CN103074429B - UU (ureaplasma urealyticum) detection kit - Google Patents
UU (ureaplasma urealyticum) detection kit Download PDFInfo
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Abstract
The invention provides a UU detection kit. The kit contains a nucleic acid releasing agent and a PCR (polymerase chain reaction) liquid, wherein the nucleic acid releasing agent comprises 0.01-0.5mM/L of surfactin, 20-300mM/L of potassium chloride, 0.01-2% of sodium dodecyl sulfate and 0.05-1% of ethanol; and the PCR liquid comprises an upstream primer, a downstream primer and a probe, the upstream primer and the downstream primer are used for amplifying a target polynucleotide, and the probe is used for detecting the target polynucleotide. No significant differences exist between detection results of a method for releasing nucleic acid by the nucleic acid releasing agent in the kit and a boiling method; when nucleic acid is extracted, a strong protein denaturation agent is adopted, a coat protein structure of a pathogen is destroyed rapidly, pathogen nucleic acid is released, and release and extraction of DNA (deoxyribonucleic acid) can be achieved without heating; the UU detection sensitivity of the kit can reach 400 copies/ml, and the detection linear range is 400-4.00E+10 copies/ml; and the kit can be used for detecting UU-DNA in unknown samples such as genital tract secretions and the like rapidly and accurately, so that a reliable experimental basis is provided for the diagnosis of ureaplasma urealyticum infection.
Description
Technical field
The invention provides a kind of ureaplasma urealyticum (UU) detection kit, specifically a kind of UU-DNA detection kit based on fluorescent PCR.
Background technology
Ureaplasma urealyticum claims again Ureaplasma urealyticum (UU), belong to the Ureaplasma of soft film guiding principle Mycoplasmas Mycoplasmataceae, the minimum prokaryotic micro-organisms of a class between bacterium and virus, mainly be distributed in human body urogenital tract, and trafficability characteristic contact and propagating, also can be by parent vertical infection to fetus.The modal parasitism in male genetic road is in urethral orifice and seminal fluid, and the modal parasitism of female genital tract is in vagina.
Ureaplasma urealyticum is one of main pathogens of nongonococcal urethritis, after infection, can cause multiple urogenital tract infection.Can cause the generation of nongonococcal urethritis, acute epididymitis, prostatitis etc. the male sex; Can cause endometritis, salpingitis, ovaritis etc. in women, intrauterine infection can cause the adverse consequencess such as miscarriage, stillborn foetus, premature labor.In recent years, UU increases as the report of main pathogens, but many infected's atypical symptoms, therefore, early stage, easy, quick, diagnoses specifically UU, the early treatment to clinical diagnosis, disease and prevent its popular etc. having great importance.
At present the laboratory diagnostic method of ureaplasma urealyticum mainly contains isolated culture, immunological method, molecular biosciences method based on detection of nucleic acids etc.Specificity and the susceptibility of isolated culture are high, but it has clinical detection overlong time (at least needing 24h ~ 48h, even the longer time), and process is loaded down with trivial details, need to use special developing medium and be subject to the defects such as miscellaneous bacteria impact, not being suitable on a large scale and detecting.Advantages such as that Immunological Method has is quick, easy, sensitivity is high, but be subject to the impact of many factors, at present domestic do not have a commercial test kit (only have a protein chip detection system get the Green Light certification) yet.In recent years, poly-ribozyme chain reaction (PCR) method has manifested its advantage on detecting gradually, and Fluorescence PCR assay is sensitiveer, more special based on normal PCR technology the one that grows up in conjunction with spectroscopic techniques, more accurate nucleic acid detection technique.Detected result is accurate, and repeatability is high, can the forward and backward pathogenic agent dynamic change of dynamic response patient treatment and with clinical relation, and in whole process, avoided normal PCR to need the problem of aftertreatment, reduced pollution.
Real-time fluorescence PCR technology has demonstrated the superiority of its clinical diagnosis by feat of the advantage such as quick, responsive, special.Use round pcr to detect and be mainly concerned with two aspects, the extraction of nucleic acid and the augmentation detection of nucleic acid.
The domestic boiling method that mainly adopts clinically extracts the nucleic acid of ureaplasma urealyticum at present, specifically first by secretory product sample concentration, washing, then adds lysate, boil, and high speed centrifugation, getting supernatant is template; The method nucleic acid extraction process is more complicated, sample process length consuming time, and in the time processing sample, through multiple steps such as boiling lysises, high speed centrifugation enrichment DNA, there is loss in the DNA in sample, especially for the sample of high density, cracking is insufficient, enrichment is incomplete, can cause a large amount of loss of DNA to cause sample quantitatively on the low side, simultaneously owing to having adopted the heat step of water-bath or metal bath, easily cause aerosol dirt; In addition, for concentrated this step, the concentrated effect of different manufacturers is different, and what have can see precipitation, and what have cannot see, can see that precipitation is because this step has all concentrated nucleic acid and albumen, can cause being like this difficult to fully mix while adding lysate below; Cannot see precipitation make operator determine to inhale while abandoning supernatant can or can not blow and beat nucleic acid.
The method that detects clinically UU-DNA is mainly technology and the improvement thereof based on real-time fluorescence quantitative PCR at present, Real-Time Fluorescent Quantitative PCR Technique is development in recent years a kind of nucleic acid detection technique rapidly, use a kind of pcr amplification instrument with fluorescence detection device, fluorescence detection device can according to certain property program loop send the exciting light of specific wavelength, collect and detect fluorescent signal, reflect in real time the level of amplification of each circulation of PCR by detecting the dynamic change of fluorescent signal, after off-test, can obtain amplification curve by software automatic analysis, according to the shape of the intersection point of amplification curve and fluorescence threshold line (being Ct value) and amplification curve, can judge yin and yang attribute result, if have quantitative reference material or the standard substance of concentration known in same reaction, can obtain typical curve by software automatic analysis, realize thus the definite value (being detection by quantitative) to unknown sample.Compare with traditional PCR, it has increased the two ends probe of mark fluorescent reporter group and quenching group respectively in reaction system.When probe structure is complete, the fluorescent energy that fluorescence report group sends is quenched group and absorbs, and presents quenching effect; If there is the existence of target sequence in amplification procedure, along with the extension of target fragment, probe molecule is cut off by Taq enzymic hydrolysis gradually, fluorescence report group and quenching group dissociate mutually, blocked the two fluorescence energy transfer effect, the fluorescent signal that fluorescence report group sends is collected by fluorescence detection device.Along with the carrying out of amplification, fluorescent signal presents linear enhancing along with the amplification of object fragment.After off-test, the software automatic analysis data that can carry by fluorescent PCR instrument, can obtain the definite value result of yin and yang attribute result and concentration of specimens, therefore, this technology is in the detection and quantitative analysis of target polynucleotide sample, replace gradually traditional PCR method, obtain applying very widely.
The existing test kit based on Real-Time Fluorescent Quantitative PCR Technique detection UU-DNA is applied in clinical detection both at home and abroad at present, but these test kits extract nucleic acid with boiling method mostly, and its detection sensitivity is not high, about 500 ~ 1000copies/ml left and right; In addition, these test kits lack perfect system of quality control mostly, also need further improve and improve technical level, and make this series products more meet the needs of clinical Accurate Diagnosis.
Summary of the invention
The invention provides the application of a kind of nucleic acid releasing agent in ureaplasma urealyticum UU detects, described nucleic acid releasing agent comprises Buddhist Sha graceful (surfactin) 0.01 ~ 0.5mM/L, Repone K 20 ~ 300mM/L, sodium laurylsulfonate 0.01 ~ 2% and ethanol 0.05 ~ 1%.
In nucleic acid releasing agent of the present invention, its solvent can be conventional for this area, for example, be sterilized water or TE damping fluid.The present invention is disclosed in first in ureaplasma urealyticum UU detection and uses the nucleic acid releasing agent containing strong protein denaturant, simplified to a great extent the step of this detection of nucleic acids, and detection sensitivity is greatly improved.
The invention provides a kind of ureaplasma urealyticum UU detection kit, described test kit comprises nucleic acid releasing agent and PCR reaction solution, described nucleic acid releasing agent comprises Buddhist Sha graceful (surfactin) 0.01 ~ 0.5mM/L, Repone K 20 ~ 300mM/L, sodium laurylsulfonate 0.01 ~ 2% and ethanol 0.05 ~ 1%; In described PCR reaction solution, comprise for upstream primer, the downstream primer of target polynucleotide amplification and the probe detecting for target polynucleotide.
Use the method for the nucleic acid releasing agent release nucleic acid in test kit of the present invention and the detected result of boiling method extraction nucleic acid there is no notable difference, and adopt strong protein denaturant while extracting nucleic acid in the present invention, rapid damage pathogenic agent coat protein structure, discharge pathogen nucleic acid, can complete release and the extraction of DNA without heating; The sensitivity that test kit of the present invention detects ureaplasma urealyticum UU can reach 400copies/ml, and detection linearity range is 400 ~ 4.00E+10copies/ml.
Particularly, be 5 '-TTTTGCTGACTCACGTATTCGTAGCCAA-3 ' for the probe sequence of target polynucleotide; Be preferred for the upstream primer of target polynucleotide and the sequence of downstream primer is respectively 5 '-CACCACTTAAATCCTAAGGTTCCAG-3 ' and 5 '-CTGATGACATAATTGAGATTGCACC-3 '.Test kit of the present invention, because adopting above-mentioned primer and/or the probe sequence for detection of target polynucleotide, has good specificity.
In the present invention, in preferred described test kit, also comprise interior mark, described interior target sequence is 5 '-GTGTCTGCGGCGTTTTATCATCTTCCTCTGTCATCCAGTGCAAGTCTTGATCCTGT CGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGT-3 '.A segment length who is designated as insertion pUC18T carrier in described in the present invention is the recombinant chou of the DNA artificial sequence synthetic of 97 base pairs, it is plasmid, it prevents as the positive internal reference in pcr amplification system the false negative causing due to the PCR interfering substance that may exist in sample.In described test kit, comprise in interior target situation, in described PCR reaction solution, also comprise the upstream primer, downstream primer and the probe that detect for interior mark; And preferably, the sequence of mark probe is 5'-TCATCCAGTGCAAGTCTTGATCCTGTC-3'.
In preferred test kit of the present invention, also comprise enzyme mixation, in described enzyme mixation, comprise the uracil dna glycosylase (UNG enzyme) of hot resistant DNA polymerase (Taq enzyme) and 0.5 ~ 2U/ μ l, in described PCR reaction solution, also comprise dUTP simultaneously.Wherein the function of UNG enzyme is the PCR product that degraded contains dU, utilizes the dUTP in UNG enzyme and PCR reaction solution can play the effect of prevention PCR product pollution, thereby prevents pattern detection false positive.
In an embodiment, in described test kit, also comprise UU positive control, UU negative control and the quantitative reference material of UU.
The present invention also provides a kind of ureaplasma urealyticum detection kit, described test kit comprises PCR reaction solution, in described PCR reaction solution, comprise for upstream primer, the downstream primer of target polynucleotide amplification and the probe detecting for target polynucleotide, and be respectively 5 '-CACCACTTAAATCCTAAGGTTCCAG-3 ' and 5 '-CTGATGACATAATTGAGATTGCACC-3 ' for the upstream primer of target polynucleotide and the sequence of downstream primer.
The present invention provides again a kind of ureaplasma urealyticum detection kit, described test kit comprises PCR reaction solution, in described PCR reaction solution, comprise for upstream primer, the downstream primer of target polynucleotide amplification and the probe detecting for target polynucleotide, and be 5 '-TTTTGCTGACTCACGTATTCGTAGCCAA-3 ' for the probe sequence of target polynucleotide.
The present invention also specifically provides a kind of ureaplasma urealyticum detection kit, and described test kit comprises nucleic acid releasing agent, PCR reaction solution, interior mark, enzyme mixation, UU positive control, UU negative control and the quantitative reference material of UU; And in described nucleic acid releasing agent, comprise Buddhist Sha graceful (surfactin) 0.01 ~ 0.5mM/L, Repone K 20 ~ 300mM/L, sodium laurylsulfonate 0.01 ~ 2%, ethanol 0.05 ~ 1% and solvent TE damping fluid; In described PCR reaction solution, comprise upstream primer, downstream primer and probe for target polynucleotide amplification and detection, for upstream primer, downstream primer and the probe of interior mark amplification and detection, 10 × PCR reaction buffer, deoxyribonucleoside triphosphate and/or ribonucleotide triphosphate dNTP; A segment length who is designated as insertion pUC18T carrier in described is the recombinant chou of the DNA artificial sequence synthetic of 97 base pairs; In described enzyme mixation, comprise archaeal dna polymerase and uracil dna glycosylase.
UU fluorescence PCR detection reagent kit operation provided by the invention fast, method is easy, detection sensitivity is high, sensing range is wide, applying this test kit can detect fast and accurately to the UU-DNA in the unknown sample such as genital secretion, for diagnosis Ureaplasma Urealyticum Infection provides reliable experimental basis.Its detected result can be used for auxiliary diagnosis that UU infects and the observation of curative effect of medication, for the early diagnosis of venereal disease and venereal disease high risk population's primary dcreening operation provide molecular diagnosis foundation.
Brief description of the drawings
2. in Fig. 1, be 1. the amplification curve of the positive sample to be tested of ureaplasma urealyticum UU-DNA detected result in embodiment, be the amplification curve of the negative sample to be tested of UU-DNA detected result in embodiment in Fig. 1.
Embodiment
Below be only the preferred embodiment of the present invention, protection scope of the present invention is not limited to this, and any those skilled in the art is in technical scope disclosed by the invention, within can being easy to the change carried out or changing and be encompassed in protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain of claims.
Embodiment 1
The present embodiment provides a kind of concrete ureaplasma urealyticum fluorescence PCR detection reagent kit, and it comprises following component:
1. nucleic acid releasing agent: comprise Buddhist Sha graceful (surfactin) 0.1mM/L, Repone K 100mM/L, the ethanol of sodium laurylsulfonate (SDS) 0.1%, 0.1%.
2. mark (positive internal reference) in: for a segment length who inserts pUC18T carrier is the recombinant chou of the DNA artificial sequence synthetic of 97 base pairs, it is plasmid, concentration is 5.00E+05copies/ml, and the sequence of 97 base pairs is: 5 '-GTGTCTGCGGCGTTTTATCATCTTCCTCTGTCATCCAGTGCAAGTCTTGATCCTGT CGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGT-3 '.
3. PCR reaction solution: comprise 10 × PCR reaction buffer, 5 μ l, the dNTP of 0.2mmol/L, upstream and downstream primer for target polynucleotide amplification is 0.3 μ mol/L, the probe detecting for target polynucleotide is 0.3 μ mol/L, upstream and downstream primer for interior mark fragment amplification is 0.3 μ mol/L, is 0.1 μ mol/L for detection of interior target probe.Wherein, described 10 × PCR reaction buffer is the 200mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride solution that comprises pH7.5,30mmol/L magnesium chloride solution, 500mmol/L Klorvess Liquid, 0.2% Triton solution and 10% formamide soln; Described dNTP comprises dATP, dCTP, dUTP and dGTP; The described upstream and downstream primer increasing for target polynucleotide and the probe detecting for target polynucleotide are primer and the probes that comes from the conservative region of ureaplasma urealyticum nucleic acid, and its base sequence is respectively: upstream primer: 5 '-CACCACTTAAATCCTAAGGTTCCAG-3 '; Downstream primer: 5 '-CTGATGACATAATTGAGATTGCACC-3 '; Probe: 5 '-TTTTGCTGACTCACGTATTCGTAGCCAA-3 '; Described is respectively for detection of interior target primer probe sequence: upstream primer: 5 '-GTGTCTGCGGCGTTTTATCAT-3 '; Downstream primer: 5 '-ACAAACGGGCAACATACCTTG-3 '; Probe: 5'-TCATCCAGTGCAAGTCTTGATCCTGTC-3'.
4. enzyme mixation: the hot resistant DNA polymerase (Taq enzyme) that comprises 5U/ μ l and the uracil dna glycosylase (UNG enzyme) of 1U/ μ l.
5. the quantitative reference material of UU: derive from the UU strong positive plasmid using after the quantitative linearity reference material L1 ~ L5 of UU enterprise definite value, the quantitative reference material of this ureaplasma urealyticum comprises the gradient reference material of A, B, C, tetra-concentration compositions of D, and its concentration is respectively 4.00E+07copies/ml(A), 4.00E+06copies/ml(B), 4.00E+05copies/ml(C), 4.00E+04copies/ml(D).
6. UU positive control: be the UU strong positive sample that clinical hospitals is collected, its concentration is 4.00E+05copies/ml.
7. UU negative control: the negative clinical sample of deactivation that does not contain ureaplasma urealyticum, gonococcus, hsv, chlamydia trachomatis and human papillomavirus etc.
Embodiment 2
The present embodiment provides described in above-described embodiment 1 test kit for detection of the operation steps of UU-DNA in the unknown sample such as genital secretion:
One, reagent is prepared
According to the quantity of sample to be tested, UU negative control, UU positive control and the quantitative reference material A ~ D of UU, get in proportion PCR reaction solution (38 μ l/ person-portion), enzyme mixation (2 μ l/ person-portion) and the interior mark 1.0 μ l/ person-portions of respective amount, fully be mixed into PCR-mix, for example, when sample to be tested is 3 person-portion, need altogether the PCR-mix of preparation 9 person-portions (people's umber of above-mentioned four is respectively 3,1,1 and 4); Instantaneous centrifugal rear for subsequent use.
Two, sample process
1. method A: sample is nucleic acid release fast directly
In each PCR reaction tubes, add shallow the beating of nucleic acid releasing agent 2～5 dark suction of μ l(suggestion, avoid occurring bubble), each pipe adds each 3 ~ 5 μ l of sample to be tested, UU negative control, UU positive control and the quantitative reference material A ~ D of UU successively, inhales to make a call to mix (inhale and beat gently, avoid occurring bubble) for 3 ~ 5 times; Interval is more than 10 minutes, and every pipe adds PCR-mix40 ~ 45 μ l, inhales to beat to mix 2-3 time lid upper tube cap (removing after bubble), centrifugal 30 seconds of 2000rpm.
2. method B: sample nucleic acid after high speed centrifugation is concentrated discharges
Get 200 ~ 500 μ l samples to be tested, 13,000rpm inhales and abandons supernatant after centrifugal 5 minutes, adds 20 ~ 50 μ l nucleic acid releasing agents, leaves standstill after 10 minutes, for subsequent use; Each PCR reaction tubes adds each 5 ~ 10 μ l of above-mentioned pretreated sample to be tested, UU negative control, UU positive control and the quantitative reference material A ~ D of UU; Each PCR reaction tubes adds PCR-mix40 ~ 45 μ l, lid upper tube cap (removing after bubble), centrifugal 30 seconds of 2000rpm.
Three, Fluorescence PCR and interpretation of result (carrying out on fluorescent quantitative PCR instrument)
1) PCR reaction tubes is put into amplification instrument sample cell, by correspondence order, sample to be tested title and quantitative reference material concentration are set.
2) fluorescence detection channel is selected: select FAM passage (Reporter:FAM, Quencher:None) to detect UU; Select HEX or VIC passage (Reporter:VIC, Quencher:None) to detect interior mark; Reference fluorescence (Passive Reference) is set to none.
3) quantitative fluorescent PCR reaction conditions is in table 1:
Table 1
4) interpretation of result
After reaction finishes, the automatic saving result of instrument, can utilize the software that instrument carries to carry out automatic analysis, and starting value, end value and threshold value that also can manual regulation baseline be analyzed, and then record sample Ct value and result.The intersection point of amplification curve and threshold line, is called Ct value (be cycle threshold, refer to the cycling numerical value that fluorescent signal in PCR reaction tubes experiences while reaching the threshold value of setting); Instrument software, according to each sample Ct value size, by the typical curve of 4 quantitative reference materials draftings of concentration gradient, can be tried to achieve the definite value result of each sample automatically.For measuring Ct value≤38.5(Ct value > 0) sample, be reported as the UU-DNA positive, now the amplification curve of sample to be tested is S-type, as in Fig. 1 1.; Show without the sample of Ct value for measuring, mark test positive (Ct value≤38.5) in simultaneously, be reported as UU-DNA feminine gender, now sample to be tested amplification curve is straight, as in Fig. 1 2.; For the sample of measuring Ct value >38.5, interior mark test positive (Ct value≤38.5) simultaneously, is reported as lower than detecting lower limit.If interior mark Ct value >38.5 or interior mark show without Ct value, the detected result of this sample is invalid, should search and get rid of reason, and this sample is carried out to revision test.
Use the specific test of test kit in the present invention to show, itself and the equal no cross reaction such as common venereal diseases pathogenic agent HPV, HSV, CT, NG, illustrate that test kit of the present invention has good specificity.
Using the yin and yang attribute coincidence rate of test kit detection enterprise work reference material in the present invention is 100%, and the detected result of quantitative linearity reference material, sensitivity reference material meets quality standard; In precision test shows batch and batch between reproducible, the variation coefficient <10% of its Ct value, concentration variation coefficient <50%.Use test kit in the present invention to show the detection test of clinical sample, the detection linearity range of this test kit is 400～4.00E+10copies/ml, and detecting lower limit is that sensitivity is 400copies/ml.
Claims (6)
1. a ureaplasma urealyticum UU detection kit, described test kit comprises nucleic acid releasing agent and PCR reaction solution, described nucleic acid releasing agent comprises Sha graceful 0.01～0.5mmol/L of ancient India, Repone K 20～300mmol/L, sodium laurylsulfonate 0.01～2% and ethanol 0.05～1%; In described PCR reaction solution, comprise for upstream primer, the downstream primer of target polynucleotide amplification and the probe detecting for target polynucleotide, upstream primer and downstream primer sequence for target polynucleotide are respectively: 5 '-CACCACTTAAATCCTAAGGTTCCAG-3 ' and 5 '-CTGATGACATAATTGAGATTGCACC-3 ', and be 5 '-TTTTGCTGACTCACGTATTCGTAGCCAA-3 ' for the probe sequence of target polynucleotide.
2. test kit according to claim 1, it is characterized in that, in described test kit, also comprise interior mark, described interior label sequence is 5 '-GTGTCTGCGGCGTTTTATCATCTTCCTCTGTCATCCAGTGCAAGTCTTGATCCTGT CGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGT-3 '.
3. detection kit according to claim 2, is characterized in that, also comprises the upstream primer, downstream primer and the probe that detect for interior mark in described PCR reaction solution; And the sequence of interior mark probe is 5'-TCATCCAGTGCAAGTCTTGATCCTGTC-3'.
4. according to the detection kit described in any one in claim 1～3, it is characterized in that, in described test kit, also comprise enzyme mixation, in described enzyme mixation, comprise the uracil dna glycosylase of Taq enzyme and 0.5～2U/ μ l, in described PCR reaction solution, also comprise dUTP simultaneously.
5. according to the detection kit described in any one in claim 1～3, it is characterized in that, in described test kit, also comprise UU positive control, UU negative control and the quantitative reference material of UU.
6. a ureaplasma urealyticum detection kit, described test kit comprises PCR reaction solution, in described PCR reaction solution, comprise for upstream primer, the downstream primer of target polynucleotide amplification and the probe detecting for target polynucleotide, and be respectively 5 '-CACCACTTAAATCCTAAGGTTCCAG-3 ' and 5 '-CTGATGACATAATT GAGATTGCACC-3 ' for the upstream primer of target polynucleotide and the sequence of downstream primer, and be 5 '-TTTTGCTGACTCACGTATTCGTAGCCAA-3 ' for the probe sequence of target polynucleotide.
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| CN105349661A (en) * | 2015-11-27 | 2016-02-24 | 湖南圣湘生物科技有限公司 | Chlamydia trachomatis and gonococcus nucleic acid detection kit |
| CN105349660A (en) * | 2015-11-27 | 2016-02-24 | 湖南圣湘生物科技有限公司 | Chlamydia trachomatis and ureaplasma urealyticum nucleic acid detection kit |
| CN110257534A (en) * | 2019-04-22 | 2019-09-20 | 深圳市国赛生物技术有限公司 | Ureaplasma urealyticum kit for detecting nucleic acid |
| CN112301137B (en) * | 2020-02-06 | 2024-03-22 | 广州普世利华科技有限公司 | RDA method and kit for rapidly detecting ureaplasma urealyticum |
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