CN101509041A - Chlamydia trachomatis nucleic acid detection kit for constant-temperature amplification by using RNA - Google Patents
Chlamydia trachomatis nucleic acid detection kit for constant-temperature amplification by using RNA Download PDFInfo
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- CN101509041A CN101509041A CNA200910047902XA CN200910047902A CN101509041A CN 101509041 A CN101509041 A CN 101509041A CN A200910047902X A CNA200910047902X A CN A200910047902XA CN 200910047902 A CN200910047902 A CN 200910047902A CN 101509041 A CN101509041 A CN 101509041A
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Abstract
The invention relates to a kit for utilizing a magnetic bead-RNA concentrating technology to extract purified target RNA as well as testing chlamydia trachomatis (CT) by using constant temperature nucleic acid simultaneous amplification detection technology (SAT). The kit comprises urine sample preservation solution, nucleic acid extracting solution, cleaning solution, CT reaction solution, CT detection solution, STA enzyme liquid, CT positive control and CT negative control. The kit has high specificity and sensitivity; furthermore, the amplified product RNA is easy for degradation in natural environment with little pollution.
Description
Technical field
The present invention relates to the external diagnosis reagent technical field, be specifically related to a kind of test kit that utilizes magnetic bead-RNA beneficiation technologies extraction purifying target RNA and the synchronous amplification detection technology of constant temperature nucleic acid (SAT) that chlamydia trachomatis (CT) is detected.
Background technology
Chlamydia trachomatis (Chlamydia trachomatis, CT) be a kind of in human body the long-term survival and the pathogenic agent of wide-scale distribution again, the about 250～450nm of size.Extensive by its diseases range that causes, can involve eye, reproductive tract and other internal organs, can cause male urethra inflammation, epididymitis, women's cervicitis, pelvic inflammatory disease etc., more increase the weight of advancing of disease and cause other complication during especially with other pathogenic agent concurrent infections such as gonococcuss, also can cause mother-to-baby transmission.Thereby the control of chlamydia trachomatis infection has crucial public health meaning.Clinically, 70%～80% women and nearly 50% the male sex often show as the infection of no clinical symptom, so laboratory diagnosis has vital role.The detection of chlamydia trachomatis has three class methods at present: the cytobiology detection method; Immunological detection method and molecular biology method.
The cytobiology detection method comprises microscopy and cellular segregation cultivation.Microscopy can be found the chlamydia trachomatis inclusion body, crosses low the recommendation clinically because of susceptibility and uses, and only limits to CT and identifies.The specificity of culture method is 100%, but susceptibility is low, and each laboratory operation step is different, does not have stdn.The histocyte culture method is " gold standard " that diagnosis CT infects at present, but because separation and Culture complicated operation, technology and equipment requirements height, required time is long, and susceptibility is subjected to collection of specimens, transports, the influence of preservation etc. is bigger, add that domestic a lot of hospital does not possess cell culture condition, thereby be unsuitable for clinical application, be used for identifying eventually of scientific research and intractable case more.
Immunological detection method comprises direct immunofluorescence, colloidal gold method and enzyme linked immunosorbent assay etc.Directly fluorescence antibody mensuration (DFA) will be at the monoclonal anti body and function fluorescent mark of CT, after CT in the sample combines, fluorescence microscopy just can be seen fluorescigenic substance (Ebs), its susceptibility is subjected to crowd infection rate's influence, and there is result of determination to have subjectivity, the easy cancellation of fluorescence is unsuitable for detecting a large amount of samples.Because this method is easy and simple to handle, expense is cheap, specificity is higher, in the hospital that does not possess the molecular biology test condition, can be in order to detecting clinical samples, but need experienced person's operation.Enzyme linked immunosorbent assay (EIA) mono-clonal of enzyme labelling or lipopolysaccharides (LPS) or the outer membrane protein (MOMP) that polyclonal antibody detects CT, enzyme reaction generates coloured product, measure its susceptibility from 64% to 98%, specificity from 93% to 98% with microplate reader.It has been generally acknowledged that its susceptibility can obtain satisfactory result in the high risk population, and infect recently and treat the monitoring in susceptibility obviously descend.Colloidal gold method combines enzyme and monoclonal anti body and function linking agent, forms enzyme labelled antibody, detects in the sample to have or not CT antigen, if any CT antigen,, add the substrate of enzyme then with enzyme labelled antibody generation specific reaction, substrate is generated solubility or insoluble product by enzyme catalysis, and is promptly positive.Can be with the naked eye or spectrophotometer qualitative or quantitative, can go out report in 10 minutes.Its disadvantage is, with other common microorganisms cross reactions, as streptococcus aureus, A group and B group streptococcus, Diplococcus gonorrhoeae etc., thereby makes specificity not high.
Molecular biology method comprise direct detection nucleic acid technology (being the gene probe technology), PCR method, connect chain reaction (LCR), strand displacement amplification (SDA method), the amplification technique (NASBA method) that relies on nucleotide sequence, the amplification (TMA method) of transcriptive intermediate, these class methods all reach 90%～100% for diagnosis urogenital tract chlamydia trachomatis infection susceptibility and specificity.
The amplification of recent development is the transcriptive intermediate TRAP (TMA) of the amplification CT of Gen-Probe company, the rRNA of amplification and detection CT.Nucleic acid amplification uses M-MLV ThermoScript II and T7RNA polymerase to realize simultaneously, adopts chemoluminescence method to carry out end point determination.There is the scholar to report that the susceptibility and the specificity of TMA detection female urine sample are respectively 93.8% and 100%, it is 95.6% and 98.7% that the male sex urinates sample, prompting TMA will become another and can utilize the urine sample to detect the amplification method of CT, but the terminal point chemiluminescence detection that this method is used is inadequate slightly in pollution control.
200810111479.0) and utilize magnetic bead-RNA beneficiation technologies to extract the method (application number: 200810111478.6) of purifying target RNA our company has applied for patent constant temperature synchronous amplification detecting process for nucleic acid (Simultaneous Amplification and Testing, SAT) (application number:; On the basis of these two technology, what chlamydia trachomatis nucleic acid detection kit of the present invention adopted is the SAT technology, nucleic acid amplification uses M-MLV ThermoScript II and T7RNA polymerase to realize simultaneously, ThermoScript II is used to produce a DNA copy of target nucleic acids (RNA), the T7RNA polymerase produces a plurality of RNA copies from the DNA copy, have the RNA copy specific combination that fluorescently-labeled optimization probe and amplification back produce, thereby produce fluorescence, this fluorescent signal can be caught by detecting instrument.This test kit can detect the CT rRNA in swab and the urine sample, has specificity height, highly sensitive and pollute the characteristics of low (amplified production RNA be easy to degraded) under physical environment.
Summary of the invention
Technical problem to be solved by this invention is that to overcome prior art specificity, susceptibility not high, the uppity shortcoming of experimental pollution provides a kind of test kit that utilizes magnetic bead-RNA beneficiation technologies extraction purifying target RNA and the synchronous amplification detection technology of constant temperature nucleic acid (SAT) that chlamydia trachomatis (CT) is detected.
The test kit that chlamydia trachomatis provided by the present invention detects comprises following composition:
(1) urine sample is preserved liquid: be tensio-active agent and the HEPES damping fluid of chlamydia trachomatis (CT) RNA in cracking and preservation urine or the reproductive tract swab washings sample;
(2) nucleic acid extraction liquid: be the magnetic bead of oligodT bag quilt and specificity one section RNA sequence in conjunction with target nucleic acids (CT RNA);
(3) washings: for containing SDS, NaCl and alcoholic acid solution;
(4) CT reaction solution: be the required components of amplification such as dNTPs and NTPs;
(5) CT detects liquid: essential primer and fluorescent probe during for constant-temperature amplification, and sequence is selected from CT 23s rRNA gene conservative district;
(6) SAT enzyme liquid: essential multienzyme components system during for constant-temperature amplification, the t7 rna polymerase that contains, M-MLV ThermoScript II and stablizer;
(7) CT positive control; For containing CT 23s rRNA gene in vitro transcribe rna dilution;
(8) CT negative control: for the urine sample that does not contain any nucleic acid is preserved liquid and physiological saline.
It is ammonium sulfate and washing agent that urine sample is preserved the main effective constituent of liquid, and the existence of high density washing agent can make the rapid inactivation of RNase, effectively preserves RNA.In addition, urine sample is preserved the existence of liquid middle and high concentration salt ion and washing agent, by protein denaturation somatic cells is broken, and target RNA has obtained release, and sample is placed in this preservation liquid, has prolonged the shelf time of sample, and RNA discharges from cell.
Nucleic acid extraction liquid is to have utilized the magnetic bead partition method to carry out nucleic acid extraction, and its main component is magnetic-particle and capture probe.In the nucleic acid extraction process, the magnetic-particle specific combination in nucleic acid that the bacterium cracking discharges and the nucleic acid extraction liquid under the situation that does not need traditional centrifugally operated, obtains purified bacterial target nucleic acid (RNA) by cleaning magnetic-particle.The extraction of pathogenic agent rRNA realizes by the specific adsorption principle.
The sample that contains in the nucleic acid extraction liquid extracts capture probe, and include following one or more sequences: sequence is seen sequence table SEQ .ID.NO1-5.
Contained t7 rna polymerase, M-MLV ThermoScript II in the SAT enzyme liquid, its stability has directly determined the use validity period of the amplification efficiency and the test kit of amplified reaction.Be added with stablizer in this SAT enzyme liquid, through long-term stable experiment, stability can reach 10 months, has guaranteed that the test kit validity period reaches 6 months.
The CT fluorescent probe is a molecular beacon in the CT detection liquid, it is the molecular probe of a class high specific, hypersensitivity, form by the single stranded nucleic acid molecule of fluorescence dye and quencher by two ends difference covalent labeling, be hair clip type or loop-stem structure, the loop section of molecular beacon and target complementation, two becomes stem owing to complementary, and molecular beacon probe is compared with the TaqMan probe of linearity, because of opening of its hairpin structure needs certain power, thereby specificity is better than linear probe.CT detects CT primer in the liquid, according to the principle design of SAT amplification 5 ' end have the downstream primer of T7 promoter sequence tail and special upstream primer, downstream primer distinguished sequence and target are complementary fully, upstream primer and target are in full accord, have guaranteed that this test kit can accurately carry out the SAT reaction that CT detects.
The primer that CT detects in the liquid includes following a pair of or several to sequence:
CTT7: sequence is seen sequence table SEQ .ID.NO6-10;
CTnT7: sequence is seen sequence table SEQ .ID.NO11-15.
The probe that CT detects in the liquid includes following one or more sequences:
CT probe: sequence is seen sequence table SEQ .ID.NO16-20.
Owing to being subject to multiple factor affecting, the SAT amplification makes the amplification failure, test kit user of service error in judgement is got the wrong sow by the ear, CT positive control in the test kit of the present invention and CT negative control, all contain urine sample preservation liquid and physiological saline that this test kit has been addressed, wherein the CT positive control also contains CT RNA, be the dilution of chlamydia trachomatis 23srRNA in-vitro transcription product, definite value is about 10
7Copies/ml.When using this test kit, should do the quality control of parallel control simultaneously, and the result should satisfy positive control dt≤35 simultaneously, negative control dt does not have numerical value or is 40 at every turn, otherwise that experiment this time is considered as is invalid.(dt represents the X-coordinate reading of sample curve and threshold line intersection point, and is similar with the ct value of general real-time fluorescence PCR experimental result.)
The CT RNA of the in-vitro transcription in the CT positive control can prepare gained by several different methods, and wherein a kind of preparation method is as follows:
(1) with the target fragment of the synthetic CT23s rRNA gene of chemical synthesis, the about 500bp of length;
(2) with fragment cloning in the pGEMb-T carrier, make up CT positive control plasmid;
(3) CT positive control plasmid is transformed in the bacillus coli DH 5 alpha, called after pGEM
b-T-CT1 bacterial strain is stored in-70 ℃;
(4) purifying is removed DNA, and quantitative, evaluation RNA.
The CT positive control contains the CT23s rRNA gene order that is about 500bp, and sequence is seen sequence table SEQ .ID.NO21.
The sensitivity for analysis that detection kit of the present invention detects CT RNA is 10
3The copies/ reaction (contains 3.2 * 10 in 1 bacterium of chlamydia trachomatis approximately
3Individual rRNA), this sensitivity is higher than the like product of present domestic listing far away, thus detection kit specificity height of the present invention, highly sensitive, and amplified production RNA is easy to degraded and pollutes low under physical environment.Test kit of the present invention can be used for the detection of urine specimen, and this Noninvasive sample mode is subjected to patient's welcome.
Description of drawings
Fig. 1. embodiment 4 test kits of the present invention detect the amplification figure of patient's swab specimen;
Fig. 2. embodiment 5 test kits of the present invention detect the amplification figure of patient's urine specimen.
Embodiment
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.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
The extraction of embodiment 1 CT nucleic acid RNA
The nucleic acid extraction concrete steps are:
In sample processing tube (1.5ml centrifuge tube), add 100 μ l nucleic acid extraction liquid, add 400 μ l urine samples or swab washing lotion, whenever add a sample and change a suction nozzle, vortex mixing; 60 ℃ are incubated 5 minutes; Room temperature was placed 10 minutes;
Sample processing tube is placed on the magnetic bead tripping device, leave standstill 5 minutes (if in the magnetic bead adsorption process, have indivedual magnetic beads to be difficult to be adsorbed to tube wall then answer the proper extension adsorption time);
After treating that magnetic bead is adsorbed in tube wall, keep sample processing tube on the magnetic bead tripping device, inhale and abandon liquid, keep magnetic bead;
With washings (if washings adularescent flocculent precipitate, with before should first 42 ℃ of heating, until clarification) washed twice, add at every turn and take off behind the 1ml washings on 30 seconds rearmounted magnetic bead tripping devices of sample processing tube concussion, left standstill 5 minutes; Keep sample processing tube on the magnetic bead tripping device, inhale and abandon liquid, keep magnetic bead;
Sample processing tube is moved apart the magnetic bead tripping device, the magnetic bead-nucleic acid complexes in the pipe standby (this step is answered high-visible magnetic bead);
In each sample processing tube, add 40 μ l augmentation detection liquid (40 μ l CT reaction solutions+2.5 μ l CT detect liquid) washing magnetic bead.
Embodiment 2 SAT nucleic acid amplifications detect
Get 30 μ l augmentation detection liquid (comprising magnetic bead) to clean micro-reaction pipe from the above-mentioned augmentation detection liquid behind the concussion mixing, 60 ℃ are incubated 10 minutes, and 42 ℃ are incubated 5 minutes; Simultaneously SAT enzyme liquid also is preheating to 42 ℃;
In the micro-reaction pipe, add the 10 μ l enzyme liquid of preheating (application of sample tip head does not contact the micro-reaction pipe, do change the tip head if any contact), enzyme-added bonnet upper tube cap 1200rpm shake 15 second mixing;
The micro-reaction pipe is gone to suitable constant-temperature fluorescence detector device fast, and 42 ℃ were reacted 40 minutes, set per 1 minute and detected first order fluorescence, detected altogether 40 times;
After reaction finishes, directly the taking-up of micro-reaction pipe is soaked in 10%84 thimerosals, gets the operation of micro-reaction pipe care should be used to, forbid to open reaction tubes (preventing to pollute reaction zone)! Experiment finishes the back with 10%84 thimerosal clean work area and apparatus, uses the clear water wiped clean at last.
Reference value:
1. threshold setting: with the vertex of threshold line just above normal negative control amplification curve.
2.dt≤35 sample is positive;
3.35 the suggestion of the sample of＜dt＜40 detects again, the sample of detected result dt＜40 is positive;
4.dt do not have numerical value or be that 40 sample is negative.
Annotate: dt represents the X-coordinate reading (similar with the ct value of general real-time fluorescence PCR experimental result) of sample curve and threshold line intersection point
Quality control: each detection all is provided with positive control and negative control, and the result should satisfy positive control dt≤35 simultaneously, and negative control dt does not have numerical value or is 40, otherwise that experiment this time is considered as is invalid.
The preparation and the assembling of embodiment 3 CT test kit each components
Test kit is divided into the A box (sample disposal unit) of 2～30 ℃ of storages and the B box (nucleic acid amplification detecting unit) of-15～-35 ℃ of storages.
A box (sample disposal unit) consists of:
Urine sample is preserved liquid: 0.1M ammonium sulfate; 0.15M HEPES;
Nucleic acid extraction liquid: 0.15uM capture probe and magnetic-particle 250mg/L;
Washings: 0.1%SDS.
B box (nucleic acid amplification detecting unit) consists of:
CT reaction solution: 4mM dNTPs and 16mM NTPs;
SAT enzyme liquid: M-MLV2000U; T7 rna polymerase 500U;
CT detects liquid: mainly contain 3.5uM CT special primer, 2.7uM specific fluorescence probe;
CT positive control: mainly contain 10
7Copies/ml CT RNA;
CT negative control: do not contain any nucleic acid, be used for the validity of checked operation process and reagent.
The detection of the clinical sample urine of embodiment 4 application models
This detecting pattern is best embodiment the of the present invention: the preparation of reagent is with embodiment 3, the production of reagent is carried out in the GMP workshop, the clinical experiment sample that chlamydia trachomatis urine clinical sample provides for Shanghai dermatopathy and venereal disease hospital, be numbered CT urine specimen 1～8, other establishes each one of negative control, positive control.The nucleic acid extraction of sample is with embodiment 1, and SAT augmentation detection and judgement are with embodiment 2, and employed fluorescent PCR instrument is Bio-Rad iQ5.The results are shown in accompanying drawing 1, identical with gold standard culture method detected result.
The detection of the clinical sample swab of embodiment 5 application models
This detecting pattern is an another application of the invention: the preparation of reagent is with embodiment 3, the production of reagent is carried out in the GMP workshop, the clinical experiment sample that chlamydia trachomatis swab clinical sample provides for Shanghai dermatopathy and venereal disease hospital, be numbered CT swab specimen 1～8 (being numbered corresponding one by one with urine specimen), other establishes each one of negative control, positive control.The nucleic acid extraction of sample is with embodiment 1, and SAT augmentation detection and judgement are with embodiment 2, and employed fluorescent PCR instrument is Bio-RadiQ5.The results are shown in accompanying drawing 2, identical with gold standard culture method detected result, also in full accord with embodiment 4 results.
According to disclosure of the present invention, those skilled in the art need not too much test and can implement the present invention chlamydia trachomatis required for protection (CT) kit for detecting nucleic acid (RNA constant-temperature amplification), and produce a desired effect.Embodiment disclosed by the invention only describes the present invention, but is not to constitute the present invention is limited.Those skilled in the art are with conspicuous similar surrogate or transformation; or substitute preparation described here at the relevant preparation of structure function chemically or on the biology with some; or associated viscera of the present invention changed; but do not exceed spirit of the present invention, scope and thought, all fall into the scope of protection of present invention.
Claims (10)
1. chlamydia trachomatis nucleic acid detection kit that utilizes the RNA constant-temperature amplification is characterized in that comprising following composition:
(1) urine sample is preserved liquid: be tensio-active agent and the HEPES damping fluid of chlamydia trachomatis (CT) RNA in cracking and preservation urine or the reproductive tract swab washings sample;
(2) nucleic acid extraction liquid: be the magnetic bead of oligodT bag quilt and specificity one section RNA sequence in conjunction with target nucleic acids (CT RNA);
(3) washings: for containing SDS, NaCl and alcoholic acid solution;
(4) CT reaction solution: be the required components of amplification such as dNTPs and NTPs;
(5) CT detects liquid: essential primer and fluorescent probe during for constant-temperature amplification, and sequence is selected from CT 23s rRNA gene conservative district;
(6) SAT enzyme liquid: essential multienzyme components system during for constant-temperature amplification contains T7 RNA polymerase, M-MLV ThermoScript II and stablizer;
(7) CT positive control; For containing CT 23s rRNA gene in vitro transcribe rna dilution;
(8) CT negative control: for the urine sample that does not contain any nucleic acid is preserved liquid and physiological saline.
2. chlamydia trachomatis nucleic acid detection kit according to claim 1 is characterized in that: described urine sample is preserved liquid and is contained ammonium sulfate and washing agent.
3. chlamydia trachomatis nucleic acid detection kit according to claim 1 is characterized in that: described nucleic acid extraction liquid is to utilize the magnetic bead partition method to carry out nucleic acid extraction, contains magnetic-particle and capture probe.
4. chlamydia trachomatis nucleic acid detection kit according to claim 3 is characterized in that: described capture probe includes following one or more sequences: sequence is seen sequence table SEQ .ID.NO1-5.
5. chlamydia trachomatis nucleic acid detection kit according to claim 1, it is characterized in that: the CT fluorescent probe is a molecular beacon in the described CT detection liquid, form by the single stranded nucleic acid molecule of fluorescence dye and quencher by two ends difference covalent labeling, be hair clip type or loop-stem structure, the loop section of molecular beacon and target complementation, two is owing to the complementary stem that becomes.
6. chlamydia trachomatis nucleic acid detection kit according to claim 5 is characterized in that: the probe that described CT detects in the liquid includes following one or more sequences: CT probe: sequence is seen sequence table SEQ .ID.NO16-20.
7. chlamydia trachomatis nucleic acid detection kit according to claim 1, it is characterized in that: the CT primer is that 5 ' end has the downstream primer of T7 promoter sequence tail and special upstream primer in the described CT detection liquid, downstream primer distinguished sequence and target are complementary fully, and upstream primer sequence and target are in full accord.
8. chlamydia trachomatis nucleic acid detection kit according to claim 7 is characterized in that: the primer that described CT detects in the liquid includes following a pair of or several to sequence:
CT T7: sequence is seen sequence table SEQ .ID.NO6-10;
CT nT7: sequence is seen sequence table SEQ .ID.NO11-15.
9. chlamydia trachomatis nucleic acid detection kit according to claim 1 is characterized in that: the CT RNA of in-vitro transcription in the described CT positive control, the preparation method comprises the following steps:
(1) with the target fragment of the synthetic CT 23s rRNA gene of chemical synthesis, the about 500bp of length;
(2) with fragment cloning in the pGEMb-T carrier, make up CT positive control plasmid;
(3) CT positive control plasmid is transformed in the bacillus coli DH 5 alpha, called after pGEM
b-T-CT1 bacterial strain is stored in-70 ℃;
(4) purifying is removed DNA, and quantitative, evaluation RNA.
10. according to claim 1 or 9 described chlamydia trachomatis nucleic acid detection kits, it is characterized in that: described CT positive control contains the CT 23s rRNA gene order of long 521bp, and sequence is seen sequence table SEQ .ID.NO21.
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| CN108588185A (en) * | 2018-05-14 | 2018-09-28 | 零零二信息科技（沧州）有限责任公司 | Based on RNA target mark SAT Mycoplasma genitalium infection molecular biology for detection |
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| CN108546746A (en) * | 2018-05-14 | 2018-09-18 | 零零二信息科技（沧州）有限责任公司 | Based on RNA target mark SAT chlamydia trachomatis infection molecular biology for detection |
| CN110923345A (en) * | 2019-12-19 | 2020-03-27 | 武汉中帜生物科技股份有限公司 | Colloidal gold chromatography kit for detecting chlamydia trachomatis and application thereof |
| CN110923345B (en) * | 2019-12-19 | 2023-06-27 | 武汉中帜生物科技股份有限公司 | Colloidal gold chromatography kit for chlamydia trachomatis detection and application thereof |
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