of human body intestinal canal flora detection parting of the present invention and quantification kit, it is at the 16S rDNA of entero-bacte, use universal primer PCR and specific probe, bifidus bacillus, lactobacterium casei kind, Lactobacterium acidophilum, faecalis, enterobacteria and Eubacterium and content thereof in the human body fecal sample.
The 16S rDNA universal primer PCR sequence of described bifidus bacillus, lactobacterium casei kind, Lactobacterium acidophilum, faecalis, enterobacteria and 6 kinds of human intestine bacterium of Eubacterium is:
The upstream: 5 '-CAGGATTAGATACCCTGGTAGT-3 '
The downstream: 5 '-TTGCGCTCGTTGCGGGACTT-3.′
The specific probe sequence of described enterococcal 16S rDNA is: the LDR probe:
F：TTTTTTTTTTTTTTTTTTTTTTTGACCACTCTAGAGATAG
R：P-AGCTTCCCCTTCGGGGGCAATTTTTTTTTTTTTTTTTTTT-FAM
The LCR downstream probe:
F：TTTTTTTTTTTTGCCCCCGAAGGGGAAGCT
R：CTATCTCTAGAGTGGTCAAATTTTTTTTTT
Enterococcal product length 80bp.
The specific probe sequence of the 16S rDNA of described enterobacteria is:
The LDR probe:
F：TTTTTTTTTTTTTTTTTTTTTTTTTGGAGGTTGTGCCCTTGAG
R：P-GCGTGGCTTCCGGAGCTAACTTTTTTTTTTTTTTTTTTTTTT-FAM
The LCR downstream probe:
F：CTCAAGGGCACAACCTCCAATTTTTTTTTT
R：TTTTTTTTTTGTTAGCTCCGGAAGCCACGC
The product length 85bp of enterobacteria.
The specific probe sequence of the 16S rDNA of described Eubacterium is:
The LDR probe:
F：TTTTTTTTTTTTTTTTTTTTTTTTTTTGACATATGGGTGAAGCGG
R：P-GGGAGACCCCGTGGCCGAGATTTTTTTTTTTTTTTTTTTTTTTTT-FAM
The LCR downstream probe:
F：CCGCTTCACCCATATGTCAATTTTTTTTTT
R：TTTTTTTTTTTCTCGGCCACGGGGTCTCCC
The product length 90bp of Eubacterium.
The specific probe sequence of the 16S rDNA of described bifid bar is:
The LDR probe:
F：TTTTTTTTTTTTTTTTTTTTTTTTTTTTGGATGTGGGGCCCGTTCCA
R：P-CGGGTTCCGTGTCGGAGCTATTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM
The LCR downstream probe:
F：TTTTTTTTTTTAGCTCCGACACGGAACCCG
R：TGGAACGGGCCCCACATCCATTTTTTTTTT
The product length 95bp of bifidus bacillus.
The specific probe sequence of the 16S rDNA of described lactobacterium casei is:
The LDR probe:
F：TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCAGGTCTTGACATCTTTTGA
R：P-TCACCTGAGAGATCAGGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM
The LCR downstream probe:
F：TTTTTTTTTTAAACCTGATCTCTCAGGTGA
R：TCAAAAGATGTCAAGACCTGTTTTTTTTTT
The product length 100bp of lactobacterium casei.
The specific probe sequence of the 16S rDNA of described Lactobacterium acidophilum is:
The LDR probe:
F：TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGTCTTGACATCTAGTGCAA
R：P-TCCGTAGAGATACGGAGTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM
The LCR downstream probe:
F：TTTTTTTTTTGAACTCCGTATCTCTACGGA
R：TTGCACTAGATGTCAAGACCTTTTTTTTTT
The product length 105bp of Lactobacterium acidophilum.
The probe hybrid plan of described bifidus bacillus, lactobacterium casei kind, Lactobacterium acidophilum, faecalis, enterobacteria and 6 kinds of human intestine bacterium of Eubacterium is:
The LCR probe pM100:100:100 of the LDR probe pM100:100:100 faecalis of enterobacteria, Eubacterium, bifidus bacillus, Lactobacterium acidophilum, lactobacterium casei.
A kind of human body intestinal canal flora detection parting of the present invention and quantification kit, it detects step and comprises: the extracting of cultivation of standard bacterium bifidus bacillus, lactobacterium casei, Lactobacterium acidophilum and standard bacterium DNA, the extracting of faecalis, enterobacteria, Eubacterium pre-treatment and DNA in the sample; Sequence fragment with the 16S rDNA of specific PCR primer amplification bifidus bacillus, lactobacterium casei, Lactobacterium acidophilum, faecalis, enterobacteria, Eubacterium; 16S rDNA fragment is inserted the T carrier, change in the competent cell that DH5 α intestinal bacteria make, choose to contain by blue hickie screening and insert segmental thalline, confirm that by order-checking inserting fragment is the special 16s rDNA of each Pseudomonas fragment again; Single bacterium colony is used for a large amount of cultivations, and extracting plasmid DNA more promptly obtains the standard substance DNA of each Pseudomonas.At last by the uv-spectrophotometric instrument measure take out standard substance DNA concentration, redilution to 10
9Copies/uL; 6 kinds of Pseudomonas standard substance that make are mixed according to the scheme of uniform distribution form design, see Table 1.
Table 1 standard substance template hybrid plan
| The factorial experiments number of times | Enterobacteria | Eubacterium | Bifidus bacillus | Faecalis | Lactobacterium casei | Lactobacterium acidophilum |
| Probe | LDR | LDR | LDR | LCR | LCR | LCR |
| 1 | 1 | 0.5 | 0.0625 | 0.01 | 0.005 | 0.000625 |
| 2 | 0.5 | 0.0625 | 0.125 | 0.005 | 0.000625 | 0.00125 |
| 3 | 0.25 | 0.25 | 0.25 | 0.0025 | 0.0025 | 0.0025 |
| 4 | 0.125 | 1 | 0.5 | 0.00125 | 0.01 | 0.005 |
| 5 | 0.0625 | 0.125 | 1 | 0.000625 | 0.00125 | 0.1 |
Annotate: 1 is the concentration unit (unit) of a demarcation, 10
5Copies utilizes 16S rDNA universal primer to increase respectively according to 5 template concentrations combinations shown in the table 1.With the PCR product as template, utilize the isoconcentration LDR probe 100pmol/L of enterobacteria, Eubacterium, bifidus bacillus, and the isoconcentration LCR probe 100pmol/L of faecalis, Lactobacterium acidophilum, lactobacterium casei, obtain fluorescent signal by sequenator at last at the somatotype detection that LDR and LCR are carried out in these 5 template concentrations combinations simultaneously, with the template concentrations ratio is X-coordinate, signal is done linear regression analysis than for ordinate zou, obtains typical curve.Extracting DNA after the sample pre-treatment as the amplification template of 16s rDNA, utilizes 16s rDNA universal primer to carry out pcr amplification then.PCR product after the amplification continues to carry out LDR and LCR reaction simultaneously as template, and used probe is the isoconcentration LCR probe 100pmol/L of the isoconcentration LDR probe 100pmol/L of enterobacteria, Eubacterium, bifidus bacillus and faecalis, Lactobacterium acidophilum, lactobacterium casei.Obtain fluorescent signal by sequenator at last, the typical curve that contrasts each Pseudomonas is converted into original template content, finishes the detection by quantitative and the somatotype of sample.
Detection method of the present invention is by a pair of universal primer of design, two similar templates are increased simultaneously, these two templates have proximate sequence, length is identical or close, so no matter influence of what and the known variable factor that maybe can not expect of cycle number, they are all with the amplification of identical speed, the reaction accurate in scale of amplified production the starting point concentration ratio of two templates.Sample to be checked is used the ligase enzyme detection reaction again after universal primer PCR is amplified (ligase detection reaction LDR) comes the bacterial classification in the sample is carried out somatotype and quantitative.Two DNA chains of dna ligase catalysis generation condensation reaction generates phosphodiester bond.Because will be under nucleotide fragment and the complete paired situation of template strand, ligase enzyme could connect breach, and particularly hold upstream pulsating 3 ', the mispairing of a base of 3 ' end, also will cause the failure of ligation, so ligation is used to the research of single base mutation.In LDR, as long as choosing and designing of upstream and downstream probe is appropriate, just can guarantee to connect the specificity of product, so the application of LDR in somatotype also is advantageous, and many probe is carried out ligation simultaneously, interference each other also is very little, so just can distinguish a plurality of sequences in a sample.
The present invention is when pattern detection, because the difference of the order of magnitude is arranged on each Pseudomonas amount in the sample, earlier with the LDR probe to increase through universal primer the PCR product carry out signal detection, as if there being some Pseudomonas LDR probe in detecting not go out signal, then using the LCR probe instead this sample PCR product heavily examined.The area detection signal of LDR has only in 2 orders of magnitude.The LCR probe can detect the template than the low 1000 times of concentration of template that LDR examines.The dynamicrange that detects expands in 10000 times.Beneficial effect
(1) detection method of the present invention effectively amplifies all target entero-bacte 16S rDNA sequences at the universal primer of entero-bacte, has guaranteed that each target sequence and the wherein low template that copies can be detected;
(2) present method makes the specific probe of each Pseudomonas further guarantee specificity, and each target sequence is distinguished fully, realizes that multiple somatotype is quantitative.
Description of drawings
Fig. 1 is the synoptic diagram of 16S rDNA sequence universal primer PCR in conjunction with the pattern detection of the quantitative human enteric bacteria test kit of LDR somatotype, wherein (A) is the sample DNA template that extracting goes out in people's the ight soil, (B) be universal primer at entero-bacte 16S rDNA sequences Design, (C) in the PCR reaction that utilizes 16S rDNA sequence universal primer and sample DNA template to carry out, (D) be special LDR probe at entero-bacte 16S rDNA sequence, (E) be special LCR probe at entero-bacte 16S rDNA sequence, what carry out (F) is the multi-LDR that is undertaken by the specific probe hybrid plan, LCR reaction, (G) fluorescent quantitation phenotypic analysis figure for obtaining on the ABI Prism 377 type sequenators.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Embodiment 1
Sample pre-treatment and DNA extracting
Testing sequence:
(1) pre-treatment
Gather normal adults fresh excreta sample, the about 4g of every this weight in wet base of increment is collected in the aseptic plastic bag, and 4 ℃ of refrigerators are preserved, and the time is no more than 12h.Every part of stool sample add aseptic PBS damping fluid that 6ml is housed (0.05mol/L, pH7.4) in the test tube fully vibration shake up the centrifugal 5min of 500rpm behind 5-10min, collect supernatant liquor.This step repeats 3 times.Supernatant liquor 5 then, the centrifugal 10min of 000rpm, and collecting precipitation places the Eppendorf pipe.Throw out suspends in 1mL distilled water (ddH2O), mixed back 14, the centrifugal 5min collecting precipitation of 000rpm.Precipitation is dissolved in 200 μ L dehydrated alcohols (20 ℃ of precoolings), and behind the vibration mixing 14, the centrifugal 2min of 000rpm abandons supernatant, and this step repeats 3 times.
(2) DNA extracting
At the TE (lysozyme concentration 20mg/mL) that in pretreated sample, adds 200 μ L lysozymes, with moving liquid point mixing.Hatch the digestion damping fluid 400 μ L that 40min adds to be provided in the DNA extraction agent box under the room temperature, mixing; Add 3 μ L Proteinase Ks again, 55 ℃ of insulation 2-3h behind the mixing.Add 260 μ L dehydrated alcohols, with moving the liquid point sample all is transferred in the DNA extracting post behind the mixing.8, the centrifugal 1min of 000rpm room temperature.Take off DNA extracting post, discard the waste liquid in the collection tube.Post is put back in the collection tube, added 500 μ L washing lotions, 10,000rpm, the centrifugal 30s of room temperature.This step repeats twice.Take off DNA extracting post, discard the whole waste liquids in the collection tube.Post is put back in the collection tube, 10,000rpm, the centrifugal 1min of room temperature is to remove residual washing lotion.Post is put into new Eppendorf pipe, and central authorities add 50 μ l elution buffers at post, room temperature or 37 ℃ of placement 2min.10,000rpm, the centrifugal 1min of room temperature.The liquid of collecting in the centrifuge tube is bacterial genomes DNA.
Embodiment 2
16s rDNA universal primer, LDR and LCR probe design
Testing sequence:
In Genebank, search each Pseudomonas 16S rDNA sequence information, all find more than 50 and compare together then, find two ends section conservative, that the centre comprises the region of variability territory to be used for design.Be the consistence of assurance pcr amplification efficient and the specificity of probe, filter out the regional and middle region of variability that contains two ends sequence in full accord all has distinguished sequence to each Pseudomonas section again.At two ends conserved regions design universal primer, guaranteed that the primer binding sequence of primer and each Pseudomonas matches fully, compare out the special sequences Design of each Pseudomonas at the middle part region of variability and go out specific probe.For the unanimity that further guarantees amplification efficiency with effectively, confirmed a section design primer, probe about 320bp at last, stride two region of variability, 3 conserved regions.Other is all guarded except the designing probe zone, and whole GC content is close, meets the requirement of cPCR sequence similarity.
Embodiment 3
The universal primer amplification of standard substance DNA and sample DNA
Testing sequence:
(1) standard DNA is identical with the used amplification system of pattern detection.
(2) universal primer: the upstream, 5 '-CAGGATTAGATACCCTGGTAGT-3 '
The downstream, 5 '-TTGCGCTCGTTGCGGGACTT-3 '
(3) amplification system (10 μ L):
Upstream primer 0.5 μ mol/L
Downstream primer 0.5 μ mol/L
10×Buffer 1×
dNTP 0.2mmol/L
Mg
2+ 2.0mmol/L
Taq polymerase 1U
Add water to 10 μ L
(4) amplification program:
95℃15min
94℃30s→55℃30s→72℃60s(40cycles)
72℃7min
Embodiment 4
The ligase enzyme detection reaction (LDR) of standard substance DNA, sample DNA and link polymerase chain reaction (LCR) testing sequence:
(1) universal primer is increased gained PCR product is used for LDR and LCR signal detection
(2) the shared reaction system of LDR and LCR
System (10 μ L):
Mixed probe 0.1 μ mol/L
Buffer 1×
Template 1 μ L
Taq 0.1μL(4unit)
H
2O is supplemented to 10 μ L (3) LDR response procedures:
95℃ 2min
94℃ 30s→60℃2min(25cycles)
(4) in pattern detection, gained PCR product after the universal primer amplification detects by following probe hybrid plan.LDR probe (pM) 100:100:100 of enterobacteria, Eubacterium, bifidus bacillus
LCR probe (pM) 100:100:100 of faecalis, Lactobacterium acidophilum, lactobacterium casei.
Claims (10)
1. human body intestinal canal flora detection parting and quantification kit, it is characterized in that: at the 16S rDNA of entero-bacte, use universal primer PCR and specific probe, bifidus bacillus, lactobacterium casei kind, Lactobacterium acidophilum, faecalis, enterobacteria and Eubacterium and content thereof in the human body fecal sample.
2. a kind of human body intestinal canal flora detection parting according to claim 1 and quantification kit is characterized in that: the 16SrDNA universal primer PCR sequence of described bifidus bacillus, lactobacterium casei kind, Lactobacterium acidophilum, faecalis, enterobacteria and 6 kinds of human intestine bacterium of Eubacterium is:
The upstream: 5 '-CAGGATTAGATACCCTGGTAGT-3 '
The downstream: 5 '-TTGCGCTCGTTGCGGGACTT-3.′
3. a kind of human body intestinal canal flora detection parting according to claim 1 and quantification kit is characterized in that: the specific probe sequence of described enterococcal 16S rDNA is:
The LDR probe:
F：TTTTTTTTTTTTTTTTTTTTTTTGACCACTCTAGAGATAG
R：P-AGCTTCCCCTTCGGGGGCAATTTTTTTTTTTTTTTTTTTT-FAM
The LCR downstream probe:
F：TTTTTTTTTTTTGCCCCCGAAGGGGAAGCT
R：CTATCTCTAGAGTGGTCAAATTTTTTTTTT
Enterococcal product length 80bp.
4. a kind of human body intestinal canal flora detection parting according to claim 1 and quantification kit is characterized in that: the specific probe sequence of the 16S rDNA of described enterobacteria is:
The LDR probe:
F：TTTTTTTTTTTTTTTTTTTTTTTTTGGAGGTTGTGCCCTTGAG
R：P-GCGTGGCTTCCGGAGCTAACTTTTTTTTTTTTTTTTTTTTTT-FAM
The LCR downstream probe:
F：CTCAAGGGCACAACCTCCAATTTTTTTTTT
R：TTTTTTTTTTGTTAGCTCCGGAAGCCACGC
The product length 85bp of enterobacteria.
5. a kind of human body intestinal canal flora detection parting according to claim 1 and quantification kit is characterized in that: the specific probe sequence of the 16S rDNA of described Eubacterium is:
The LDR probe:
F：TTTTTTTTTTTTTTTTTTTTTTTTTTTGACATATGGGTGAAGCGG
R：P-GGGAGACCCCGTGGCCGAGATTTTTTTTTTTTTTTTTTTTTTTTT-FAM
The LCR downstream probe:
F：CCGCTTCACCCATATGTCAATTTTTTTTTT
R：TTTTTTTTTTTCTCGGCCACGGGGTCTCCC
The product length 90bp of Eubacterium.
6. a kind of human body intestinal canal flora detection parting according to claim 1 and quantification kit is characterized in that: the specific probe sequence of the 16S rDNA of described bifid bar is:
The LDR probe:
F：TTTTTTTTTTTTTTTTTTTTTTTTTTTTGGATGTGGGGCCCGTTCCA
R：P-CGGGTTCCGTGTCGGAGCTATTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM
The LCR downstream probe:
F：TTTTTTTTTTTAGCTCCGACACGGAACCCG
R：TGGAACGGGCCCCACATCCATTTTTTTTTT
The product length 95bp of bifidus bacillus.
7. a kind of human body intestinal canal flora detection parting according to claim 1 and quantification kit is characterized in that: the specific probe sequence of the 16S rDNA of described lactobacterium casei is:
The LDR probe:
F：TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCAGGTCTTGACATCTTTTGA
R：P-TCACCTGAGAGATCAGGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM
The LCR downstream probe:
F：TTTTTTTTTTAAACCTGATCTCTCAGGTGA
R：TCAAAAGATGTCAAGACCTGTTTTTTTTTT
The product length 100bp of lactobacterium casei.
8. a kind of human body intestinal canal flora detection parting according to claim 1 and quantification kit is characterized in that: the specific probe sequence of the 16S rDNA of described Lactobacterium acidophilum is:
The LDR probe:
F：TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGTCTTGACATCTAGTGCAA
R：P-TCCGTAGAGATACGGAGTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-FAM
The LCR downstream probe:
F：TTTTTTTTTTGAACTCCGTATCTCTACGGA
R：TTGCACTAGATGTCAAGACCTTTTTTTTTT
The product length 105bp of Lactobacterium acidophilum.
9. a kind of human body intestinal canal flora detection parting according to claim 1 and quantification kit is characterized in that: the probe hybrid plan of described bifidus bacillus, lactobacterium casei kind, Lactobacterium acidophilum, faecalis, enterobacteria and 6 kinds of human intestine bacterium of Eubacterium is:
The LDR probe pM 100:100:100 of enterobacteria, Eubacterium, bifidus bacillus
The LCR probe pM 100:100:100 of faecalis, Lactobacterium acidophilum, lactobacterium casei.
10. a kind of human body intestinal canal flora detection parting according to claim 1 and quantification kit, it detects step and comprises: the extracting of cultivation of standard bacterium bifidus bacillus, lactobacterium casei, Lactobacterium acidophilum and standard bacterium DNA, the pre-treatment of sample and the extracting of DNA; Sequence fragment with the 16S rDNA of specific PCR primer amplification bifidus bacillus, lactobacterium casei, Lactobacterium acidophilum, faecalis, enterobacteria, Eubacterium; 16S rDNA fragment is inserted the T carrier, change in the competent cell that DH5 α intestinal bacteria make, choose to contain by blue hickie screening and insert segmental thalline, confirm that by order-checking inserting fragment is the special 16s rDNA of each Pseudomonas fragment again; Single bacterium colony is used for a large amount of cultivations, and extracting plasmid DNA more promptly obtains the standard substance DNA of each Pseudomonas.At last by the uv-spectrophotometric instrument measure take out standard substance DNA concentration, redilution to 10
9Copies/uL; 6 kinds of Pseudomonas standard substance that make are mixed according to the scheme of uniform distribution form design,
According to 5 template concentrations combinations shown in the last table, utilize 16S rDNA universal primer to increase respectively, with the PCR product as template, the somatotype that carries out LDR and LCR according to the probe hybrid plan in the claim 9 simultaneously detects, obtaining fluorescent signal by sequenator at last, is X-coordinate with the template concentrations ratio, and signal is than being ordinate zou, do linear regression analysis, obtain typical curve; The detection of sample and standard substance carry out simultaneously, and the relative concentration of above-mentioned bacterium is read or calculated to last reference standard curve.
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