CN101748197A - Target sequence for detection of yersinia enterocolitica and kit - Google Patents
Target sequence for detection of yersinia enterocolitica and kit Download PDFInfo
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- CN101748197A CN101748197A CN200810204456A CN200810204456A CN101748197A CN 101748197 A CN101748197 A CN 101748197A CN 200810204456 A CN200810204456 A CN 200810204456A CN 200810204456 A CN200810204456 A CN 200810204456A CN 101748197 A CN101748197 A CN 101748197A
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Abstract
The invention provides a genetic marker and a method for detection of yersinia enterocolitica, in particular a nucleotide sequence for detection of the ail gene of the yersinia enterocolitica, oligonucleotide primers and a probe. The oligonucleotide primers and the probe are designed based on the nucleotide sequence. The invention also provides a method for rapid and specific detection of the yersinia enterocolitica with the primers. Moreover, the invention can detect the yersinia enterocolitica conveniently, rapidly, accurately, qualitatively and quantitatively.
Description
Technical field
The invention belongs to biological technical field, particularly relate to and a kind ofly utilize the pathogenic ail gene of polymerase chain reaction (PCR) amplification, thus method of the gloomy bacterium of enterocolitis Ye Ershi and the real-time fluorescence PCR assay kit that utilizes this method to obtain in the special detection clinical sample in high sensibility ground.
Background technology
The gloomy Salmonella disease of enterocolitis Ye Ershi (yersinia enterocolitica disease) is a kind of new infectious intestinal disease that just comes into one's own the eighties, all there is discovery different continents, be main diseases kind of some country diarrhoea of Europe, more than gastro-enteritis that many regional yersinia enterocoliticas cause and the serious diarrhoea, dysentery.Except that the enteron aisle symptom, can also cause respiratory tract, cardiovascular systems, bone, reticular tissue and general disease, case fatality rate reaches more than 30% when septicemia occurring.China just found this disease in 1981, caused whole nation attention, and had carried out national survey and research, isolated pathogenic bacteria from crowd, animal and external environment respectively, proved that the yersinia enterocolitica disease is very widely in the distribution of China.
China has found 58 serotypes, and wherein 11 types are reported first in the world, and they are O:7,19, O:7,8,19, O:10,16, O:17,19, O:18,19, O:26,35, O:25,45, O:29, O:36,46, O:42,51.China is verified to have the serotype of virulence that O:3, O:9, O:5,27, O:6,30, O:7,8 are arranged to the people, O:15, O:40 is identical with external report, having the O:8 type only is that virulence is very strong abroad, and the bacterial strain that China is separated at present all lacks virulence factor.Divide from biotype in addition to have 6 types, wherein the 1A type is non-pathogenic strain, and 1B, 2,3,4,5 types are pathogenic strain.Isolated in China is biological 3 types to O:3 and O:9 serological type strain, from domestic and international various pathogenic strain, the pathogenic inV that all is positioned at of the gloomy Salmonella of enterocolitis Ye Ershi, ail, on the gene locus such as yst and virF, the ail site is relevant with the invasiveness of bacterium, and yst and the virF gene locus that is positioned on the pYv plasmid are relevant with the virulence of bacterium.
Eliminating the gloomy Salmonella of enterocolitis Ye Ershi need make great efforts in many ways, and as the development fast diagnosis method, develop new drug, carry out Study on Molecular Mechanism and vaccine research etc., it is the most urgent wherein to develop fast diagnosis method.Because the yersinia entero-colitica poor growth identifies that by culture method the gloomy Salmonella of enterocolitis Ye Ershi needs the time long, therefore, culture method is little to clinical help.In order to check out quickly the gloomy Salmonella of enterocolitis Ye Ershi in the sample, augmentation detection is carried out to the nucleic acid of the gloomy Salmonella of enterocolitis Ye Ershi in needs development and use polymerase chain reactions (PCR).
The quantitative fluorescent PCR that development in recent years is got up (Fluorescence Quantitative PCR, FQ-PCR) technology is highly sensitive with it, specificity is good, advantages such as speed is fast are used widely at aspects such as the qualitative detection of gene expression dose analysis, pathogenic agent and detection by quantitative, and have become the quantitative main method of current viral nucleic acid.Domestic also existing at present test kit listing about hepatitis B, acquired immune deficiency syndrome (AIDS), tuberculosis detection by quantitative.
For polymerase chain reaction (PCR), select the base sequence of target DNA to be amplified the most important.At the gloomy Salmonella of enterocolitis Ye Ershi, this base sequence is necessary for that the gloomy Salmonella of enterocolitis Ye Ershi is special to be had and can not be present in for human or other biological DNA genome and may take place in other species of concurrent infection.
From domestic and international various pathogenic strain, the pathogenic inV that all is positioned at of yersinia entero-colitica, ail, on the gene locus such as yst and virF, the ail site is relevant with the invasiveness of bacterium, and yst and the virF gene locus that is positioned on the pYv plasmid are relevant with the virulence of bacterium, because plasmid is easily lost in culturing process, virF is prone to false negative by PCR amplification, and the specificity in inV and vst site is not strong, therefore, this area presses for method and the test kit that exploitation newly detects yersinia entero-colitica with high specificity, so that can effectively detect yersinia entero-colitica.
Summary of the invention
Technical problem to be solved
Purpose of the present invention just provides a kind of method and test kit of effective detection yersinia entero-colitica.
In a first aspect of the present invention, a kind of genetic marker that detects the gloomy bacterium of enterocolitis Ye Ershi is provided, it has 75～537 Nucleotide of successive among the SEQ ID NO:1.
In another preference, described genetic marker has the 1st～150 Nucleotide among the SEQ ID NO:2.
In a second aspect of the present invention, a kind of test kit that detects the gloomy bacterium of enterocolitis Ye Ershi is provided, it is right that it contains the primer of the gloomy bacterium genetic marker of specific amplification enterocolitis Ye Ershi, described primer length is 15～30 Nucleotide, and the sequence of a primer is identical with the sequence shown in the SEQ ID NO:1, and the sequence complementation shown in the sequence of another primer and the SEQ ID NO:1, and amplified production length is 75～537 Nucleotide.
In another preference, described test kit also contains probe, and the length of described probe is 18～30 Nucleotide, and the sequence of this probe is identical or complementary with the sequence shown in the SEQ ID NO:1.
In another preference, a primer of described primer centering is selected from down group: SEQ ID NO:3 or 6; Another primer is selected from down group: SEQ ID NO:4 or 7; And described probe is the TaqMan probe, and its sequence is selected from SEQ ID NO:5 or 8.
In another preference, this test kit also comprises a) nucleic acid extraction liquid, b) PCR damping fluid, c) quantitative calibration object, d) positive control, e) negative control.
In a third aspect of the present invention, the method for the gloomy bacterium of enterocolitis Ye Ershi in a kind of test sample is provided, it comprises operation steps:
1. sample DNA extracts;
2. with the DNA that extracts as template, with the primer of the gloomy bacterium genetic marker of specific amplification enterocolitis Ye Ershi to increasing, wherein said primer length is 15～30 Nucleotide, the sequence of a primer is identical with the sequence shown in the SEQ ID NO:1, and the sequence complementation shown in the sequence of another primer and the SEQ ID NO:1.
3. detect amplified production and whether exist, exist amplified production to represent that there is the gloomy bacterium of enterocolitis Ye Ershi in sample.
In another preference, 3. described step is to come the gloomy bacterium of enterocolitis Ye Ershi in the detection by quantitative sample by the power that detects fluorescent signal.
In a fourth aspect of the present invention, a kind of purposes of pathogenic ail gene is provided, it is characterized in that it is used as the genetic marker whether gloomy bacterium of vitro detection enterocolitis Ye Ershi exists.
In another preference, pathogenic ail gene has the nucleotide sequence shown in the SEQ ID NO:1.
Embodiment
The inventor is extensive studies through going deep into, the gene of one section about 500 base pair from the gloomy bacterium of enterocolitis Ye Ershi, finding-pathogenic ail gene (S.Thisted Lambertz, C.Nilsson, S.Hallanvuo, and M.Lindblad.Real-Time PCR Method for Detection of Pathogenic Yersinia enterocolitiea inFood.Applied and Environmental Microbiology, October 2008, p.6060-6067, Vol.74, No.19) the suitable extension increasing sequence of middle discovery.This gene order is the gloomy bacterium pathogenic strain of an enterocolitis Ye Ershi functions peculiar gene, and the ail gene locus is relevant with the invasiveness of bacterium, therefore, selects this ad hoc structure, functioning gene sequence as primer, has good characteristic.
As used herein, the nucleotide sequence of ail locus gene, its GenBank accession number AJ605740, concrete sequence is shown in SEQ ID NO:1.
Can in the gloomy bacterium pathogenic strain of enterocolitis Ye Ershi, expand according to the primer of ail gene design and corresponding D NA fragment, and, this primer does not amplify respective segments on other relevant bacterial strains, illustrate with the ail gene order to be that the PCR reactive system of basic design has good specificity.
Though relatively by homology, a plurality of zones are arranged as suitable specificity marker zone in the full-length gene of ail gene, yet the nucleotide sequence shown in the SEQ ID NO:2 is a kind of particularly preferred sequence that is suitable as the gloomy bacterium specificity of enterocolitis Ye Ershi genetic marker.
Therefore, based on SEQ ID NO:1 and 2, the invention provides a kind of method of utilizing the gloomy bacterium of polymerase chain reaction (PCR) amplification ail gene test enterocolitis Ye Ershi; Also provide a kind of fast quantification to detect the PCR kit for fluorescence quantitative of the gloomy bacterium of enterocolitis Ye Ershi.Key wherein has been to use the primer of the gloomy bacterium genetic marker of specific amplification enterocolitis Ye Ershi right, described primer length is 15～30 Nucleotide, and a primer sequence is identical with the sequence shown in the SEQ ID NO:1, the sequence of another primer and SEQ ID NO:1 complementation, and the length of amplified production is 75～537 Nucleotide.For example, SEQ ID NO:3 is identical with the sequence shown in the SEQ ID NO:1, and the sequence complementation shown in another primer SEQ ID NO:4 and the SEQ IDNO:1, and the length of amplified production is 150bp.SEQ ID NO:3 and 4 corresponds respectively to 2389 and 2518 positions of SEQ IDNO:1.
The probe of primer of the gloomy bacterium of specific amplification enterocolitis Ye Ershi of the present invention and detection usefulness, can design according to the sequence (SEQ ID NO:1 or 2) of the genetic marker of inventing, and obtain (as can be synthetic) with business-like automatic dna synthesizer by conventional DNA synthetic method.
These primers of the present invention can carry out mark with radio isotope, vitamin H, enzyme, fluorescein or other chemiluminescent substance.
The gloomy bacterium specificity of enterocolitis Ye Ershi of the present invention nucleic acid molecule primer has fabulous specific specificity.Carry out conventional PCR reaction with primer of the present invention, the result can amplify size and be 150bp specific PCR product from the DNA extraction thing of the material that contains the gloomy bacterium of enterocolitis Ye Ershi.Therefore, carry out conventional PCR reaction with primer of the present invention, and by judge having or not of corresponding big or small PCR product can be accurately, the gloomy bacterium of rapid detection enterocolitis Ye Ershi, and required sample size is considerably less.
In order to reduce false positive, also can hybridize with the gloomy bacterium specific probe of enterocolitis Ye Ershi amplified production.A kind of preferred probes is the TaqMan probe, it can be in PCR reaction directly whether and the height of quantity the existence by fluorescent signal reflection amplified production in real time.
Probe of the present invention can carry out mark with radio isotope, vitamin H, enzyme, fluorescein or other chemiluminescent substance.5 ' end report fluorophor such as Fluoresceincarboxylic acid (Carboxyfluorescein with this sequence, mark such as FAM), 3 ' end reacts with promptly can be used for the PCR that the gloomy bacterium of enterocolitis Ye Ershi carries out qualitative and quantitative analysis behind the marks such as quenching group such as TAMRA or Dabcyl.
Though the complete complementation of primer and probe and template sequence is preferred, but those skilled in the art will know that, there be under the situation of certain not complementary (especially 5 ' of primer end) also can increase specifically (promptly only amplifying required fragment) at primer and template.The method that contains the test kit of these primers and use these primers is all within the scope of the invention, as long as the product that this primer amplification goes out contains fragment of the present invention.
Though the length of amplified production is not particularly limited, the length of amplified production is 70～1000bp usually, preferably is 75～500bp, more preferably is 100～300bp.
Primer of the present invention is combined with the TaqMan probe technique, just obtained of the present inventionly simultaneously the gloomy bacterium of enterocolitis Ye Ershi being carried out qualitative detection and quantitative counting analytical procedure, this analytical procedure has not only overcome the error in the conventional quantifying PCR method, and it is few to analyze required sample size, detection limit is low, and is highly sensitive.
In a preference, a kind of fast quantification detects the fluorescent PCR kit of the gloomy bacterium of enterocolitis Ye Ershi, and this test kit comprises a) dna cleavage liquid, b) fluorescent quantitation reaction solution, and c) quantitative calibration object, d) positive reference substance, e) negative control product is characterized in that:
1) the fluorescent quantitation reaction solution contains PCR damping fluid, dNTPs solution, Taq archaeal dna polymerase, aseptic double-distilled water, MgCl
2Solution, (as primer sequence is SEQ ID NO:3 and SEQ ID NO:4 for the primer of specific amplification ail gene genetic marker and fluorescent probe, the fluorescent probe sequence is SEQ ID NO:5), the fluorescence report group of fluorescent probe 5 ' end mark is FAM, and the fluorescent quenching group of 3 ' end mark is TAMRA.
2) quantitatively calibration object is to contain SEQ ID NO:2 totally 150 pGEM-T carriers that nucleotide fragments constitutes, and this carrier can be bred in intestinal bacteria.Positive reference substance is the positive culture of the gloomy bacterium of enterocolitis Ye Ershi.
In a preferred version of the present invention, the fluorescent quantitation reaction solution comprises 50mM KCl, 10mM Tris-HClPH8.3 (25 ℃), 2mM MgCl
2, 3% methane amide, each 3pmol of forward primer and reverse primer, fluorescent probe 2pmol, 0.2mM dNTPs, the Taq enzyme of aseptic double-distilled water and 1.5 units (Roche, Switzerland).The nucleotide sequence shown in primer SEQ ID NO:3 and the SEQ ID NO:4 wherein, the nucleotide sequence shown in the fluorescent probe SEQ ID NO:5, the fluorescence report group of fluorescent probe 5 ' end mark is FAM, the fluorescent quenching group of 3 ' end mark is TAMRA.
In a preferred version of the present invention, quantitatively calibration object is to contain SEQ ID NO:2 to have the pGEM-T carrier that 150 nucleotide fragments constitute, and this carrier can be bred in intestinal bacteria.Storing concentration is 5 * 10
9Copies/ μ l uses preceding 10 times of gradient dilutions.After containing the segmental plasmid transformation escherichia coli propagation of purpose, use alkaline lysis method of extracting, through DNA purification kit purifying, with spectrophotometric instrumentation A
260Quantitatively and be diluted to 5 * 10
9Copies/ μ l ,-20 ℃ of preservations.
In a priority scheme of the present invention, dna cleavage liquid comprises 10mM Tris-HCl pH 8.0,0.05mM EDTA, 1%NP-40,1%Triton, 0.05M NaCl.
In the invention provides the PCR kit for fluorescence quantitative that detects the gloomy bacterium of enterocolitis Ye Ershi, there are two ends to be marked with the specific probe of fluorophor, when probe is complete, two groups distance on space structure is close, the fluorescence that 5 ' end reporter group produces is FRET (fluorescence resonance energy transfer) (FRET) and by the group cancellation of going out of 3 ' end quenching, thus in the system detection less than fluorescence.In PCR annealing and extension process, probe and template specificity combination, extension along with primer, the TaqDNA polysaccharase utilizes it at 5 ' → 3 ' 5 prime excision enzyme activity fluorescent probe cutting to be discharged reporter group, destroyed the FRET between the group like this, the fluorescence that reporter group discharged can be built in the fluoroscopic examination in the quantitative PCR instrument, the proportional example relation of the accumulation volume of the increase of fluorescence volume and PCR product, to the gloomy bacterium of enterocolitis Ye Ershi quantitatively can by with the cycle threshold (Ct of quantitative calibration object, Threshold Cycle) compares and draw, the Ct value is in the PCR process, and the accumulation of fluorescence volume surpasses substrate fluorescence volume cycle number.The Ct value is big more, and the starting template number is few more.Utilize the Ct value of the quantitative calibration object of gradient to make typical curve, again according to the initial copy number that can accurately measure this sample for the Ct value of test sample product.
The PCR kit for fluorescence quantitative of the gloomy bacterium of detection enterocolitis Ye Ershi provided by the invention, process is to each component, as primer concentration, fluorescent probe concentration, Mg
2+Optimization such as concentration, annealing temperature (comprises round pcr and real-time fluorescence PCR system PE7000/7300/7500, LightCycler) combines.By prioritization scheme, experiment repeatedly, test kit can satisfy the actual requirement of the gloomy bacterium of quick special detection enterocolitis Ye Ershi fully.
The present invention also provides a kind of method of utilizing test kit of the present invention to detect the gloomy bacterium of enterocolitis Ye Ershi, and this method comprises following detection step:
I. from positive reference substance and basis such as test sample such as grade, extract DNA with dna cleavage liquid;
Ii. get respectively to go on foot in the quantitative calibration object adding of DNA, the gradient the extracted fluorescent reaction liquid and carry out augmentation detection with the real-time fluorescence PCR instrument;
Iii. the initial copy number to testing sample carries out quantitatively by the cycle threshold that compares sample to be tested and standard substance.
The present invention compares with existing culture method commonly used has following major advantage and effect:
A) detection speed is fast, adds the extraction of DNA, finishes in 2～3 hours;
B) existence of fluorescent probe has guaranteed the specificity and the sensitivity that detect;
C) simple to operate;
D) quantitatively accurately;
E) can carry out the sample detection that height passes through simultaneously.
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.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Hardor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1 separates the gloomy bacterium DNA of enterocolitis Ye Ershi that is used for pcr analysis
To be used for the gloomy bacterium DNA of enterocolitis Ye Ershi that the polymerase chain reaction is detected in order from sample to be tested, separating, must use not influence the simple method of polymerase chain reaction.At first, with sterilization physiology suspension clinical sample (as ight soil etc.), with rinsing liquid 15,000rpm abandons supernatant after centrifugal 15 minutes, may contain the gloomy bacterium of enterocolitis Ye Ershi in the throw out; After adding dna cleavage liquid in the precipitation, vibration mixing, 100 ℃ of boiling water baths 15 minutes, the gloomy bacterium released dna of cracking enterocolitis Ye Ershi.Last 15, centrifugal 15 minutes of 000rpm, DNA is dissolved in the supernatant liquor, gets supernatant liquor as the gloomy bacterium DNA of enterocolitis Ye Ershi that is used for pcr analysis.
The preparation of embodiment 2 detection kit
Prepare the fluorescent PCR kit that a kind of fast quantification detects the gloomy bacterium of enterocolitis Ye Ershi with ordinary method, this test kit comprises a) dna cleavage liquid, b) fluorescent quantitation reaction solution, c) quantitative calibration object, d) positive reference substance, e) negative control product.Wherein:
1) the fluorescent quantitation reaction solution contains PCR damping fluid, dNTPs solution, Taq archaeal dna polymerase, aseptic double-distilled water, MgCl
2Solution, the primer sequence of specific amplification ail gene genetic marker is SEQ ID NO:3 and 4, and the fluorescent probe sequence is SEQ ID NO:5, and the fluorescence report group of fluorescent probe 5 ' end mark is FAM, and the fluorescent quenching group of 3 ' end mark is TAMRA.(the amplified production size is 150bp)
Particularly, the fluorescent quantitation reaction solution comprises 50mM KCl, 10mM Tris-HCl PH8.3 (25 ℃), 2mM MgCl
2, 3% methane amide, each 3pmol of forward primer and reverse primer, fluorescent probe 2pmol, 0.2mM dNTPs, the Taq enzyme of aseptic double-distilled water and 1.5 units (Roche, Switzerland).
2) quantitatively calibration object is to contain SEQ ID NO:2 to have the pGEM-T carrier (this carrier is available from Promego company, and SEQ ID NO:2 inserts the β-Nei Xiananmei coding region) that 150 nucleotide fragments constitute, and this carrier can be bred in intestinal bacteria.
Particularly, quantitatively calibration object is 5 * 10 storing concentration
9Copies/ μ l uses preceding 10 times of gradient dilutions.After containing the segmental plasmid transformation escherichia coli propagation of purpose, use alkaline lysis method of extracting, through DNA purification kit purifying, with spectrophotometric instrumentation A
260Quantitatively and be diluted to 5 * 10
9Copies/ μ l ,-20 ℃ of preservations.
3) dna cleavage liquid comprises 10mM Tris-HCl pH 8.0,0.05mM EDTA, 1%NP-40,1%Triton, 0.05M NaCl.
The gloomy bacterium of embodiment 3 usefulness enterocolitis Ye Ershi carries out the polymerase chain reaction
Each the 26 μ l of fluorescent reaction liquid that get respectively among the embodiment 2 join in the different PCR reaction tubess, the DNA and the quantitative calibration object of gained among the embodiment 1 are added 4 μ l in the fluorescent quantitation reaction solution, cumulative volume 30 μ l begin augmentation detection on real-time fluorescence quantitative PCR instrument (ABI 7500).The amplification parameter be set to 50 ℃ 2 minutes, 94 ℃ 5 minutes, by 94 ℃ 10 seconds → 60 ℃ 45 seconds the circulation 40 times.At each round-robin annealing steps as a result the time, the detection wavelength is 530nm the programdesign of fluoroscopic examination.
After the loop ends, utilization instrument software kit reads sample copy number to be checked.The result is: quantitatively accurate product 5 * 10
6Copies/ μ l, 5 * 10
5Copies/ μ l, 5 * 10
422.13,25.65,29.08 of copies/ μ l; The Ct value scope of sample to be tested is 21～36, is 8.26 * 10 through the typical curve quantitative scope that converts
6Copies/ μ l～1.18 * 10
1Copies/ μ l.Concrete outcome such as following table:
| Sample to be tested | The Ct value | Virus quantity (copies/ μ l) |
| ??1 | ??31.13 | ??5.72×10 3 |
| ??2 | ??26.38 | ??2.51×10 5 |
| ??3 | ??30.96 | ??9.37×10 3 |
| ??4 | ??21.25 | ??8.26×10 6 |
| ??5 | ??36.12 | ??1.18×10 1 |
| ??6 | ??24.98 | ??7.51×10 5 |
| ??7 | ??33.47 | ??2.64×10 2 |
This shows that test kit of the present invention can be 8.26 * 10
6Copies/ μ l～1.18 * 10
1Extremely sensitivity amplify the gloomy bacterium of enterocolitis Ye Ershi in the vast scope of copies/ μ l.
The various pathogenic agent of embodiment 4 usefulness are carried out the polymerase chain reaction
Carry out augmentation detection with embodiment 1～3 identical method, difference is that the reaction template of present embodiment chooses the DNA of other close all kinds of pathogenic agent, as Chlamydia pneumoniae, mycoplasma genitalium, streptococcus pneumoniae, streptococcus aureus, human leukocyte DNA.
Detected result such as following table:
| The template source | The Ct value |
| Chlamydia pneumoniae | ??- |
| The template source | The Ct value |
| Mycoplasma genitalium | ??- |
| Streptococcus pneumoniae | ??- |
| Streptococcus aureus | ??- |
| Human leukocyte DNA | ??- |
As mentioned above, present embodiment can amplify the gloomy bacterium of enterocolitis Ye Ershi specifically to other close pathogen gene no cross reactions.
Therefore, the present invention has disclosed the advantage of the amplification ail gloomy bacterium of gene test enterocolitis Ye Ershi aspect susceptibility, specificity for the first time, and a kind of quick on this basis special detection enterocolitis Ye Ershi is provided the PCR kit for fluorescence quantitative of gloomy bacterium simultaneously.This test kit only needs 2～3 hours to sensitivity, high specificity, the good reproducibility of the gloomy bacterium of enterocolitis Ye Ershi, has shortened detection time greatly.The entire operation process only needs one, once can detect the individual sample of 32～96 (decisions of real-time fluorescence PCR instrument model), has reduced waste of manpower resource.
The various pathogenic agent of embodiment 5 usefulness are carried out the polymerase chain reaction
Repeat the same procedure of embodiment 1～4, difference only is to replace the primer shown in the SEQID NO:3 and 4 with the primer shown in the SEQ ID NO:6 and 7, replaces the probe shown in the SEQ ID NO:5 with the probe shown in the SEQ ID NO:8.
Detected result shows that this primer is to amplifying the gloomy bacterium of enterocolitis Ye Ershi specifically equally with probe.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.In addition, should be understood that those skilled in the art can make various changes or modifications the present invention after pronunciation has been read above-mentioned teachings of the present invention, these equivalent form of values fall within application appended claims restricted portion equally.
The nucleotides sequence tabulation
SEQUENCE?LISTING
＜110〉Shanghai Fosun Pharmaceutical (Group) Co., Ltd.
Fuxing Medical Science-Technology Development Co., Ltd., Shanghai
＜120〉a kind of target sequence and test kit that is used to detect Yersinia enterocolitica
<130>05
<160>8
<170>PatentIn?version?3.3
<210>1
<211>3865
<212>DNA
<213>Yersinia?enterocolitica
<220>
<221>SEQ?ID?NO：1
<222>(1)..(3865)
<400>1
gcgtaaaaag?ttttcccgca?ctcagctgat?tcattttctc?ggttcctgtc?aggcggcgac????60
cgtggtgatg?gaggcctgca?gcggtgcgca?atttatggct?cgccagattg?ctgatttggg????120
ccatgagccg?aagttgattt?atccgcagtt?cgtccgccct?tttgttaaga?gtaacaagaa????180
cgattttgtc?gatgcggaag?ctatctgcga?ggccgcttcc?cggccctcta?tgcgttttgt????240
aaaaccccgc?aggcaatggc?cgccctgcat?cgggtcaggg?actcgttgat?acggcaacgc????300
gttaaaacca?ccaaccagat?gcacgctttt?ttgctggagt?tcagcgtcag?cctgccgcgc????360
ggtattgccg?tgatacggcg?tctgagcgct?gtgctggaag?aaaacgactt?cccgccttat????420
cttgcaaggg?ttctcagcag?gcttcaccgc?cattatgttt?acctctgcgg?ggaaattgat????480
gaggtcgata?aggaactgaa?aacccacctt?gcagaggatg?aaaacgcaca?gcgcctgttg????540
accattcccg?gcgtcgggcc?ggtaacggcg?agtctgttag?ccacgcggct?gggtgacgga????600
aaaaactatg?ccagcagtcg?tgactttgcg?gcgtcgacag?gtctggttcc?tcgccagtac?????660
aagcacaggt?ggaaaaacaa?cgctgatggg?catcagcaag?cgaggtgata?aaaatctgcg?????720
gcgacttctg?gtacagtgtg?cacgggtctt?tatacagcgg?ctggagcaca?atccagggcg?????780
tctggcagag?tgggttagaa?agcagcttgc?caaccatcat?tcaaacgtgg?tggcgtgcgc?????840
actggcaaac?aaactggccc?gcatagcctg?ggcagtgacg?gcacatcagg?cggcgttcag?????900
tcaataaaag?agccaacgaa?aaactcgtca?gctcagttaa?ttaacacgtt?actttctggt?????960
tttgcgacga?cagataattg?atgacatgaa?cggcatatcg?gcctgctgat?gaacctgaca????1020
taaaaaatgg?ctctctgaag?ccgggcggct?ttttaaggtt?cggcaggcgc?ggaactcatc????1080
gtggcgcggg?cattaatgct?caccagacgc?cgaatagatt?tacgcaagcc?catcataaat????1140
caaaatctgc?gttgcaaaaa?cgggggtgac?catagatttt?atggcagtaa?atcaccgcgc????1200
tctcaccgcg?cattttaaat?ccgagaccat?tcacaaaagt?gacctctgag?aacgccatcg????1260
cagcatggtg?cgctcgggca?tggtcgttct?ggtgagcaga?ggtctctttt?gagggtaatt????1320
accatgcaat?atccaacagt?atctgtaaac?ggagtctctg?ttcgtattga?cgaagagggg????1380
cgttacaacc?tcaacgatct?gcacgctgcc?gctgtagcta?atggagaagc?aacggagtca????1440
caaagaccga?gtgtatttct?tcggagtgcg?cagataaaac?gctttgtaaa?agcgcttgag????1500
gtcaaagcac?aaaaaaagtt?acttgtaaac?aaatcaatca?cttagggtta?ttaaaggtgg????1560
tgatgagctt?ggagtatggg?gtgtcgaatt?gttggctatc?cgatatgcag?catgaattaa????1620
gccggagttt?gaaatcgaag?tttacgacgt?tttcagaaca?gtcgtccgtc?ttcgggtgag????1680
cgccatgtct?cacctgaaca?aactggatca?catcatcaac?accgaaacca?aagagataag????1740
ccagtacgcc?agaaaaatgg?ctcgatgggg?tgttggggga?aggaagcaaa?tattgcttat????1800
ggcacgaaaa?cgtatgatat?atgagattca?gatgtatttg?ccggggctgg?gaagtaacgc????1860
gggtaattac?tgtgacattc?agtaataaaa?tagagcctct?attaaaggag?cttcccgatt????1920
tgaaatcgga?aaaattacat?cataaacatg?ggtgtccaga?agtcagttat?cggcgatata????1980
tccatttaaa?gagtattgag?ctatgaccag?tattcatcaa?taactacaga?acaaaaatac????2040
aggaataagc?gactgatggg?ataaagctga?ggtaagctca?tggtactgta?tcaatatcca????2100
tatttacata?tgtatcatgg?acttggcatt?atatcactcg?ccgtgtcagt?gatatggtta????2160
ttgtattagt?attgttataa?caattagggt?gattttgatg?aaaaagacat?tactaactag????2220
ttctttaata?gcctgtttat?caattgcgtc?tgttaatgtg?tacgctgcga?gtgaaaatag????2280
tatttctatt?ggttatgcac?aaagccatgt?aaaagaaaat?gggtatacat?tggataatga????2340
ccctaaaggt?tttaacctga?agtaccgtta?tgaactcgat?gataactggg?gagtaatagg????2400
ttcgtttgct?tatacccatc?agggatacga?tttcttctat?ggcagtaata?agtttggtca????2460
cggtgatctt?gattactatt?cagtaacaat?ggggccatct?ttccgcatta?acgaatatgt????2520
tagcctttat?ggattactgg?gtgctgctca?cggaaaggtt?aagtcatctg?tatttgatgg????2580
gtcagtcagt?acaagtaaga?cgtcaatggc?atacggggca?ggggtgcaat?tcaacccgct????2640
tccaaatttt?gtcattgacg?cttcatatga?atactccaaa?ctcgatagcg?taaaatttgg????2700
cacttggatg?cttggtgcag?ggtatcgatt?ctaattatct?cagatagtga?aaacccacct????2760
aaatggaggg?ttcagacggc?tgacaaagtc?ctgacccttg?ggccgggact?ttgatataat????2820
agggacaggc?taaaaaacga?gcttatccct?gcgctaaaag?aatctgctcc?gcaacaatat????2880
caactcgaaa?tggtcaccct?cgaagagctg?gtgccgcaac?atcatctggt?gcgcaaagtc????2940
gatgccgcca?tagattttga?atttatccgc?gatgccgtcg?ctcacctcta?ttgtcctgac????3000
aatggccgtc?cggccgtcga?ccctgtccgc?ctgattaaaa?tgatgctacc?cggcggccgg????3060
gtgctctaca?ccgacagcac?gcatctcaag?gccagtgcca?acccgcgtaa?agccaaaaac????3120
attccccagc?cggtcaaagc?cagtgcttac?attgattcgc?tgaatgcggc?gattgatgaa????3180
gaccgggccg?ctgccggaaa?aaagccgcta?acgcccgcga?cgacggcgaa?aatgaaggac????3240
accaaagtca?gcaccactga?cccggaaagc?ggttttatgc?accgggataa?caagccgaaa????3300
ggcttcttct?ggctcgacca?tcgcacagag?gatggcaaat?acggaattat?cactgacacc????3360
tatactaccc?cccgtaatgt?ccatgacagc?cagccctata?tcgcccggct?ggagcgccag????3420
ttaagccgtt?ttaaactcaa?ccccattgcg?gtcgggctgg?acacgggtta?cttcaccgcc????3480
ccggtctgtc?acatgacaga?acagattgat?attattgcgg?ttatgggtta?ccggtggcca????3540
aataaaggcc?aaaatgtact?gcaaaaaaac?acttcattta?cgatgataaa?caggatgttt????3600
atcaatgccc?acaagggcag?caacttatct?acaaaaccac?aagccgggaa?ggctatcggc????3660
actatcactc?ttcatctgaa?atctgtgggc?aatgcccgca?cttaattaac?tgtacacaaa????3720
gcaaaaacag?ccaaaaagtc?gtgacgaaga?aatgtgtggg?aggggagcaa?agagcgggca????3780
aatcagactc?ggctgacggc?atggggcaag?aaagtttacg?cgaggcgaaa?agagacggtg????3840
gaaggagctt?cgcggatgca?aaaca??????????????????????????????????????????3865
<210>2
<211>150
<212>DNA
<213>Yersinia?enterocolitica
<220>
<221>SEQ?ID?NO：2
<222>(1)..(150)
<400>2
gggagtaata?ggttcgtttg?cttataccca?tcagggatac?gatttcttct?atggcagtaa?????60
taagtttggt?cacggtgatc?ttgattacta?ttcagtaaca?atggggccat?ctttccgcat????120
taacgaatat?gttagccttt?atggattact?????????????????????????????????????150
<210>3
<211>20
<212>DNA
<213>Yersinia?enterocolitica
<220>
<221>SEQ?ID?NO：3
<222>(1)..(20)
<400>3
gggagtaata?ggttcgtttg?????????????????????????????????????????????????20
<210>4
<211>21
<212>DNA
<213>Yersinia?enterocolitica
<220>
<221>SEQ?ID?NO：4
<222>(1)..(21)
<400>4
agtaatccat?aaaggctaac?a????????????????????????????????????????????21
<210>5
<211>24
<212>DNA
<213>Yersinia?enterocolitica
<220>
<221>SEQ?ID?NO：5
<222>(1)..(24)
<223>“n＝FAM”，“y＝TAMRA”
<220>
<221>misc_feature
<222>(1)..(1)
<400>5
nacaatgggg?ccatctttcc?gcay?????????????????????????????????????????24
<210>6
<211>20
<212>DNA
<213>Yersinia?enterocolitica
<220>
<221>SEQ?ID?NO：1
<222>(1)..(20)
<400>6
ctggggagta?ataggttcgt????????????????????????????????????????????????20
<210>7
<211>20
<212>DNA
<213>Yersinia?enterocolitica
<220>
<221>SEQ?ID?NO：7
<222>(1)..(20)
<400>7
cccagtaatc?cataaaggct????????????????????????????????????????????????20
<210>8
<211>24
<212>DNA
<213>Yersinia?enterocolitica
<220>
<221>SEQ?ID?NO：8
<222>(1)..(24)
<223>“n＝FAM”，“y＝TAMRA”
<220>
<221>misc_feature
<222>(1)..(1)
<400>8
ntggtcacgg?tgatcttgat?tacy???????????????????????????????????????????24
Claims (10)
1. a genetic marker that detects the gloomy bacterium of enterocolitis Ye Ershi is characterized in that, it has 75～537 Nucleotide of successive among the SEQ ID NO:1.
2. genetic marker according to claim 1 is characterized in that, it has the 1st～150 Nucleotide among the SEQ ID NO:2.
3. test kit that detects the gloomy bacterium of enterocolitis Ye Ershi, it is characterized in that, it is right that it contains the primer of the gloomy bacterium genetic marker of specific amplification enterocolitis Ye Ershi, described primer length is 15～30 Nucleotide, and the sequence of a primer is identical with the sequence shown in the SEQ ID NO:1, and the sequence complementation shown in the sequence of another primer and the SEQ ID NO:1, and amplified production length is 75～537 Nucleotide.
4. test kit according to claim 3 is characterized in that, also contains probe, and the length of described probe is 18～30 Nucleotide, and the sequence of this probe is identical or complementary with the sequence shown in the SEQ ID NO:1.
5. test kit according to claim 3 is characterized in that, a primer of described primer centering is selected from down group: SEQID NO:3 or 6; Another primer is selected from down group: SEQ ID NO:4 or 7; And described probe is the TaqMan probe, and its sequence is selected from SEQ ID NO:5 or 8.
6. test kit according to claim 3 is characterized in that, this test kit also comprises a) nucleic acid extraction liquid, b) PCR damping fluid, c) quantitative calibration object, d) positive control, e) negative control.
7. the method for the gloomy bacterium of enterocolitis Ye Ershi in the test sample is characterized in that it comprises operation steps:
1. sample DNA extracts;
2. with the DNA that extracts as template, with the primer of the gloomy bacterium genetic marker of specific amplification enterocolitis Ye Ershi to increasing, wherein said primer length is 15～30 Nucleotide, the sequence of a primer is identical with the sequence shown in the SEQ IDNO:1, and the sequence complementation shown in the sequence of another primer and the SEQ ID NO:1.
3. detect amplified production and whether exist, exist amplified production to represent that there is the gloomy bacterium of enterocolitis Ye Ershi in sample.
8. method according to claim 7 is characterized in that, 3. described step is to come the gloomy bacterium of enterocolitis Ye Ershi in the detection by quantitative sample by the power that detects fluorescent signal.
9. the purposes of a pathogenic ail gene is characterized in that, it is used as the genetic marker whether gloomy bacterium of vitro detection enterocolitis Ye Ershi exists.
10. purposes according to claim 9 is characterized in that, pathogenic ail gene has the nucleotide sequence shown in the SEQ ID NO:1.
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| CN200810204456A CN101748197A (en) | 2008-12-12 | 2008-12-12 | Target sequence for detection of yersinia enterocolitica and kit |
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|---|---|---|---|
| CN200810204456A CN101748197A (en) | 2008-12-12 | 2008-12-12 | Target sequence for detection of yersinia enterocolitica and kit |
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| CN200810204456A Pending CN101748197A (en) | 2008-12-12 | 2008-12-12 | Target sequence for detection of yersinia enterocolitica and kit |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434987A (en) * | 2016-11-29 | 2017-02-22 | 百奥森（江苏）食品安全科技有限公司 | Quick detection method for Yersinia enterocolitica |
| CN106755497A (en) * | 2017-01-23 | 2017-05-31 | 北京康宝利华生物科技有限公司 | A kind of detection method of yersinia enterocolitica and its application |
| CN110872632A (en) * | 2018-08-30 | 2020-03-10 | 深圳华大生命科学研究院 | Specific gene sequence of streptococcus pharyngolaris, detection primer and application thereof |
| CN110923349A (en) * | 2019-12-30 | 2020-03-27 | 广东省微生物研究所（广东省微生物分析检测中心） | Species-specific detection molecular tags 3283 and 3316 of yersinia enterocolitica and rapid detection method thereof |
| CN113755612A (en) * | 2021-04-14 | 2021-12-07 | 吉林大学 | Specific primer pair for identifying pathogenic yersinia enterocolitica, detection kit and application thereof |
| CN115261494A (en) * | 2021-04-30 | 2022-11-01 | 上海旺旺食品集团有限公司 | System and method for detecting yersinia enterocolitica and application of system and method |
-
2008
- 2008-12-12 CN CN200810204456A patent/CN101748197A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434987A (en) * | 2016-11-29 | 2017-02-22 | 百奥森（江苏）食品安全科技有限公司 | Quick detection method for Yersinia enterocolitica |
| CN106755497A (en) * | 2017-01-23 | 2017-05-31 | 北京康宝利华生物科技有限公司 | A kind of detection method of yersinia enterocolitica and its application |
| CN106755497B (en) * | 2017-01-23 | 2019-03-12 | 江西华牧生物科技有限公司 | A kind of detection method and its application of yersinia enterocolitica |
| CN110872632A (en) * | 2018-08-30 | 2020-03-10 | 深圳华大生命科学研究院 | Specific gene sequence of streptococcus pharyngolaris, detection primer and application thereof |
| CN110923349A (en) * | 2019-12-30 | 2020-03-27 | 广东省微生物研究所（广东省微生物分析检测中心） | Species-specific detection molecular tags 3283 and 3316 of yersinia enterocolitica and rapid detection method thereof |
| CN113755612A (en) * | 2021-04-14 | 2021-12-07 | 吉林大学 | Specific primer pair for identifying pathogenic yersinia enterocolitica, detection kit and application thereof |
| CN115261494A (en) * | 2021-04-30 | 2022-11-01 | 上海旺旺食品集团有限公司 | System and method for detecting yersinia enterocolitica and application of system and method |
| CN115261494B (en) * | 2021-04-30 | 2024-09-17 | 上海旺旺食品集团有限公司 | System, method and application for detecting yersinia enterocolitica |
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Open date: 20100623 |