CN108179209A - For detecting the specific primer of proteus mirabilis and probe and real-time fluorescence quantitative PCR kit - Google Patents
For detecting the specific primer of proteus mirabilis and probe and real-time fluorescence quantitative PCR kit Download PDFInfo
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Abstract
The present invention is provided to detect the specific primer of proteus mirabilis and probe, probe sequence is：5’‑TCTTGACCGTGGCTTCTG‑3’；Specific primer be following sequences or be following sequences complementary strand sequence：Upstream primer sequence is 5 ' CTAAATGTCGATTTGAGGTT 3 '；Downstream primer sequence is 5 ' GCATTCTGATCTACGATTAC 3 '.Primer and probe provided by the invention, by extracting the genomic DNA of proteus mirabilis, with reference to real-time fluorescence quantitative PCR detection technique, can in accurate quantitative analysis sample to be measured proteus mirabilis content, there is highly sensitive and high specific.The primer and probe of the present invention can carry out qualitative and quantitative analysis to proteus mirabilis content, and the researchs such as content and secondary infection to judging proteus mirabilis active bacteria are of great significance.
Description
Technical field
The invention belongs to molecular biology and external diagnosis reagent technical field, and in particular to for detecting unusual deformed rod
The specific primer and probe of bacterium and real-time fluorescence quantitative PCR kit.
Background technology
Proteus mirabilis (Proteus mirabilis, PM) is a kind of more typical opportunist, which deposits extensively
It is in the enteron aisle of water, the organic matter of soil corruption and humans and animals, is conditioned pathogen, mostly secondary infection is such as chronic
Tympanitis, wound infection etc. can also cause cystitis, infantile diarrhea, food poisoning etc..Traditional diagnosis proteus mirabilis
Method is mainly classified by Bacterial Physiological biochemical characteristic, and the result of a large amount of time progress biochemical reactions is needed to sentence
It is fixed, it is unfavorable for finding cause of disease in time, diagnoses the cause of disease.Round pcr can selectively expand in vitro as a kind of biology tool
Increase DNA or RNA segments, easy to operate, high specificity, the features such as quick, sensibility is high.Taq Man sonde methods detection specificity
By force, high sensitivity facilitates optimization reaction condition, can be used in the quick detection of proteus mirabilis.
Invention content
It is an object of the invention to design one group of proteus mirabilis specific primer and probe sequence, it is wide to establish a kind of energy
General quick, sensitive, the specific good method with fluorescence quantitative PCR detection applied to proteus mirabilis detection.The present invention
Using gene clone technology, proteus mirabilis 16SrDNA genetic fragments are inserted into carrier pMD18-T, are contained
The recombinant plasmid of 16SrDNA genetic fragments, in this, as standard items.It is encoded according to proteus mirabilis 16SrDNA genetic fragments
Gene order designs and synthesizes a group-specific primers and probe, optimizes PCR reaction conditions, establishes and polymerize with real time fluorescent quantitative
Enzyme chain reaction is the detection method of platform, and the method established is assessed.
First purpose of the present invention is to provide specific primer and probe for detecting proteus mirabilis, the spy
Needle sequence is：5’-TCTTGACCGTGGCTTCTG-3’；
The specific primer be following sequences or be following sequences complementary strand sequence：
Upstream primer sequence is 5 '-CTAAATGTCGATTTGAGGTT-3 '；
Downstream primer sequence is 5 '-GCATTCTGATCTACGATTAC-3 '.
Preferably, the fluorescent reporter group of the end of probe 5 ' label is FAM, the fluorescent quenching group of 3 ' end labels is BHQ.
Preferably, the sense primer and the downstream primer are to 5 ' ends and/or 3 ' extreme directions extension one to several
Base deletes a sequence obtained to several bases.
Second object of the present invention is to provide a kind of real-time fluorescence quantitative PCR reagent for being used to detect proteus mirabilis
Box, the real-time fluorescence quantitative PCR kit include claim 1-3 any one of them specific primer and probe.
Preferably, in 20 μ l PCR reaction systems, the dosage of the sense primer is 0.2 μ l, the downstream primer
Dosage for 0.2 μ l, the dosage of the probe is 0.2 μ l.
Preferably, the real-time fluorescence quantitative PCR kit further includes series concentration standard items and positive control；It is described
Series concentration standard items are to connect proteus mirabilis 16SrDNA genes with carrier, convert into competent cell induction table
It reaches, extracts recombinant plasmid, diluted after recombinant plasmid is quantified, obtain series concentration standard items.
Preferably, the nucleotides sequence of the standard items is classified as sequence 2 in sequence table.
Third object of the present invention is to provide above-mentioned specific primer and probe, real-time fluorescence quantitative PCR kit exist
It is following it is any in application, the application is not for the purpose of the diagnose and treat of disease：
(1) qualitatively or quantitatively detection or auxiliary detect proteus mirabilis；
(2) product of qualitative or quantitative detection or auxiliary detection proteus mirabilis is prepared.
Fourth object of the present invention is to provide in a kind of detection or auxiliary detection measuring samples whether contain unusual deformation
The method of bacillus, this method is not for the purpose of the diagnose and treat of disease, respectively with series concentration standard items and sample to be tested DNA
For template, real-time fluorescence quantitative PCR is carried out using specific primer and probe, draws standard curve, by standard curve and is treated
The Ct values judgement result of sample.
Preferably, the reaction condition of the real-time fluorescence quantitative PCR is：94 DEG C of pre-degenerations 2 minutes, 94 DEG C 10 seconds, 60
DEG C 30s simultaneously collects fluorescence signal, 40 cycles.
It is unusual by extracting the present invention is provided to the primer and probe that proteus mirabilis carries out qualitative and quantitative analysis
The genomic DNA of proteus with reference to real-time fluorescence quantitative PCR detection technique, can reach unusual in accurate quantitative analysis sample to be measured
The purpose of proteus content has highly sensitive and high specific：Sensitivity is 1.00 × 102Copies/ml, and can be special
The opposite sex from Lactobacillus rhamnosus, the primary bacterium of pneumonia gram, escherichia coli, haemophilus influenzae, enterococcus faecalis, Neisseria meningitdis
Bacterium, staphylococcus aureus, enterobacter cloacae, beta hemolytic streptococcus, clostridium tetani, pseudomonas aeruginosa, cheese breast
Bacillus, vibrio parahaemolytious, streptococcus thermophilus, clostridium perfringen, salmonella typhimurium, NEISSERIA GONORRHOEAE, streptococcus pneumonia, good fortune
Proteus mirabilis is detected in family name's shigella dysenteriae, salmonella, streptococcus pyogenes, helicobacter pylori and proteus mirabilis.
Primer and probe provided by the present invention can carry out qualitative and quantitative analysis to proteus mirabilis content, to judging unusual deformation
So that follow-up study secondary infection is of great significance, the present invention will be led the content of bacillus active bacteria in microorganism clinical detection
Domain plays a significant role.
Below in conjunction with the accompanying drawings and the present invention is described in detail in specific embodiment.
Description of the drawings
Attached drawing is used to provide further understanding of the present invention, and a part for constitution instruction, the reality with the present invention
Example is applied together for explaining the present invention, is not construed as limiting the invention.In the accompanying drawings：
Fig. 1 is ncbi database blast comparison results.
Fig. 2 is upstream and downstream primer sequence through Primer-Blast comparison results in NCBI.
Fig. 3 is the standard curve of standard items of the present invention.
Fig. 4 is sensitivity experiment result of the present invention.
Fig. 5 is specificity experiments result of the present invention.
Fig. 6 is specificity experiments gel imaging proof diagram of the present invention.
Specific embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is conventional method unless otherwise specified.The primer, probe and sequencing efforts used are by raw work biology work
Journey (Shanghai) limited company synthesizes and completes.
The preparation of 1 16SrDNA gene standard items of embodiment
Establish real time fluorescence quantifying PCR method it may first have to which the external standards needed for preparation method, standard items should wrap
Containing highly conserved, special sequence, it is ensured that the high specific of reaction.16SrDNA genes are widely present in proteus mirabilis
In, there is very high conservative.The present invention is using proteus mirabilis 16SrDNA genes as target sequence.The present embodiment is main
Proteus mirabilis 16SrDNA genes are expanded using round pcr, plasmid vector is connected to using gene recombination technology
In pMD18-T, recombinant plasmid pMD18-T-16SrDNA is constructed, and carries out corresponding PCR identifications and sequencing identification, is most passed through afterwards
It quantifies as the standard items for treating method for building up, lays the foundation for the method for next step and assessment.
First, the preparation of template DNA
Proteus mirabilis (reference culture) genomic DNA is extracted, the template as the amplification of 16SrDNA gene PCRs.Using
The bacterial genomes extracts kit of Beijing hundred Imtech production is extracted, and specific extracting method is as follows：
1. taking 1ml bacteria suspensions, 1.5ml centrifuge tubes are added in, 8000r/min is centrifuged 2 minutes, abandons supernatant；
2. adding in 400 μ l Buffer Digestion suspension bacteria liquids precipitation, it is complete to cell to be put into 65 DEG C of water-bath 1h after mixing
It totally cleaves solution；
3. add in -20 DEG C of standing 5min of 200 μ l Buffer PB mixings, centrifuging and taking supernatant；
4. add in -20 DEG C of standing 5min of 600 μ l isopropanols mixing.Supernatant is abandoned in centrifugation；
5. adding in -20 DEG C of standing 5min of 75% ethyl alcohol of 1ml, supernatant is abandoned in centrifugation；
6. precipitation is washed with 75% ethyl alcohol, dry；
7. it is dissolved in 50 μ l TE Buffer, -20 DEG C of preservations.
2nd, the PCR amplification of 16SrDNA genetic fragments
1st, the design and synthesis of primer
16SrDNA identifications are to carry out Species estimation to bacterium using the method for bacterial 16 S rDNA sequences.It is a kind of fast
The method that speed obtains bacterium kind information.The present invention detects proteus mirabilis 16SrDNA genes using real-time fluorescence quantitative PCR
Expression specifies meanings and specificity of the 16SrDNA in proteus mirabilis detects.
The present invention to proteus mirabilis 16SrDNA genes complete sequence in ncbi database by carrying out bioinformatics ratio
To analysis, choose and be suitble to the conservative fragments sequence of design primer and probe for target, using 3 softwares of Primer express,
7 software of 5 softwares of Primer Premier and Beacon Designer, devise one group of real-time fluorescence quantitative PCR primer and
The peripheral primer of probe and one group of correlated series.
The extension increasing sequence that the present invention chooses is as follows：
5’-GAGCCTAAATGTCGATTTGAGGTTGTGGTCTTGACCGTGGCTTCTGGAGCTAACGCGTTAAATCGA
CCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGT
TTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGCGAATCCTTTAGAGATAGAGGAGTGCCTTCG
GGAACGCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGC
AACCCTTATCCTTTGTTGCCAGCACGTAATGGTGGGAACTCAAAGGAGACTGCCGGTGATAAACCGGAGGAAGGTGG
GGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCAGATACAAAGAGAAGCGAC
CTCGCGAGAGCAAGCGGAACTCATAAAGTCTGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGA
ATCGCTAGTAATCGTAGATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGG
GAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAA
GTCGTACAGGGGAAACCCGTAAAAGGGGAGGGA-3’
This sequence is shown in Fig. 1 by being shown to ncbi database blast results.
Fig. 1 is ncbi database blast comparison results.
As shown in Figure 1：This sequence pair proteus mirabilis has specificity.
The periphery primer sequence is as follows：
Sense primer：16SrDNA-5F 5'-CTAAATGTCGATTTGAGGTT-3'
Downstream primer：16SrDNA-555R 5'-GCATTCTGATCTACGATTAC-3'
Amplified fragments size is：551bp.
Upstream and downstream primer sequence is shown in Fig. 2 through Primer-Blast comparison results in NCBI.
Fig. 2 is upstream and downstream primer sequence through Primer-Blast comparison results in NCBI.
As shown in Figure 2：This has specificity to primer pair proteus mirabilis.
2nd, PCR reaction systems and reaction condition
Using DNA as template, using above-mentioned periphery primer 16SrDNA-5F/16SrDNA-555R as amplimer, use is following
System and reaction condition carry out PCR amplification.
PCR system：
Wherein primer uses 16SrDNA-5F/16SrDNA-555R, and Taq enzyme uses hundred Tyke Power Taq of Beijing
PlusDNA Polymerse, PCR amplification instrument are the serial 96 grads PCR instrument of Hangzhou Lang Ji scientific instrument Co., Ltd MG.
Amplification program/reaction condition：
94 DEG C of pre-degeneration 5min；94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min, 30 cycles；72℃10min.5 μ L is taken to expand
Product carries out 2% agarose electrophoresis, detects PCR product size, then recycles examination using the DNA gel of Shanghai Sangon Biotech Company's production
Remaining pcr amplification product is recycled in the purifying of agent box.
3rd, the structure of recombinant plasmid pMD18-T-16SrDNA and conversion
1st, coupled reaction：The pcr amplification product that above-mentioned purifying obtains and pMD18-T (Dalian treasured biotech firm) are connected
It connects, is prepared using following linked system：
Preparation completion is placed on 16 DEG C and carries out staying overnight coupled reaction.
2nd, the conversion of pMD18-T-16S plasmids and PCR identifications
1. taking out the DH5 α competent cells frozen from -70 DEG C of ultra low temperature freezer, being placed on ice chest makes it solve naturally
Freeze；
2. 10 μ L of connection product is taken to add in the DH5 α competent cells of 100 μ L；
3. heat shock 90s in 42 DEG C of water-baths puts cooled on ice 30min after heat shock immediately；
After 4. LB fluid nutrient mediums (without ampicillin) mixing of 800 μ l of precooling is added in into 1.5ml EP pipes,
37 DEG C of 140rpm jog cultures 1h；
5. above-mentioned culture solution 8000rpm is centrifuged 1min, supernatant is abandoned, cell resuspension absorption residue is coated on and is contained
It on the LB tablets of 0.1ngAmp, faces up and places 30min, after bacterium solution is cultured base absorption completely, be inverted 37 DEG C of culture dish
Insulating box overnight incubation；
6. next day is observed, picking monoclonal bacterium colony is in 100 μ L LB fluid nutrient mediums (containing ampicillin) from tablet
In PCR pipe, 37 DEG C of shaken cultivations 2-3 hours.It draws 2 μ L and carries out PCR identifications as template, remaining bacterium solution is added to 20ml's
It carries out expanding in LB fluid nutrient mediums and shake；
7. expand above-mentioned dilution bacterium solution, PCR with proteus mirabilis specific primer 16SrDNA-5F/16SrDNA-555R
Product uses 2% agarose gel electrophoresis, and positive transformant is identified by detecting PCR product size.
Primer 16SrDNA-5F/16SrDNA-555R sequences are as follows：
Sense primer；16SrDNA-5F 5'-CTAAATGTCGATTTGAGGTT-3'
Downstream primer；16SrDNA-555R 5'-GCATTCTGATCTACGATTAC-3'
PCR reaction systems are as follows：
Amplification program/reaction condition：94 DEG C of pre-degeneration 5min；94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min, 30 cycles；72
℃10min。
Positive recombinant plasmid pMD18-T-16S is extracted using the Plasmid Preparation kit of hundred Imtech production, is measured dense
Degree and purity, while draw a part of plasmid purification and supreme marine growth Engineering Co., Ltd is given to be sequenced, determine Insert Fragment
Gene order it is consistent with aim sequence.
4th, the acquisition of standard items and quantitative
1st, the 100 μ L transferred speciess of bacillus coli DH 5 alpha containing recombinant plasmid pMD18-T-16S that step 3 obtains are taken in 5mL's
LB fluid nutrient mediums, 37 DEG C of 200rpm shake training overnight；
2nd, the bacterium solution transferred speciess of 1ml overnight incubations is taken in 10ml LB fluid nutrient mediums, 200rpm Zengjing Granules 2-3 hours, so
Afterwards using the Plasmid Preparation kit extraction plasmid of hundred Imtech of Beijing production；
3rd, using hundred Tyke bio tech ltd ultramicron ultraviolet-uisible spectrophotometer (ND5000) of Beijing to carrying
The plasmid taken is measured, and measures A260、A280, according to A260/A280Judge the purity of plasmid；
4th, plasmid pMD18-T-16S concentration (copy number) calculates
(1) molecular weight=2842bp × 660 (average molecular weight of each pair of base) of plasmid
(2) plasmid concentration is measured as 146.82ng/ μ l, plasmid purity A260/A280=1.86, because determining in progress real-time fluorescence
It when measuring PCR, needs with " copy number " for unit, it is therefore desirable to by Conversion of measurement unit into copies/ml.
Plasmid copies/ml=Avgadro constants × plasmid molal quantity
Wherein Avgadro constant=6.02 × 1023copies/mol。
Therefore plasmid concentration copies/ml=146.82 × 10 of extraction-9g/ml×6.02×1023copies/mol÷
(2842bp × 660g/bpmol)=4.71 × 1010copies/ml
10 μ l plasmids add 37.1 sterile water just to obtain a concentration of 1.00 × 1010The plasmid of copies/ml, then by the plasmid
A series of plasmid that 10 doubling dilutions just can obtain concentration is carried out, and is saved backup in -20 DEG C.
2 real-time fluorescence quantitative PCR kit of embodiment
First, the design and synthesis of specific primer and probe
The present invention to proteus mirabilis 16SrDNA genes complete sequence in ncbi database by carrying out bioinformatics ratio
To analysis, it is target (see embodiment 1), and further apply to choose the conservative fragments sequence for being suitble to design primer and probe
7 software of 3 softwares of Primer express, 5 softwares of Primer Premier and Oligo, devises multigroup real-time fluorescence and determines
PCR primer and probe are measured, is screened by Preliminary Experiment, is finally determined one group for detecting the fluorescent quantitation of proteus mirabilis
PCR primer and probe.
As the core of the present invention, one group of primer and probe nucleosides for being used for proteus mirabilis real-time fluorescence PCR detection
Acid sequence is as follows：
Sense primer：16SrDNA-5F 5'-CTAAATGTCGATTTGAGGTT-3'
Downstream primer：16SrDNA-555R 5'-GCATTCTGATCTACGATTAC-3'
Probe：16SrDNA5-555 5'-TCTTGACCGTGGCTTCTG-3'.
The fluorescent reporter group of the end of probe 5 ' label is FAM, and the fluorescent quenching group of 3 ' end labels is BHQ.
The primer and probe expands Target Nucleotide Sequence：
5’-CTAAATGTCGATTTGAGGTTGTGGTCTTGACCGTGGCTTCTGGAGCTAACGCGTTAAATCGACCGC
CTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAA
TTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGCGAATCCTTTAGAGATAGAGGAGTGCCTTCGGGAA
CGCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACC
CTTATCCTTTGTTGCCAGCACGTAATGGTGGGAACTCAAAGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGAT
GACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCAGATACAAAGAGAAGCGACCTCG
CGAGAGCAAGCGGAACTCATAAAGTCTGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCG
CTAGTAATCGTAGATCAGAATGC-3’。
2nd, the foundation of real-time fluorescence quantitative PCR kit
1st, real-time fluorescence quantitative PCR kit includes following components：
(2 × premix is produced 2 × premix for hundred Imtech of Beijing, and full name is 2 × real-time PCR
Premixture (probe)), it is final concentration of 10 μM of sense primer, final concentration of 10 μM of downstream primer, 10 μM final concentration of
Probe, (series concentration of series concentration standard items is the 16SrDNA gene series concentration standards that prepare of embodiment 1：1.00
×1010copies/ml、1.00×109copies/ml、1.00×108copies/ml、1.00×107copies/ml、1.00×
106copies/ml、1.00×105copies/ml、1.00×104copies/ml、1.00×103copies/ml、1.00×
102Copies/ml), positive control (a concentration of 4.71 × 1010Recombinant plasmid pMD18- prepared by the embodiment 1 of copies/ml
) and ddH T-16SrDNA2O, ddH2O is used as reagent and negative control.
Sense primer：16SrDNA-5F 5'-CTAAATGTCGATTTGAGGTT-3'
Downstream primer：16SrDNA-555R 5'-GCATTCTGATCTACGATTAC-3'
Probe：16SrDNA5-555 5'-TCTTGACCGTGGCTTCTG-3'.
2nd, using the standard curve of real-time fluorescence quantitative PCR kit measurement standard items
Using embodiment 1 prepare 16SrDNA gene series concentration standards (series concentration of series concentration standard items as：
1.00×1010copies/ml、1.00×109copies/ml、1.00×108copies/ml、1.00×107copies/ml、
1.00×106copies/ml、1.00×105copies/ml、1.00×104copies/ml、1.00×103copies/ml、
1.00×102Copies/ml it is) template, using the primer and probe in kit, carries out fluorescent quantitative PCR, set simultaneously
Put positive control and negative control.
PCR reaction systems are as follows：
Reaction condition：94 DEG C of pre-degenerations 2 minutes, 94 DEG C 10 seconds, 60 DEG C of 30s and collect fluorescence signal, 40 cycles.
The preparation method of standard curve：Using the logarithm of the plasmid concentration of standard items as abscissa, obtained using ct values as ordinate
To standard curve, as a result with reference to figure 3.Standard curve original equation is y=a+bx, and the equation of this standard curve is Y=-
2.434x+37.87。
Fig. 3 is the standard curve of standard items of the present invention.From the figure 3, it may be seen that standard items standard curve is smooth, related coefficient is high,
Specially R2=0.997, meet the requirement of real-time fluorescence quantitative PCR detection.
3 real-time fluorescence quantitative PCR kit of embodiment is to the quantitative detecting method of sample to be tested
16SrDNA gene series concentration standards (the series concentration marks prepared respectively with sample to be tested DNA and embodiment 1
The series concentration of quasi- product is：1.00×1010copies/ml、1.00×109copies/ml、1.00×108copies/ml、
1.00×107copies/ml、1.00×106copies/ml、1.00×105copies/ml、1.00×104copies/ml、
1.00×103copies/ml、1.00×102Copies/ml it is) template, using the primer and probe in kit, carries out fluorescence
Quantitative pcr amplification, while positive control and negative control are set.
PCR reaction systems are as follows：
Reaction condition：94 DEG C of pre-degenerations 2 minutes, 94 DEG C 10 seconds, 60 DEG C of 30s and collect fluorescence signal, 40 cycles.It draws
Standard curve carries out Quantitative detection by the Ct values of standard curve and sample to be tested.
The performance test of 4 real-time fluorescence quantitative PCR kit of embodiment
1st, sensitivity experiment
The plasmid that embodiment 1 is prepared is subjected to 10 doubling dilutions and obtains a series of plasmid of concentration, is chosen a concentration of
1.00×108copies/ml、1.00×107copies/ml、1.00×106copies/ml、1.00×105copies/ml、
1.00×104copies/ml、1.00×103copies/ml、1.00×102copies/ml、1.00×101Copies/ml and
Negative control is as template.Reaction system is as follows：
Reaction condition：94 DEG C of pre-degenerations 2 minutes, 94 DEG C 10 seconds, 60 DEG C of 30s and collect fluorescence signal, 40 cycles.According to
Fluorescence signal detected by instrument is handled through software obtains fluorescence curve, observes the signal of fluorescence curve, as a result reference Fig. 4,
Data are shown when plasmid concentration reaches 1.00 × 102Still there is fluorescence signal during copies/ml.Therefore the method for the present invention is sensitive
Spend is 1.00 × 102copies/ml。
Fig. 4 is sensitivity experiment result of the present invention.Wherein, a represents plasmid concentration as 1.00 × 108Copies/ml, b generation
Table plasmid concentration is 1.00 × 107Copies/ml, c represent plasmid concentration as 1.00 × 106Copies/ml, d represent plasmid concentration
It is 1.00 × 105Copies/ml, e represent plasmid concentration as 1.00 × 104Copies/ml, f represent plasmid concentration as 1.00 ×
103Copies/ml, g represent plasmid concentration as 1.00 × 102Copies/ml, h represent plasmid concentration as 1.00 × 101copies/
Ml and negative control.
2nd, specificity experiments
In order to confirm the specificity of the invention detected to proteus mirabilis, it is micro- that we have chosen the infection of other common clinicals
Biologic specificity is tested, and the microorganism of selection includes：The primary bacterium of Lactobacillus rhamnosus, pneumonia gram, escherichia coli, the bloodthirsty bar of influenza
Bacterium, enterococcus faecalis, Neisseria meningitidis, staphylococcus aureus, enterobacter cloacae, beta hemolytic streptococcus, lockjaw
Clostridium, pseudomonas aeruginosa, Lactobacillus casei, vibrio parahaemolytious, streptococcus thermophilus, clostridium perfringen, salmonella typhimurium,
NEISSERIA GONORRHOEAE, streptococcus pneumonia, Shigella flexneri, salmonella, streptococcus pyogenes, helicobacter pylori.
It is experiment that template carries out that the specific test, which is included with the genomic DNA of above-mentioned sample,.The DNA of mentioned microorganism
Extraction is using Shanghai life work bacterial genomes DNA rapid extractions kit (C317KA2364), using the 2 of the production of Bo companies
× real-time PCR Premixture (probe) real-time fluorescence quantitative PCR kit is tested, and reaction system is as follows：
Primer and probe therein is the primer and probe in the embodiment of the present invention 2.
Reaction condition：94 DEG C of pre-degenerations 2 minutes, 94 DEG C 10 seconds, 60 DEG C of 30s and collect fluorescence signal, 40 cycles.According to
Fluorescence signal detected by instrument is handled through software obtains fluorescence curve, observes the signal of fluorescence curve, analysis specificity.Knot
Fruit is with reference to figure 5：Using genomic DNA as template only proteus mirabilis test positive, remaining microorganism is feminine gender, shows this
Invention has specificity well.Testing result is as shown in table 1.
Table 1
Fig. 5 is specificity experiments result of the present invention.Wherein a standard amplification curves are proteus mirabilis genome, b generations
It is respectively Lactobacillus rhamnosus, the primary bacterium of pneumonia gram, escherichia coli, haemophilus influenzae, excrement that table, which does not occur standard amplification curve,
Enterococcus, Neisseria meningitidis, staphylococcus aureus, enterobacter cloacae, beta hemolytic streptococcus, clostridium tetani,
Pseudomonas aeruginosa, Lactobacillus casei, vibrio parahaemolytious, streptococcus thermophilus, clostridium perfringen, salmonella typhimurium, gonorrhoea
Neisseria, streptococcus pneumonia, Shigella flexneri, salmonella, streptococcus pyogenes, helicobacter pylori, negative control.
3rd, specificity experiments gel imaging proof diagram
In order to confirm the specificity of the invention detected to proteus mirabilis, it is micro- that we have chosen the infection of other common clinicals
Biology carries out specificity experiments, and the microorganism of selection includes：The primary bacterium of Lactobacillus rhamnosus, pneumonia gram, escherichia coli, influenza are thermophilic
Blood bacillus, Neisseria meningitidis, staphylococcus aureus, enterobacter cloacae, beta hemolytic streptococcus, is broken enterococcus faecalis
Catch cold clostridium, pseudomonas aeruginosa, Lactobacillus casei, vibrio parahaemolytious, streptococcus thermophilus, clostridium perfringen, mouse typhus sramana
Salmonella, NEISSERIA GONORRHOEAE, streptococcus pneumonia, Shigella flexneri, salmonella, streptococcus pyogenes, helicobacter pylori.
Prepare 2% agarose gel detection proteus mirabilis specific primer probe fluorescent quantitation result.With specificity
The amplified production of experiment is template.
Fig. 6 is specificity experiments gel imaging proof diagram of the present invention.Wherein, swimming lane 1-25 be followed successively by Lactobacillus rhamnosus,
It is the primary bacterium of pneumonia gram, escherichia coli, haemophilus influenzae, proteus mirabilis, enterococcus faecalis, Neisseria meningitidis, golden yellow
Color staphylococcus, enterobacter cloacae, beta hemolytic streptococcus, clostridium tetani, pseudomonas aeruginosa, DL2000
Marker, Lactobacillus casei, vibrio parahaemolytious, streptococcus thermophilus, clostridium perfringen, salmonella typhimurium, NEISSERIA GONORRHOEAE,
Streptococcus pneumonia, Shigella flexneri, salmonella, streptococcus pyogenes, helicobacter pylori, negative control.
As a result it shows：Using the amplified production of specificity experiments as template, only proteus mirabilis detection amplified band is big
Small correct purpose band, remaining microorganism do not amplify purpose band, show that the present invention has specificity well.
Finally it should be noted that：The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention,
Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be used
To modify to the technical solution recorded in foregoing embodiments or carry out equivalent replacement to which part technical characteristic.
All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in the present invention's
Within protection domain.
Sequence table
<110>Suzhou Bai Yuan gene technology Co., Ltd
<120>For detecting the specific primer of proteus mirabilis and probe and real-time fluorescence quantitative PCR kit
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 715
<212> DNA
<213>Proteus mirabilis (Proteus mirabilis)
<400> 1
gagcctaaat gtcgatttga ggttgtggtc ttgaccgtgg cttctggagc taacgcgtta 60
aatcgaccgc ctggggagta cggccgcaag gttaaaactc aaatgaattg acgggggccc 120
gcacaagcgg tggagcatgt ggtttaattc gatgcaacgc gaagaacctt acctactctt 180
gacatccagc gaatccttta gagatagagg agtgccttcg ggaacgctga gacaggtgct 240
gcatggctgt cgtcagctcg tgttgtgaaa tgttgggtta agtcccgcaa cgagcgcaac 300
ccttatcctt tgttgccagc acgtaatggt gggaactcaa aggagactgc cggtgataaa 360
ccggaggaag gtggggatga cgtcaagtca tcatggccct tacgagtagg gctacacacg 420
tgctacaatg gcagatacaa agagaagcga cctcgcgaga gcaagcggaa ctcataaagt 480
ctgtcgtagt ccggattgga gtctgcaact cgactccatg aagtcggaat cgctagtaat 540
cgtagatcag aatgctacgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac 600
catgggagtg ggttgcaaaa gaagtaggta gcttaacctt cgggagggcg cttaccactt 660
tgtgattcat gactggggtg aagtcgtaca ggggaaaccc gtaaaagggg aggga 715
<210> 2
<211> 551
<212> DNA
<213>Proteus mirabilis (Proteus mirabilis)
<400> 2
ctaaatgtcg atttgaggtt gtggtcttga ccgtggcttc tggagctaac gcgttaaatc 60
gaccgcctgg ggagtacggc cgcaaggtta aaactcaaat gaattgacgg gggcccgcac 120
aagcggtgga gcatgtggtt taattcgatg caacgcgaag aaccttacct actcttgaca 180
tccagcgaat cctttagaga tagaggagtg ccttcgggaa cgctgagaca ggtgctgcat 240
ggctgtcgtc agctcgtgtt gtgaaatgtt gggttaagtc ccgcaacgag cgcaaccctt 300
atcctttgtt gccagcacgt aatggtggga actcaaagga gactgccggt gataaaccgg 360
aggaaggtgg ggatgacgtc aagtcatcat ggcccttacg agtagggcta cacacgtgct 420
acaatggcag atacaaagag aagcgacctc gcgagagcaa gcggaactca taaagtctgt 480
cgtagtccgg attggagtct gcaactcgac tccatgaagt cggaatcgct agtaatcgta 540
gatcagaatg c 551
Claims (10)
1. for detecting the specific primer and probe of proteus mirabilis, it is characterised in that：The probe sequence is：5’-
TCTTGACCGTGGCTTCTG-3’；
The specific primer be following sequences or be following sequences complementary strand sequence：
Upstream primer sequence is 5 '-CTAAATGTCGATTTGAGGTT-3 '；
Downstream primer sequence is 5 '-GCATTCTGATCTACGATTAC-3 '.
2. specific primer according to claim 1 and probe, it is characterised in that：The fluorescence report base of the end of probe 5 ' label
Group is FAM, and the fluorescent quenching group of 3 ' end labels is BHQ.
3. specific primer according to claim 1 and probe, it is characterised in that：The sense primer and the downstream are drawn
Object is to 5 ' ends and/or 3 ' extreme directions extension one to several bases or deletes a sequence obtained to several bases.
4. a kind of real-time fluorescence quantitative PCR kit for being used to detect proteus mirabilis, it is characterised in that：The real-time fluorescence
Quantitative PCR kit includes claim 1-3 any one of them specific primer and probe.
5. real-time fluorescence quantitative PCR kit according to claim 4, it is characterised in that：In 20 μ l PCR reaction systems
In, the dosage of the sense primer is 0.2 μ l, and the dosage of the downstream primer is 0.2 μ l, and the dosage of the probe is 0.2 μ l.
6. real-time fluorescence quantitative PCR kit according to claim 4 or 5, it is characterised in that：The real time fluorescent quantitative
PCR kit further includes series concentration standard items and positive control；The series concentration standard items are by proteus mirabilis
16SrDNA genes are connect with carrier, convert into competent cell induced expression, extract recombinant plasmid, recombinant plasmid is quantified
After dilute, obtain series concentration standard items.
7. real-time fluorescence quantitative PCR kit according to claim 6, it is characterised in that：The nucleotide of the standard items
Sequence is sequence 2 in sequence table.
8. any specific primers of claim 1-3 and any real time fluorescent quantitative of probe, claim 4-7
PCR kit it is following it is any in application, the application is not for the purpose of the diagnose and treat of disease：
(1) qualitatively or quantitatively detection or auxiliary detect proteus mirabilis；
(2) product of qualitative or quantitative detection or auxiliary detection proteus mirabilis is prepared.
9. it is a kind of detection or auxiliary detection measuring samples in whether the method containing proteus mirabilis, this method is not with disease
For the purpose of diagnose and treat, it is characterised in that：Respectively using series concentration standard items and sample to be tested DNA as template, using special
Property primer and probe carry out real-time fluorescence quantitative PCR, draw standard curve, pass through the Ct values of standard curve and sample to be tested judge
As a result.
10. according to the method described in claim 9, it is characterized in that：The reaction condition of the real-time fluorescence quantitative PCR is：94
DEG C pre-degeneration 2 minutes, 94 DEG C 10 seconds, 60 DEG C of 30s and collect fluorescence signal, 40 cycles.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108841985A (en) * | 2018-06-29 | 2018-11-20 | 苏州百源基因技术有限公司 | It is a kind of for detecting nucleotide sequence group, kit and the method for Fusarium solani |
| CN110257540A (en) * | 2019-07-23 | 2019-09-20 | 中国疾病预防控制中心传染病预防控制所 | Detect PCR primer, primer sets, probe and its kit and detection method of Proteus and proteus mirabilis |
| CN110551835A (en) * | 2019-10-16 | 2019-12-10 | 四川农业大学 | specific primer and method for detecting bovine proteus mirabilis |
| CN110951895A (en) * | 2019-12-24 | 2020-04-03 | 重庆市畜牧科学院 | System and method for detecting and distinguishing proteus mirabilis, proteus vulgaris and proteus pani |
| CN114317788A (en) * | 2021-12-30 | 2022-04-12 | 漳州傲农现代农业开发有限公司 | Probe primer combination for detecting swine proteus mirabilis, kit and method thereof |
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2018
- 2018-03-13 CN CN201810205796.2A patent/CN108179209A/en not_active Withdrawn
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108841985A (en) * | 2018-06-29 | 2018-11-20 | 苏州百源基因技术有限公司 | It is a kind of for detecting nucleotide sequence group, kit and the method for Fusarium solani |
| CN110257540A (en) * | 2019-07-23 | 2019-09-20 | 中国疾病预防控制中心传染病预防控制所 | Detect PCR primer, primer sets, probe and its kit and detection method of Proteus and proteus mirabilis |
| CN110551835A (en) * | 2019-10-16 | 2019-12-10 | 四川农业大学 | specific primer and method for detecting bovine proteus mirabilis |
| CN110951895A (en) * | 2019-12-24 | 2020-04-03 | 重庆市畜牧科学院 | System and method for detecting and distinguishing proteus mirabilis, proteus vulgaris and proteus pani |
| CN114317788A (en) * | 2021-12-30 | 2022-04-12 | 漳州傲农现代农业开发有限公司 | Probe primer combination for detecting swine proteus mirabilis, kit and method thereof |
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Application publication date: 20180619 |