CN107151704A - A kind of quantitative detecting method of Intestine of Broiler microorganism - Google Patents
A kind of quantitative detecting method of Intestine of Broiler microorganism Download PDFInfo
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- CN107151704A CN107151704A CN201710486964.5A CN201710486964A CN107151704A CN 107151704 A CN107151704 A CN 107151704A CN 201710486964 A CN201710486964 A CN 201710486964A CN 107151704 A CN107151704 A CN 107151704A
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- C12Q1/6851—Quantitative amplification
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Abstract
The invention discloses a kind of quantitative detecting method of Intestine of Broiler microorganism, comprise the following steps：Using excrement genome extracts kit, by broiler chicken cecal content Genome DNA extraction in 1.5mLEP pipes, its nucleic acid concentration is surveyed, sterilizing ultra-pure water is diluted to 20ng/ μ L, 20 DEG C of refrigerators are stored in together with stoste, stand-by；Synthesize Intestine of Broiler microorganism PCR primer；Build Intestine of Broiler microorganism plasmid；Make standard items；Carry out sample detection.The present invention has specificity high, reproducible, the features such as sensitivity is high；The method for building plasmid by using specific conservative's genetic fragment insertion vector of gut flora, improves the accuracy of identification, solves the limitation that reference culture does standard curve；Pass through the absolute quantitation to Intestine of Broiler microorganism, it may be determined that the absolute copy number of certain bacterium, the analysis to gut flora diversity and abundance provides reference frame.
Description
Technical field
The invention belongs in poultry intestinal tract technical field of microbial detection, specifically, be related to a kind of micro- life of Intestine of Broiler
The quantitative detecting method of thing.
Background technology
Animal alimentary canal has huge number, metastable microbiologic population.The diversity of flora ensure that intestines
Road microflora balance.Microbiologic population is able to maintain that gastrointestinal tract environment is stablized relatively, promotes nutrient digestion to absorb.Family
The large intestine of fowl is very short, and chyme can enter caecum quickly, so caecum is the important place of microbe survival and activity.
Poultry intestinal tract microbial profile, the detection method of type and quantity uses two methods at present, and one kind is flat board meter
Number method, the method error is big, easily pollution, and testing result is viable count, and Detection results are bad, and time-consuming consumption labour.It is another to be
Quantitative real-time PCR, this method sensitivity is high, pollutes small, but dependent on standard items, detection strain selection is few, specificity
It is not strong.
Absolute quantitation PCR (absolute quantification PCR, AQ-PCR) technology is to utilize known starting copies
Several standard samples make standard curve, by determining the Ct values of unknown sample, from the sample is calculated on standard curve
Beginning concentration, realizes the absolute quantitation of the molecule copy number to determining sample gene.The relative fluorescence being widely used at present is quantified
Round pcr is to determine change of the target sequence relative to sample for reference amount in sample, and can not measure original template starting and copy
The absolute magnitude of shellfish number.The preparation of standard curve is the committed step of absolute fluorescence quantitative PCR experiment, current poultry intestinal tract microorganism
Bacterial strain known to absolute quantitation PCR method more options is standard items, extracts genome and prepares standard curve, during determination sample, standard items
PCR cycle is carried out simultaneously with unknown sample, according to the Ct values of unknown sample, combined standard curve tries to achieve unknown sample cDNA's
Starting copy number.This method can be limited by standard items, and detection strain selection is few, and specificity is not strong, and standard items bacterial strain
It is to determine viable count using colony counting method, there is larger error.
The content of the invention
In view of this, the present invention presently, there are error greatly for poultry intestinal tract microorganism detection method, take, Detection results
It is bad, detection strain limitation, there is provided a kind of quantitative detecting method of Intestine of Broiler microorganism for the problem of specificity is not strong.
In order to solve the above-mentioned technical problem, the invention discloses a kind of quantitative detecting method of Intestine of Broiler microorganism, bag
Include following steps：
Step 1, DNA are extracted：Using excrement genome extracts kit, by broiler chicken cecal content Genome DNA extraction in
In 1.5mLEP pipes, its nucleic acid concentration is surveyed, sterilizing ultra-pure water is diluted to 20ng/ μ L, -20 DEG C of refrigerators are stored in together with stoste, are treated
With；
Step 2, synthesis Intestine of Broiler microorganism PCR primer；
Step 3, structure Intestine of Broiler microorganism plasmid；
Step 4, making standard items；
Step 5, progress sample detection.
Further, Intestine of Broiler microorganism plasmid is built in the step 3 is specially：
1) using Intestine of Broiler content STb gene as template, enter performing PCR with the primer in step 1 and expand, reacted according to PCR
System amplified production, agarose gel electrophoresis, with a small amount of QIAquick Gel Extraction Kit recovery purifying purpose fragments of Ago-Gel DNA, is surveyed
OD values, -20 DEG C of preservations；
2) purpose fragment of 1) middle purifying is attached on carrier and converted with kit, 10 μ L companies are prepared to specifications
Connect mixed liquor, 16 DEG C, 30min；1 μ L mixed liquors add 10 μ L DH5 α 30min on ice, do a repeating groups；42 DEG C of heat shock 90s,
3min on ice；500 μ L LB culture mediums, 37 DEG C, recovery 45-60min, 220RPM/min；4000RPM/min is centrifuged, and 2min is abandoned
400 μ L, are resuspended, coated plate, 37 DEG C of incubated overnights, no more than 16h on LA flat boards；
3) picking single bacterium colony hickie, in 10uL liquid LA culture mediums, takes 1uL to carry out bacterium colony PCR, remaining plus 500uL
Liquid LA culture mediums；The positive bacterium solution sequencing of PCR results；
4) sequencing result is compared on blast, and kit extracts plasmid after the fermentation of positive findings bacterium solution；
5) plasmid order-checking, the upper blast comparison results of NCBI select the plasmid matched with purpose bacterial strain, detect DNA concentration,
- 20 DEG C of refrigerators are stored in, it is standby.
Further, preparing standard items in the step 4 is specially：Plasmid is pressed into decimal dilution method, with sterilizing ultra-pure water
It is diluted to 10-1-10-9Concentration gradient, take 9 sterile 1.5mLEP pipes, according to the descending number consecutively 1-9 of concentration, and
Each ultra-pure water for adding 90 μ L sterilizings, takes the μ L of Intestine of Broiler microbial standard plasmid stock 10 in test tube, quick soft piping and druming
20-30 times, repeat the step and managed to No. 9 EP, obtain including the standard items of 10 gradients of stoste.
Further, sample detection is specially in the step 5：3-7 standard items are chosen together with sample diluting liquid to enter
Row quantitative fluorescent PCR reacts；Experiment device therefor is the hole quantitative real time PCR Instruments of Bio-Rad 96.
Further, the reaction system of PCR reactions is sense primer and each 0.8 μ L of anti-sense primer；RNase-Free
ddH2O 7.4μL；SYBR GREEN Supermix 10μL；The μ L of DNA profiling 1.
Further, the reaction condition of the quantitative fluorescent PCR reaction：The pre-degeneration stage, 1 circulation, 95 DEG C of temperature, in advance
30s is denatured, fluorescence signal is not gathered；The PCR stages of reaction, 40 circulations, 95 DEG C of temperature, denaturation 5s, 50-60 DEG C of annealing temperature,
Time 30s, 72 DEG C of temperature extends 20s, gathers fluorescence signal；Solubility curve, rises 0.5 DEG C per 2s by 72 DEG C to 95 DEG C.
Further, the Intestine of Broiler microorganism is one kind in total bacterium, lactobacillus or Escherichia coli.
Compared with prior art, the present invention can be obtained including following technique effect：
1) method for the Intestine of Broiler microorganism detection that the present invention is provided uses fluorescence quantifying PCR method, with spy
It is different in nature high, reproducible, the features such as sensitivity is high；
2) method that the present invention builds plasmid by using specific conservative's genetic fragment insertion vector of gut flora, is carried
The high accuracy of identification, solves the limitation that reference culture does standard curve；
3) present invention passes through the absolute quantitation to Intestine of Broiler microorganism, it may be determined that the absolute copy number of certain bacterium, right
The analysis of gut flora diversity and abundance provides reference frame.The present invention can be used for other animal intestinal tract microorganism levels
Detection.
Certainly, any product for implementing the present invention it is not absolutely required to while reaching all the above technique effect.
Brief description of the drawings
Accompanying drawing described herein is used for providing a further understanding of the present invention, constitutes the part of the present invention, this hair
Bright schematic description and description is used to explain the present invention, does not constitute inappropriate limitation of the present invention.In the accompanying drawings：
Fig. 1 is the Intestine of Broiler lactobacillus absolute quantitation PCR standard curves in the embodiment of the present invention 1；
Fig. 2 is the lactobacillus plasmid standard real-time quantitative PCR solubility curve in the embodiment of the present invention 1；
Fig. 3 is the amplification curve in the embodiment of the present invention 1；
Fig. 4 is the Intestine of Broiler total bacteria count absolute quantitation PCR standard curves in the embodiment of the present invention 2；
Fig. 5 is total bacteria plasmid standard items real-time quantitative PCR solubility curve in the embodiment of the present invention 2；
Fig. 6 is the amplification curve in the embodiment of the present invention 2；
Fig. 7 is the Intestine of Broiler enterococcus absolute quantitation PCR standard curves in the embodiment of the present invention 3；
Fig. 8 is the enterococcus plasmid standard real-time quantitative PCR solubility curve in the embodiment of the present invention 3；
Fig. 9 is the amplification curve in the embodiment of the present invention 3.
Embodiment
Describe embodiments of the present invention in detail below in conjunction with embodiment, thereby to the present invention how application technology hand
Section can fully understand and implement according to this to solve technical problem and reach the implementation process of technology effect.
The quantitative detecting method of the Intestine of Broiler lactobacillus of embodiment 1
This method comprises the following steps：
Step 1, DNA are extracted：Using excrement genome extracts kit, by broiler chicken cecal content Genome DNA extraction in
In 1.5mLEP pipes, its nucleic acid concentration is surveyed, sterilizing ultra-pure water is diluted to 20ng/ μ L, -20 DEG C of refrigerators are stored in together with stoste, are treated
With；
Step 2, primer synthesis：Intestine of Broiler lactobacillus PCR primer is synthesized, its sense primer is
CGATGAGTGCTAGGTGTTGGA, nucleotide sequence is as shown in SEQ ID NO.1；Anti-sense primer is
CAAGATGTCAAGACCTGGTAAG, sequence is as shown in SEQ ID NO.2；
Step 3, structure plasmid：
1) using Intestine of Broiler content STb gene as template, enter performing PCR with the primer in step 2 and expand, according to the PCR of table 1
Reaction system amplified production, agarose gel electrophoresis, with a small amount of QIAquick Gel Extraction Kits of Ago-Gel DNA (magen, D2111-02)
Recovery purifying purpose fragment, surveys OD values, -20 DEG C of preservations.
2) kit pMD is usedTM19-T Vector Cloning Kit (takara, 6013) are by the purpose piece of purifying in 1)
Section is attached on carrier and converted, and 10 μ L connection mixed liquors, 16 DEG C, 30min are prepared to specifications；1 μ L mixed liquors add 10 μ
L DH5 α 30min on ice, can do a repeating groups；42 DEG C of heat shock 90s, on ice 3min；500 μ L LB culture mediums, 37 DEG C, recovery
45-60min, 220RPM/min；4000RPM/min is centrifuged, and 2min abandons 400 μ L, is resuspended, coated plate on LA flat boards, and 37 DEG C are trained overnight
Support, no more than 16h.
3) picking single bacterium colony hickie, in 10uL liquid LA culture mediums, takes 1uL to carry out bacterium colony PCR, remaining plus 500uL
Liquid LA culture mediums.The positive bacterium solution sequencing of PCR results.
4) sequencing result is compared on blast, and kit extracts plasmid (20ml volume bacterium solutions after the fermentation of positive findings bacterium solution
Reclaim one to manage).
5) plasmid order-checking, the upper blast comparison results of NCBI select the plasmid matched with purpose bacterial strain, detect DNA concentration,
- 20 DEG C of refrigerators are stored in, standby, Intestine of Broiler lactobacillus plasmid is as shown in gene order SEQ ID NO.3.
The making of step 4, standard items：Plasmid is pressed into decimal dilution method, 10 are diluted to sterilizing ultra-pure water-1-10-9It is dense
Gradient is spent, 9 sterile 1.5mLEP pipes is taken, according to the descending number consecutively 1-9 of concentration, and respectively adds the super of 90 μ L sterilizings
Pure water, takes the μ L of lactobacillus standard plasmid stoste 10 in test tube, quick softly piping and druming 20-30 times, repeats the step to No. 9 EP
Pipe, obtains including the standard items of 10 gradients of stoste；
Step 5, sample detection：Choose 3-7 standard items reaction system and table according to table 1 together with sample diluting liquid
2 reaction condition carries out quantitative fluorescent PCR reaction.Experiment device therefor is the hole quantitative real time PCR Instruments of Bio-Rad 96.
The PCR reaction systems of table 1
| Reagent | Volume |
| Sense primer | 0.8μL |
| Anti-sense primer | 0.8μL |
| RNase-Free ddH2O | 7.4μL |
| SYBR GREEN Supermix | 10μL |
| DNA profiling | 1μL |
| Total | 20μL |
The PCR reaction conditions of table 2
The present embodiment 1 is successfully established Intestine of Broiler lactobacillus absolute quantitation PCR standard curves, sees Fig. 1.The present embodiment 1 leads to
Cross the absolute quantitation to Intestine of Broiler lactobacillus, it may be determined that the absolute copy number of the bacterium, its microorganism level is analyzed and provided
Reference frame.Using the logarithm of original template amount as abscissa, Ct values are ordinate, draw out the quantitative fluorescent PCR mark of lactobacillus
Directrix curve, is in preferable linear relationship between initial template concentration and Ct values, coefficient correlation is 1.000, and slope is -3.553, is expanded
Increasing Efficiency is 91.2%.Pass through the detection of lactobacillus plasmid standard real-time quantitative PCR solubility curve (see Fig. 2), it is seen that for list
Peak, therefore amplified production specificity of the present invention is good.Amplification curve (see Fig. 3) shows that detection sample Ct values are in standard items Ct value models
In enclosing, the copy number accuracy drawn is high.
The quantitative detecting method of the Intestine of Broiler total bacteria count of embodiment 2
PCR primer used in this method includes sense primer and anti-sense primer, wherein, sense primer is
CGGCAACGACGCAACCC, nucleotide sequence is as shown in SEQ ID NO.4；Anti-sense primer is
CCATTGTAGCACGTGTGTAGCC, nucleotide sequence is as shown in SEQ ID NO.5；The annealing temperature of quantitative fluorescent PCR reaction
For 60 DEG C；The total bacteria plasmid sequence of Intestine of Broiler prepared is as shown in SEQ ID NO.6, remaining step be the same as Example 1.
The present embodiment 2 is successfully established the total bacterium absolute quantitation PCR standard curves of Intestine of Broiler, sees Fig. 4.The present embodiment 2 passes through
To the absolute quantitation of the total bacterium of Intestine of Broiler, it may be determined that the absolute copy number of total bacterium, reference is provided to the analysis of its microorganism level
Foundation.Using the logarithm of original template amount as abscissa, Ct values are ordinate, draw out the quantitative fluorescent PCR standard curve of total bacterium,
It is in preferable linear relationship between initial template concentration and Ct values, coefficient correlation is 0.996, slope is -3.456, amplification efficiency
For 94.7%.Pass through the detection of total bacteria plasmid standard items real-time quantitative PCR solubility curve (see Fig. 5), it is seen that be unimodal, therefore this
Invention amplified production specificity is good.Amplification curve (see Fig. 6) shows that detection sample Ct values are drawn in the range of standard items Ct values
Copy number accuracy it is high.
The quantitative detecting method of the Intestine of Broiler coliform count of embodiment 3
PCR primer used in this method includes sense primer and anti-sense primer, wherein, sense primer is
GTTAATACCTTTGCTCATTGA, nucleotide sequence is as shown in SEQ ID NO.7；Anti-sense primer is
ACCAGGGTATCTAATCCTGTT, nucleotide sequence is as shown in SEQ ID NO.8；Quantitative fluorescent PCR reaction annealing temperature be
50℃；The Intestine of Broiler escherichia coli plasmid sequence prepared is as shown in SEQ ID NO.9, remaining step be the same as Example 1.
The present embodiment 3 is successfully established Intestine of Broiler Escherichia coli absolute quantitation PCR standard curves, sees Fig. 7.The present embodiment 3
Pass through the absolute quantitation to Intestine of Broiler Escherichia coli, it may be determined that the absolute copy number of Escherichia coli, to its microorganism level
Analysis provides reference frame.Using the logarithm of original template amount as abscissa, Ct values are ordinate, draw out the fluorescence of Escherichia coli
Quantitative PCR standard curve, is in preferable linear relationship between initial template concentration and Ct values, coefficient correlation is 0.999, slope
For -3.907, amplification efficiency is 80.3%.Pass through escherichia coli plasmid standard items real-time quantitative PCR solubility curve (see Fig. 8)
Detection, it is seen that be unimodal, therefore amplified production specificity of the present invention is good.Amplification curve (see Fig. 9) shows that detection sample Ct values exist
In the range of standard items Ct values, the copy number accuracy drawn is high.The present invention can be used for the detection of other animal intestinal tract microorganisms.
Some preferred embodiments of invention have shown and described in described above, but as previously described, it should be understood that invention is not
Form disclosed herein is confined to, the exclusion to other embodiment is not to be taken as, and available for various other combinations, modification
And environment, and can be carried out in invention contemplated scope described herein by the technology or knowledge of above-mentioned teaching or association area
Change., then all should be in the appended power of invention and the change and change that those skilled in the art are carried out do not depart from the spirit and scope of invention
In the protection domain that profit is required.
SEQUENCE LISTING
<110>Animal science research institute of Guangdong Academy of Agricultural Sciences
<120>A kind of quantitative detecting method of Intestine of Broiler microorganism
<130> 2017
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence
<400> 1
cgatgagtgc taggtgttgg a 21
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence
<400> 2
caagatgtca agacctggta ag 22
<210> 3
<211> 1091
<212> DNA
<213>Intestine of Broiler lactobacillus plasmid
<400> 3
aaagttccat ggcctcattc ggtggtaagt tcttagcatc gggcggatgt aggagcaaac 60
cattaggaca tggtcacgac gacggtcacg ctattcagcc caaatggccc acctgagttc 120
tgctatcaat ggcctattcc gcgtcgccag cccgacttgc cccccaagca cgtgtgtcgg 180
gtcgaacctc gcttgctgga tgtggcttga ctctatggat gtcgcactcg atactctttc 240
gcggtgcgaa gggcttccct ctttccgcct gtccataggc cattcgccgt cccagccttg 300
tcctctcgcg tgctccctcg aaggtccccc tttgcggacc atagaaatat caggacagcc 360
caaagcggtg gagactgaac tcgcagctaa aaacactacg agcagtcccc ccgcctcgga 420
tacctttttg cggtcgttgc gccggaaaaa tgccaaggac cggaaaacga ccggaaaacg 480
agtgtacaag aaaggacgca ataggggact aagacaccta ttggcataat ggcggaaact 540
cactcgacta tggcgagcgg cgtcggcttg ctggctcgcg tcgctcagtc actcgctcct 600
tcgccttctc gcgggttatg cgtttggcgg agaggggcgc gcaaccggct aagtaattac 660
gtcgaccgtg ctgtccaaag ggctgacctt tcgcccgtca ctcgcgttgc gttaattaca 720
ctcaatcgag tgagtaatcc gtggggtccg aaatgtgaaa tacgaaggcc gagcatacaa 780
cacaccttaa cactcgccta ttgttaaagt gtgtcctttg tcgatactgg tactaatgcg 840
gttcgaacgt acggacgtcc agctgctaag ttctacagtt ctggaccatt ccaagaagcg 900
caacgaagct taatttggtg tatgaggtga cgaacacgcc cgggggcagt taaggaaact 960
caaagttgga acgccagcat gaggggtcca cctaatgaat aacacaattg acgccgtgac 1020
ttccccagtt aggaggttgt ggatcgtgag tagcttagag atctcctagg ggcccatggc 1080
tcgagcttaa g 1091
<210> 4
<211> 17
<212> DNA
<213>Artificial sequence
<400> 4
cggcaacgac gcaaccc 17
<210> 5
<211> 22
<212> DNA
<213>Artificial sequence
<400> 5
ccattgtagc acgtgtgtag cc 22
<210> 6
<211> 1094
<212> DNA
<213>The total bacteria plasmid of Intestine of Broiler
<400> 6
ccaagtgtct ccggtctagg tttatgcaaa aaaatccatc ggcatcatcc ggtggtaagt 60
tctgaagcat cgtggcggat gtatggagcg aaaccattag gacaatggtc accgacgacg 120
gtcaccgcta ttcagcacag aatggcccaa cctgagttct gctatcaatg gcctattccg 180
cgtcgccagc ccgacttgcc ccccaagcac gtgtgtcggg tcgaacctcg cttgctggat 240
gtggcttgac tctatggatg tcgcactcga tactctttcg cggtgcgaag ggcttccctc 300
tttccgcctg tccataggcc attcgccgtc ccagccttgt cctctcgcgt gctccctcga 360
aggtccccct ttgcggacca tagaaatatc aggacagccc aaagcggtgg agactgaact 420
cgcagctaaa aacactacga gcagtccccc cgcctcggat acctttttgc ggtcgttgcg 480
ccggaaaaat gccaaggacc ggaaaacgac cggaaaacga gtgtacaaga aaggacgcaa 540
taggggacta agacacctat tggcataatg gcggaaactc actcgactat ggcgagcggc 600
gtcggcttgc tggctcgcgt cgctcagtca ctcgctcctt cgccttctcg cgggttatgc 660
gtttggcgga gaggggcgcg caaccggcta agtaattacg tcgaccgtgc tgtccaaagg 720
gctgaccttt cgcccgtcac tcgcgttgcg ttaattacac tcaatcgagt gagtaatccg 780
tggggtccga aatgtgaaat acgaaggccg agcatacaac acaccttaac actcgcctat 840
tgttaaagtg tgtcctttgt cgatactggt actaatgcgg ttcgaacgta cggacgtcca 900
gctgctaagg taacatcgtg cacacatcgg gttctgtatt ccccgtacta ctaaactgca 960
gtaggggtgg aaggaggcac aataggtgcc gtcagagagg tctcacgggt tgatttacta 1020
ccgatgactt ctattcccaa cgcagcaacg gcttagagat ctcctagggg cccatggctc 1080
gagcttagtg atgg 1094
<210> 7
<211> 21
<212> DNA
<213>Artificial sequence
<400> 7
gttaatacct ttgctcattg a 21
<210> 8
<211> 21
<212> DNA
<213>Artificial sequence
<400> 8
accagggtat ctaatcctgt t 21
<210> 9
<211> 1094
<212> DNA
<213>Escherichia coli plasmid
<400> 9
ccccaagcag tggttggggt cgaacctccc ttgctgaagg gggctttacc ctagggaggt 60
cgcactcgaa accctttcgc ggtgcgaagg gcttccctct ttccgcctgt ccaaaggcca 120
ttcgccgtcc cagccttgtc ctctcgcgtg ctccctcgaa ggtccccctt tgcggaccat 180
agaaatatca ggacagccca aagcggtgga gactgaactc gcagctaaaa acactacgag 240
cagtcccccc gcctcggata cctttttgcg gtcgttgcgc cggaaaaatg ccaaggaccg 300
gaaaacgacc ggaaaacgag tgtacaagaa aggacgcaat aggggactaa gacacctatt 360
ggcataatgg cggaaactca ctcgactatg gcgagcggcg tcggcttgct ggctcgcgtc 420
gctcagtcac tcgctccttc gccttctcgc gggttatgcg tttggcggag aggggcgcgc 480
aaccggctaa gtaattacgt cgaccgtgct gtccaaaggg ctgacctttc gcccgtcact 540
cgcgttgcgt taattacact caatcgagtg agtaatccgt ggggtccgaa atgtgaaata 600
cgaaggccga gcatacaaca caccttaaca ctcgcctatt gttaaagtgt gtcctttgtc 660
gatactggta ctaatgcggt tcgaacgtac ggacgtccag ctgctaacaa ttatggaaac 720
gagtaactgc aatgggcgtc ttcttcgtgg ccgattgagg cacggtcgtc ggcgccatta 780
tgcctcccac gttcgcaatt agccttaatg acccgcattt cgcgtgcgtc cgccaaacaa 840
ttcagtctac actttagggg cccgagttgg acccttgacg tagactatga ccgttcgaac 900
tcagagcatc tccccccatc ttaaggtcca catcgccact ttacgcatct ctagacctcc 960
ttatggccac cgcttccgcc gggggacctg cttctgactg cgagtccacg ctttcgcgcc 1020
cctcgtttgt cctaatctat gggaccatta gagatctcct aaggggccca tggctcgagc 1080
ttagtgaccg gagc 1094
Claims (7)
1. a kind of quantitative detecting method of Intestine of Broiler microorganism, it is characterised in that comprise the following steps：
Step 1, DNA are extracted：Using excrement genome extracts kit, by broiler chicken cecal content Genome DNA extraction in 1.5mLEP
Guan Zhong, surveys its nucleic acid concentration, sterilizing ultra-pure water is diluted to 20ng/ μ L, -20 DEG C of refrigerators are stored in together with stoste, stand-by；
Step 2, synthesis Intestine of Broiler microorganism PCR primer；
Step 3, structure Intestine of Broiler microorganism plasmid；
Step 4, making standard items；
Step 5, progress sample detection.
2. the quantitative detecting method of Intestine of Broiler microorganism according to claim 1, it is characterised in that in the step 3
Building Intestine of Broiler microorganism plasmid is specially：
1) using Intestine of Broiler content STb gene as template, enter performing PCR with the primer in step 1 and expand, according to PCR reaction systems
Amplified production, agarose gel electrophoresis, with a small amount of QIAquick Gel Extraction Kit recovery purifying purpose fragments of Ago-Gel DNA, surveys OD
Value, -20 DEG C of preservations；
2) purpose fragment of 1) middle purifying is attached on carrier and converted with kit, 10 μ L connections are prepared to specifications and are mixed
Close liquid, 16 DEG C, 30min；1 μ L mixed liquors add 10 μ L DH5 α 30min on ice, do a repeating groups；42 DEG C of heat shock 90s, on ice
3min；500 μ L LB culture mediums, 37 DEG C, recovery 45-60min, 220RPM/min；4000RPM/min is centrifuged, and 2min abandons 400 μ
L, is resuspended, coated plate, 37 DEG C of incubated overnights, no more than 16h on LA flat boards；
3) picking single bacterium colony hickie, in 10uL liquid LA culture mediums, takes 1uL to carry out bacterium colony PCR, remaining plus 500uL liquid
Body LA culture mediums；The positive bacterium solution sequencing of PCR results；
4) sequencing result is compared on blast, and kit extracts plasmid after the fermentation of positive findings bacterium solution；
5) plasmid order-checking, the upper blast comparison results of NCBI select the plasmid matched with purpose bacterial strain, detect DNA concentration, preserve
It is standby in -20 DEG C of refrigerators.
3. the quantitative detecting method of Intestine of Broiler microorganism according to claim 1, it is characterised in that in the step 4
Preparing standard items is specially：Plasmid is pressed into decimal dilution method, 10 are diluted to sterilizing ultra-pure water-1-10-9Concentration gradient, take 9
Individual sterile 1.5mLEP pipes, according to the descending number consecutively 1-9 of concentration, and respectively add the ultra-pure water of 90 μ L sterilizings, take broiler chicken
μ L are in test tube for enteric microorganism standard plasmid stoste 10, quick softly piping and druming 20-30 times, repeat the step and managed to No. 9 EP, are obtained
To the standard items of 10 gradients including stoste.
4. the quantitative detecting method of Intestine of Broiler microorganism according to claim 3, it is characterised in that in the step 5
Sample detection is specially：Choose 3-7 standard items and quantitative fluorescent PCR reaction is carried out together with sample diluting liquid；Set used in experiment
Standby is the hole quantitative real time PCR Instruments of Bio-Rad 96.
5. the quantitative detecting method of the Intestine of Broiler microorganism according to claim 2 or 4, it is characterised in that PCR reactions
Reaction system is sense primer and each 0.8 μ L of anti-sense primer；RNase-Free ddH2O 7.4μL；SYBR GREEN
Supermix 10μL；The μ L of DNA profiling 1.
6. the quantitative detecting method of Intestine of Broiler microorganism according to claim 4, it is characterised in that the fluorescent quantitation
The reaction condition of PCR reactions：Pre-degeneration stage, 1 circulation, 95 DEG C of temperature, pre-degeneration 30s does not gather fluorescence signal；PCR is anti-
Answer the stage, 40 circulations, 95 DEG C of temperature, be denatured 5s, 50-60 DEG C of annealing temperature, time 30s, 72 DEG C of temperature extends 20s, collection
Fluorescence signal；Solubility curve, rises 0.5 DEG C per 2s by 72 DEG C to 95 DEG C.
7. the quantitative detecting method of Intestine of Broiler microorganism according to claim 1, it is characterised in that the microorganism is
One kind in total bacterium, lactobacillus or Escherichia coli.
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| CN117178950B (en) * | 2023-09-21 | 2024-03-22 | 江苏省家禽科学研究所 | Microecological preparation-based broiler chicken intestinal tract adjusting raising method |
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