CN102329864A - Fluorescence PCR (polymerase chain reaction) kit for detecting yersinia enterocolitica - Google Patents
Fluorescence PCR (polymerase chain reaction) kit for detecting yersinia enterocolitica Download PDFInfo
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Abstract
The invention relates to a fluorescence PCR (polymerase chain reaction) kit for detecting yersinia enterocolitica, reagents in the kit comprise a front primer, a rear primer, a TaqMan probe, a fluorescence PCR premixed solution, an ROX (roxithromycin) solution, pure water and a positive standard solution, wherein the front primer, the rear primer and the TaqMan probe are DNA (deoxyribonucleic acid) fragments of 20-21 bases, which are synthesized according to a specific and conserved gene nucleotide sequence of the yersinia enterocolitica and can be specifically combined with the yersinia enterocolitica. The invention further provides a method for detecting the yersinia enterocolitica, and the method comprises the following steps: performing pretreatment on samples; using the reagents of the kit for detection; and analyzing the detection result. The kit is simple, fast, sensitive, accurate and suitable for detecting a large number of samples.
Description
Technical field
The present invention relates to real-time fluorescence PCR check and analysis technical field, related in particular to a kind of real-time fluorescence PCR reagent and method that detects yersinia entero-colitica.
Background technology
Yersinia entero-colitica can cause the people and with other multiple animal diseases such as gastro-enteritis, sacroiliitis, septicemia and erythema nodosum take place, and is very important infecting both domestic animals and human disease pathogen.This bacterium is very extensive in the distribution of China, can isolate pathogenic bacteria from crowd, animal, external environment and food.This disease and people's daily life are in close relations, and epidemiological study shows, yersinia entero-colitica can be at growth and breeding under the refrigerating temperature, so the refrigerator important contagium that is yersinia enterocolitis is commonly called as " refrigerator bacterium ".
Traditional isolation and identification method check yersinia entero-colitica, therefrom temperature increases bacterium approximately needs time in 1 week to the serotype of confirming bacterium, biotype, not only requires great effort but also needs the time long.Characteristics such as the quantitative fluorescent PCR that on the PCR basis, grows up has in recent years not only been inherited the advantage of conventional P CR, has avoided the easy contaminate environment of its testing process to cause false-positive shortcoming easily, and it is easy fast to have testing process, and the result is responsive special.PCR is the polymerase chain reaction english abbreviation; Its English full name is Polymerase Chain Reaction; Be a kind of method of the synthetic specific DNA fragment of external enzymatic, form one-period by several steps reactions such as high-temperature denatured, low-temperature annealing and thermophilic extensions, circulation is carried out; Make target DNA be able to rapid amplification, have high specificity, highly sensitive, easy and simple to handle, characteristics such as save time.The preparation and the method for use of yersinia genus rapid detection reagent kit are disclosed like publication number CN101200760A (application number 200710115174.2).Comprise loop-mediated isothermal amplification liquid A, BstDNA polysaccharase B, developer C in the disclosed test kit of this patent; Reaction solution A contains reaction buffer, dNTP, sal epsom, upper reaches inner primer 5-CCGGTTTGATCGGTTTCGCCCACAAGATGGGTGTGCC-3, downstream inner primer 5-GTGCGTTTCTGGCCGAGCTTGCAGACGTTTTGCCAGGATT-3, upper reaches outer primer 5-CGCCGTGAAGGTAAAGTTCA-3, downstream outer primer 5-CAGAGTTCAGGAACGACAGC-3 and trimethyl-glycine; Detection method comprises the extraction of testing sample or DNA of bacteria, loop-mediated isothermal amplification and the color developing detection of yersinia." processing of farm products " 2008 (11) .-37-37 report, the researchist of Sweden and Finland has set up a kind of novel food product detection mode, and the germ that can carry food is quickly and accurately detected, and this will provide effective help for the supervision of food safety.The researchist utilizes TaqMan probe real-time quantitative PCR (polymerase chain reaction) method that the yersinia entero-colitica in the food is detected, but does not have concrete research contents.
Summary of the invention
The objective of the invention is to the deficiency to prior art, provide a kind of easy quick, sensitive and accurate, be applicable to yersinia entero-colitica test kit and detection method that great amount of samples detects.
To achieve these goals, the technical scheme taked of the present invention is:
Detect the yersinia entero-colitica fluorescent PCR kit, the reagent in the test kit comprises preceding primer, back primer, TaqMan probe, fluorescent PCR premixed liquid, ROX correcting fluid, pure water and positive criteria liquid; The nucleotide sequence of primer is 5 '-AATGCTGTCTTCATTTGGAGC-3 ' before said; The nucleotide sequence of said back primer is 5 '-ATCCCAATCACTACTGACTTC-3 ', and the nucleotide sequence of said TaqMan probe is 5 '-CAAGCAAGCTTG TGATCCTCCG-3 '; The mole ratio of primer, back primer and TaqMan probe is 1:1:1 before said;
Before described primer, back primer and TaqMan probe be special according to yersinia entero-colitica, the conservative gene nucleotide sequence synthetic and the dna fragmentation of 20～21 bases of specific combination with it; TaqMan probe 5 ' end is fluorescein-labelled with the acid of the different sulphur nitrile of fluorophor, and 3 ' end is used the quenching group mark.
5 ' end affinity tag of said TaqMan probe is a 6-carboxyl succinimide ester, and 3 ' end affinity tag is a 6-carboxyl tetramethylrhodamin.
Said fluorescent PCR premixed liquid comprises the PCR amplification buffer, DNA polysaccharase and mg ion and dATP, dCTP, dGTP, 4 kinds of deoxynucleoside mixtures of dTTP.This fluorescent PCR premixed liquid can be bought on market and obtain.
The mole number proportioning that said fluorescent PCR premixed liquid contains 4 kinds of dNTP is dATP:dCTP:dGTP:dTTP=l:l:l:l.
The To Template of the fluorescent PCR amplification of said test kit is an enterocolitis yersinia genus DNA of bacteria.
After the PCR response procedures of said test kit is 95 ℃ of 30 seconds enzyme activations, 95 ℃ of sex change in 5～10 seconds, 60 ℃ of annealing in 30～60 seconds, extension, fluorescent signal collection, 35～40 circulations.
Said pure water is tri-distilled water or deionized water, and the cell concentration of said positive criteria liquid is 10
7Cfu/ml; The staple of said ROX correcting fluid is a rhodamine; Rhodamine is can be used as the od-ray staining agent a kind of by tritane deutero-dyestuff, and its another name has: rose-red 123 and 2-(6-Amino-3-imino-3H-xanthen-9-yl) benzoic acid methyl ester.
The testing sample of said test kit is food, environmental sample or movement.
Primer, back primer, TaqMan probe are dry powder before in the test kit according to the invention; Add 100 microlitres (μ L) pure water before using; Other reagent is working fluid and can directly uses, and the test kit condition of storage is-20 ℃, is valid for one year; Transport condition is 4 ℃～8 ℃, and the time is no more than 72 hours.
Detection principle of the present invention: according to distinctive, a stable gene fragment of yersinia entero-colitica, the forward and backward primer of synthetic specific combination with it and the dna probe that is marked with fluorescence-quenching group are when having target gene to exist in the sample; After the front and back primer combines with it, under the effect of archaeal dna polymerase, target gene is duplicated, fluorescent probe also combines with target gene simultaneously; Under the effect of the circumscribed function of archaeal dna polymerase; Probe is by enzymolysis, is combined in fluorescence and quenching group on the probe two ends and is released and comes, and resorcinolphthalein breaks away from quenching group and luminous; Increase along with the gene replication geometricprogression; The fluorescence that discharges is the accumulation of geometricprogression ground also, through judging of change in fluorescence in the monitoring reaction liquid whether target gene is arranged in the reaction solution, or gene content what.Stipulate that certain fluorescence intensity is a threshold value, used cycle number was the Ct value when fluorescence intensity that produces when reaction reached this threshold value, and this value and sample target gene content are negative correlation, relatively can draw the concentration of yersinia entero-colitica with typical curve.
Fluorescent PCR is a modernized laboratory detection technique commonly used, and being increases by one group of responsive optical detection device on the regular-PCR basis, and direct signal in the detection reaction pipe need not the regular-PCR electrophoresis step, saves time and reagent environmentally safe.It is following that test kit according to the invention carries out detection method:
1. from-20 ℃ of freezing environment, take out required reagent, put in the room temperature (20～25 ℃), notes to shake up before every kind of liquid reagent use until melting fully.
2. DNA of bacteria extracts purifying: with phenol-chloroform cracking bacterium, isolating nucleic acid washs purification of nucleic acid with alcohol, promptly can be used for fluorescent PCR with ultrapure water dissolving nucleic acid at last and detects.
3. dosing: draw fluorescent PCR premixed liquid 10 (n+1) μ L according to positive criteria liquid and sample total amount n; Preceding primer, back primer, TaqMan probe, each 0.4 (n+1) μ L of ROX liquid; Pure water 6.4 (n+1) μ L; With above liquid abundant mixing in centrifuge tube, divide in the fluorescent PCR reaction tubes of packing into 18 μ L/ pipe (so because of 1 part of liquid subpackage error polygamy) again.
4. number: yin and yang attribute standard and sample respective tube are numbered according to the order of sequence, and the position at record standard hole and sample aperture place.
5. application of sample: add yin and yang attribute standard/sample 2 μ l/ holes in reaction tubes separately, cover tight lid or plate film shrouding, the liquid of tube wall is got rid of down.
6. go up machine: open the fluorescent PCR appearance, press the ejection sample bin, the reaction tubes that will add reaction solution inserts draw-in groove, shuts door.
7. parameter setting: TaqMan probe patterns, luminophore FAM, quenching group TAMRA, reaction volume 20 μ L.
Response procedures is provided with: behind 95 ℃ of 30 seconds enzyme activations, and 95 ℃ of sex change in 5～10 seconds, 60 ℃ of annealing in 30～60 seconds, extension, fluorescent signal collection, 35～40 circulations.
8. detect: after starting response procedures, instrument gets into reaction-detected state, and shows detected result in real time.
9. the result judges
The result judges has two kinds of methods, and yin and yang attribute is judged available (1) kind method, quantitatively judges with the 2nd kind of method.Notice that sample Ct value and its yersinia entero-colitica concentration are inversely proportional to.
(1) yin and yang attribute is judged
Amplification curve is serpentine, and Ct value≤38 are judged to be the positive, and no Ct value is judged to feminine gender, the Ct value>38 be judged to suspiciously, suspicious specimen is carried out the second time and is detected, and amplification curve is serpentine and Ct value≤40 are judged to the positive, and no Ct value is judged to feminine gender.
(2) quantitative analysis
Ct value with standard substance is an ordinate zou, is X-coordinate with the concentration logarithm of standard substance, and the drawing standard graphic representation is seen Fig. 1.In the Ct value substitution typical curve with sample, read the pairing concentration of sample, multiply by its corresponding extension rate and be yersinia entero-colitica actual concentrations in the sample from typical curve.
Compared with prior art, advantage of the present invention:
1. testing process is simple and efficient, and whole process only needs 2 hours, and traditional bacteria is cultivated rule and taken 4 days.
2. stopped pipe reaction and detection are free from environmental pollution, and reaction process is monitoring result in real time, has avoided regular-PCR test kit complex operation, and contaminate environment causes false positive results easily.
3. highly sensitive, detection sensitivity is 100 times of regular-PCR test kit.
Description of drawings
Fig. 1 is the examination criteria graphic representation of yersinia entero-colitica, and X-coordinate is represented bacterium standard substance concentration logarithm, and ordinate zou is represented bacterium standard substance Ct value.
Fig. 2 is a yersinia entero-colitica fluorescent PCR kit specificity test chart.
Embodiment
Below in conjunction with accompanying drawing the present invention is further specified.
Embodiment 1:
Yersinia entero-colitica fluorescent PCR kit of the present invention; Reagent in the test kit comprises that nucleotide sequence is the preceding primer of 5 '-AATGCTGTCTTCATTTGGAGC-3 ', the back primer that nucleotide sequence is 5 '-ATCCCAATCACTACTGACTTC-3 ', TaqMan probe, the PCR amplification buffer that nucleotide sequence is 5 '-CAAGCAAGCTTGTGATCCTCCG-3 ', and the mixing liquid, deionized water and the cell concentration that contain DNA polysaccharase, mg ion and dATP, dCTP, dGTP, 4 kinds of deoxynucleosides of dTTP are 10
7The positive criteria liquid of cfu/ml.And 5 ' end affinity tag of TaqMan probe is a 6-carboxyl succinimide ester, and 3 ' end affinity tag is a 6-carboxyl tetramethylrhodamin; The mole ratio of wherein preceding primer, back primer and TaqMan probe is 1:1:1; The mole number proportioning of 4 kinds of dNTP is dATP:dCTP:dGTP:dTTP=l:l:l:l.This is the yersinia entero-colitica fluorescent PCR kit that is used to detect of the present invention.
Embodiment 2: the detection by quantitative of yersinia entero-colitica in the faecal samples
1. DNA of bacteria extracts
(1) adds the phosphate buffered saline buffer (PBS) and the abundant mixing of faecal samples of 4 times of amounts of sample, 100 ℃ of water-baths 15 minutes, 8000rpm (rotations per minute); Centrifugal 5min; Get 500 μ L supernatant, add 50 μ L, 10% sodium lauryl sulphate (SDS), 20 μ L Proteinase Ks; 50 ℃ of water-bath 1h, 15min shakes once.
(2) add equivalent phenol, shake up 10000rpm, centrifugal 5min.
(3) get supernatant, add phenol, each 300 μ L of chloroform, shake up 10000rpm, centrifugal 5min.
(4) get supernatant (can not suck foreign material), add the equal amounts of chloroform mixing, 10000rpm, centrifugal 5min.
(5) get supernatant (can not suck foreign material), add 1000 μ L refrigerated absolute ethyl alcohols, add 1/10 volume again, the NaAc of 3mol/L puts-20 ℃ of deposition 30min.
(6) 10000rpm, centrifugal 15min, deposit D NA gently abandons supernatant, with twice, 37 ℃ of oven dry of 75% alcohol, 500 μ L flushing, adds the dissolving of 30 μ L distilled waters afterwards and is used for the application of sample detection.
2. detect with test kit
From-20 ℃ of freezing environment, take out required reagent, put in the room temperature (20～25 ℃), notes all need shaking up before every kind of liquid reagent use until melting fully.
Dosing: draw fluorescent PCR premixed liquid 10 (n+1) μ L according to yin and yang attribute standard and sample total amount n; Preceding primer, back primer, TaqMan probe, each 0.4 (n+1) μ L of ROX liquid; Pure water 6.4 (n+1) μ L; With above liquid abundant mixing in centrifuge tube, divide in the fluorescent PCR reaction tubes of packing into 18 μ L/ pipe (so because liquid subpackage error can polygamy 1 part) again.
Numbering: yin and yang attribute standard and the corresponding micropore of sample are numbered according to the order of sequence, and the position at record standard hole and sample aperture place.
Application of sample: add yin and yang attribute standard/sample 2 μ l/ holes in reaction tubes separately, cover tight lid or plate film shrouding, the liquid of tube wall is got rid of down.
Last machine: open the fluorescent PCR appearance, press the ejection sample bin, the reaction tubes that will add reaction solution inserts draw-in groove, shuts door.
Parameter is set: TaqMan probe patterns, luminophore FAM, quenching group TARAMA, reaction volume 20 μ L.
Response procedures: behind 95 ℃ of 30 seconds enzyme activations, 95 ℃ of sex change in 5 seconds, are extended, are detected 35 circulations at 60 ℃ of annealing in 30 seconds.
Detect: after starting response procedures, instrument gets into reaction-detected state, and shows detected result in real time.
3. detected result analysis
Ct value with standard substance is an ordinate zou; Concentration logarithm with standard substance is an X-coordinate; Drawing standard graphic representation (Fig. 1); In the Ct value substitution typical curve with sample, read the pairing concentration of sample, multiply by its corresponding extension rate and be yersinia entero-colitica actual concentrations in the sample from typical curve.
Embodiment 3: the detection by quantitative of yersinia entero-colitica in the food samples
1. DNA of bacteria extracts
(1) phosphate buffered saline buffer (PBS) that adds 4 times of amounts of sample grinds homogenate with food samples, 100 ℃ of water-baths 15 minutes, 8000rpm (rotations per minute); Centrifugal 5min; Get 500 μ L supernatant, add 50 μ L, 10% sodium lauryl sulphate (SDS), 20 μ L Proteinase Ks; 50 ℃ of water-bath 1h, 15min shakes once.
(2) add equivalent phenol, shake up 10000rpm, centrifugal 5min.
(3) get supernatant, add phenol, each 300 μ L of chloroform, shake up 10000rpm, centrifugal 5min.
(4) get supernatant (can not suck foreign material), add the equal amounts of chloroform mixing, 10000rpm, centrifugal 5min.
(5) get supernatant (can not suck foreign material), add 1000 μ L refrigerated absolute ethyl alcohols, add 1/10 volume again, the NaAc of 3mol/L puts-20 ℃ of deposition 30min.
(6) 10000rpm, centrifugal 15min, deposit D NA gently abandons supernatant, with twice, 37 ℃ of oven dry of 75% alcohol, 500 μ L flushing, adds the dissolving of 30 μ L distilled waters afterwards and is used for the application of sample detection.
2. detect with test kit
From-20 ℃ of freezing environment, take out required reagent, put in the room temperature (20～25 ℃), notes all need shaking up before every kind of liquid reagent use until melting fully.
Dosing: draw fluorescent PCR premixed liquid 10 (n+1) μ L according to yin and yang attribute standard and sample total amount n; Preceding primer, back primer, TaqMan probe, each 0.4 (n+1) μ L of ROX correcting fluid; Pure water 6.4 (n+1) μ L; With above liquid abundant mixing in centrifuge tube, divide in the fluorescent PCR reaction tubes of packing into 18 μ L/ pipe (so because liquid subpackage error can polygamy 1 part) again.
Numbering: yin and yang attribute standard and the corresponding micropore of sample are numbered according to the order of sequence, and the position at record standard hole and sample aperture place.
Application of sample: add yin and yang attribute standard/sample 2 μ l/ holes in reaction tubes separately, cover tight lid or plate film shrouding, the liquid of tube wall is got rid of down.
Last machine: open the fluorescent PCR appearance, press the ejection sample bin, the reaction tubes that will add reaction solution inserts draw-in groove, shuts door.
Parameter is set: TaqMan probe patterns, luminophore FAM, quenching group TARAMA, reaction volume 20 μ L.
Response procedures: behind 95 ℃ of 30 seconds enzyme activations, 95 ℃ of sex change in 7 seconds, are extended, are detected 38 circulations at 60 ℃ of annealing in 45 seconds.
Detect: after starting response procedures, instrument gets into reaction-detected state, and shows detected result in real time.
3. detected result analysis
Ct value with standard substance is an ordinate zou; Concentration logarithm with standard substance is an X-coordinate; Drawing standard graphic representation (Fig. 1); In the Ct value substitution typical curve with sample, read the pairing concentration of sample, multiply by its corresponding extension rate and be yersinia entero-colitica actual concentrations in the sample from typical curve.
Embodiment 4: the detection by quantitative of yersinia entero-colitica in the environmental sample (like soil, refrigerator dirt)
1. DNA of bacteria extracts
(1) adds sample 4 times of amounts phosphate buffered saline buffer (PBS) and fully grind homogenate, 100 ℃ of water-baths 15 minutes, 8000rpm (rotations per minute) with environmental sample; Centrifugal 5min; Get 500 μ L supernatant, add 50 μ L, 10% sodium lauryl sulphate (SDS), 20 μ L Proteinase Ks; 50 ℃ of water-bath 1h, 15min shakes once.
(2) add equivalent phenol, shake up 10000rpm, centrifugal 5min.
(3) get supernatant, add phenol, each 300 μ L of chloroform, shake up 10000rpm, centrifugal 5min.
(4) get supernatant (can not suck foreign material), add the equal amounts of chloroform mixing, 10000rpm, centrifugal 5min.
(5) get supernatant (can not suck foreign material), add 1000 μ L refrigerated absolute ethyl alcohols, add 1/10 volume again, the NaAc of 3mol/L puts-20 ℃ of deposition 30min.
(6) 10000rpm, centrifugal 15min, deposit D NA gently abandons supernatant, with twice, 37 ℃ of oven dry of 75% alcohol, 500 μ L flushing, adds the dissolving of 30 μ L distilled waters afterwards and is used for the application of sample detection.
2. detect with test kit
From-20 ℃ of freezing environment, take out required reagent, put in the room temperature (20～25 ℃), notes all need shaking up before every kind of liquid reagent use until melting fully.
Dosing: draw fluorescent PCR premixed liquid 10 (n+1) μ L according to yin and yang attribute standard and sample total amount n; Preceding primer, back primer, TaqMan probe, each 0.4 (n+1) μ L of ROX correcting fluid; Pure water 6.4 (n+1) μ L; With above liquid abundant mixing in centrifuge tube, divide in the fluorescent PCR reaction tubes of packing into 18 μ L/ pipe (so because of 1 part of liquid subpackage error polygamy) again.
Numbering: yin and yang attribute standard and the corresponding micropore of sample are numbered according to the order of sequence, and the position at record standard hole and sample aperture place.
Application of sample: add yin and yang attribute standard/sample 2 μ l/ holes in reaction tubes separately, cover tight lid or plate film shrouding, the liquid of tube wall is got rid of down.
Last machine: open the fluorescent PCR appearance, press the ejection sample bin, the reaction tubes that will add reaction solution inserts draw-in groove, shuts door.
Parameter is set: TaqMan probe patterns, luminophore FAM, quenching group TARAMA, reaction volume 20 μ L.
Response procedures: behind 95 ℃ of 30 seconds enzyme activations, 95 ℃ of sex change in 10 seconds, are extended, are detected 40 circulations at 60 ℃ of annealing in 60 seconds.
Detect: after starting response procedures, instrument gets into reaction-detected state, and shows detected result in real time.
3. detected result analysis
Ct value with standard substance is an ordinate zou; Concentration logarithm with standard substance is an X-coordinate; Drawing standard graphic representation (Fig. 1); In the Ct value substitution typical curve with sample, read the pairing concentration of sample, multiply by its corresponding extension rate and be yersinia entero-colitica actual concentrations in the sample from typical curve.
Embodiment 5: the specificity test of test kit:
Specificity is meant primer, the probe recognition capability to determinand, stresses the specificity of association reaction between primer, probe and determinand and the separating capacity of or related substances close to structure.Specificity depends on the cross reaction of determinand and other materials.
The detection method of pressing this test kit is respectively to yersinia entero-colitica; Intestinal bacteria, Salmonellas, Shigellae; Vessel used to hold grain at the imperial sacrifice coccus (all available from Chinese medicine bacterium preservation administrative center) pure growth carries out the fluorescent PCR detection after extracting DNA, and the result sees table 1 and accompanying drawing 2.
The specificity of table 1 test kit
| Bacterium | Yersinia entero-colitica | Intestinal bacteria | Salmonellas | Shigellae | The vessel used to hold grain at the imperial sacrifice coccus |
| Fluorescent PCR detects | + | - | - | - | - |
Test kit only to the yersinia entero-colitica reaction, with common entero-bacte intestinal bacteria, Salmonellas, Shigellae, vessel used to hold grain at the imperial sacrifice coccus no cross reaction, shows that this test kit has high degree of specificity.
Embodiment 6: the sensitivity test of test kit
The bacterium liquid that will pass through counting is diluted to 10
8～10
08 series concentration of cfu/ml are extracted DNA with DNA extraction method among the embodiment 1 and are used for fluorescent PCR and detect, and use the sensitivity as test kit of bacterium minimum concentration that positive reaction occurs.The result sees table 2.
The susceptibility of table 2 test kit
| Bacteria concentration cfu/ml | 10 7 | 10 6 | 10 5 | 10 4 | 10 3 | 10 2 | 10 1 | 10 0 |
| The Ct value | ＋ | ＋ | ＋ | ＋ | ＋ | ＋ | ＋ | - |
Test shows, the low energy of test kit detects the yersinia entero-colitica of concentration 1cfu/ml.
Claims (7)
1. one kind is detected the yersinia entero-colitica fluorescent PCR kit, it is characterized in that: the reagent in the test kit comprises preceding primer, back primer, TaqMan probe, fluorescent PCR premixed liquid, ROX correcting fluid, pure water and positive criteria liquid; The nucleotide sequence of primer is 5 '-AATGCTGTCTTCATTTGGAGC-3 ' before said; The nucleotide sequence of said back primer is 5 '-ATCCCAATCACTACTGACTTC-3 ', and the nucleotide sequence of said TaqMan probe is 5 '-CAAGCAAGCTTGTGATCCTCCG-3 '; The mole ratio of primer, back primer and TaqMan probe is 1:1:1 before said;
Before described primer, back primer and TaqMan probe be special according to yersinia entero-colitica, the conservative gene nucleotide sequence synthetic and the dna fragmentation of 20～21 bases of specific combination with it; TaqMan probe 5 ' end is fluorescein-labelled with the acid of the different sulphur nitrile of fluorophor, and 3 ' end is used the quenching group mark.
2. according to the said detection yersinia entero-colitica of claim 1 fluorescent PCR kit, it is characterized in that: 5 ' end affinity tag of said TaqMan probe is a 6-carboxyl succinimide ester, and 3 ' end affinity tag is a 6-carboxyl tetramethylrhodamin.
3. according to the said detection yersinia entero-colitica of claim 1 fluorescent PCR kit; It is characterized in that: said fluorescent PCR premixed liquid comprises the PCR amplification buffer; DNA polysaccharase and mg ion and dATP, dCTP, dGTP, 4 kinds of deoxynucleoside mixtures of dTTP.
4. according to the said detection yersinia entero-colitica of claim 1 fluorescent PCR kit, it is characterized in that: the mole number proportioning that said fluorescent PCR premixed liquid contains 4 kinds of dNTP is dATP:dCTP:dGTP:dTTP=l:l:l:l.
5. according to the said detection yersinia entero-colitica of claim 1 fluorescent PCR kit, it is characterized in that: the To Template of the fluorescent PCR amplification of said test kit is an enterocolitis yersinia genus DNA of bacteria.
6. according to the said detection yersinia entero-colitica of claim 1 fluorescent PCR kit; It is characterized in that: after the PCR response procedures of said test kit is 95 ℃ of 30 seconds enzyme activations; 95 ℃ of sex change in 5～10 seconds; 60 ℃ of annealing in 30～60 seconds, extension, fluorescent signal collection, 35～40 circulations.
7. according to the said detection yersinia entero-colitica of claim 1 fluorescent PCR kit, it is characterized in that: said pure water is tri-distilled water or deionized water, and the cell concentration of said positive criteria liquid is 10
7Cfu/ml.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102534023A (en) * | 2012-02-11 | 2012-07-04 | 山东出入境检验检疫局检验检疫技术中心 | RCA (rolling circle amplification) rapid detection primer and kit for Yersinia enterocolitica |
| CN103468811A (en) * | 2013-09-17 | 2013-12-25 | 北京卓诚惠生生物科技有限公司 | Yersinia enterocolitica virulence gene multiplex-PCR (Polymerase Chain Reaction) detection primer group and kit |
| CN105087576A (en) * | 2015-09-29 | 2015-11-25 | 西南民族大学 | Detection kit for Yersinia rohdei causing enterocolitis and detection method |
| CN107663545A (en) * | 2017-09-19 | 2018-02-06 | 温和心 | Detect primer sets and the application of yersinia enterocolitica |
| CN109008958A (en) * | 2018-06-14 | 2018-12-18 | 中南大学湘雅医院 | A kind of study on intestinal flora method for filtering and transplanting based on excrement |
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| CN105087576A (en) * | 2015-09-29 | 2015-11-25 | 西南民族大学 | Detection kit for Yersinia rohdei causing enterocolitis and detection method |
| CN107663545A (en) * | 2017-09-19 | 2018-02-06 | 温和心 | Detect primer sets and the application of yersinia enterocolitica |
| CN109008958A (en) * | 2018-06-14 | 2018-12-18 | 中南大学湘雅医院 | A kind of study on intestinal flora method for filtering and transplanting based on excrement |
| CN109008958B (en) * | 2018-06-14 | 2021-10-26 | 中南大学湘雅医院 | Intestinal flora research method based on fecal filtration and transplantation |
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