CN102329866B - PCR (polymerase chain reaction) fluorescence quantitative rapid test kit and method for chlamydia trachomatis - Google Patents
PCR (polymerase chain reaction) fluorescence quantitative rapid test kit and method for chlamydia trachomatis Download PDFInfo
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Abstract
The invention aims at providing a kit which is suitable for rapid test of chlamydia trachomatis in clinical samples and can be used for auxiliary diagnosis and efficacy monitoring of chlamydia trachomatis infection. The technical scheme of the invention is as follows: an PCR (polymerase chain reaction) fluorescence quantitative rapid test kit of the chlamydia trachomatis is provided and comprisesa PCR reaction solution, wherein the PCR reaction solution contains primers and a fluorescence probe; and the primers comprise an upstream primer and a downstream primer, and the kit further comprises a DNA (deoxyribonucleic acid) polymerase, a strong positive quality control product, a weak positive quality control product, a negative quality control product, a positive quantitative reference product and a DNA extraction solution. According to the CT (chlamydia trachomatis) fluorescence PCR quantitative test kit and method provided by the invention, a Taqman core technology platform and an arabidopsis internal reference system are utilized, thus the test sensitivity is higher. Furthermore, the accuracy, specificity, repeatability, stability, sensitivity and precision are improved compared with those of the existing product.
Description
Technical field
The present invention relates to a kind of disease pathogen technique of gene detection, relate in particular to PCR fluorescent quantitation quick detection kit and the method thereof of a kind of chlamydia trachomatis.
Background technology
Chlamydia trachomatis is that a class can be passed through bacterial filter, and strict eukaryotic cell endoparasitism has the prokaryotic microorganism of unique growth cycle.It can cause trachoma, inclusion body chitonitis, urogenital infections and lymphogranuloma venereum.Behind the injection chlamydia trachomatis vaccine, can bring out local specific immunity, but its provide protection only can be kept 1-2.The record of " Bai Shi system handbook " (1984), chlamydia trachomatis Chlamydia trachomatis is divided into 3 biotypes, be mouse biotype (biovar mouse), trachoma biotype (biovar trachoma) and lymphogranuloma venereum biotype (biovarlymphogranulomavenereum, LGV), the latter two are relevant with human diseases.Use the micro-indirect immunofluorescent test, the trachoma biotype is divided again A.B.Ba.C.D.Da.E.F.G.H.I.Ia.J.K14 serotype, and the LGV biotype has again L1.L2.L2a.L34 serotype.Trachoma biovariety D～K serotype and chlamydia trachomatis LGV biovariety can cause male sex's non gonococcal urethritis. women's cervicitis. and salpingitis. Infertility etc.Choamydiae infection has become a serious social concern at present.Chlamydozoan has become a kind of pathogenic agent the most common in the venereal disease on the one hand, is that it serious persistent infection phenomenon can occur on the other hand.The highest the male urethra infection scorching and 50% of 70% woman uterus is arranged is asymptomatic carrier, and because of no conscious sympton, these people are a high-risk Potential infection source socially.There is the scholar to report, through the baby of chlamydozoan Positive Mothers birth canal birth, has 50%～70% chance can obtain choamydiae infection.In view of this, the legal choamydiae infection of western countries is the antenatal conventional project that must look into of pregnant woman.So diagnosis of chlamydial infection has great meaning to prenatal and postnatal care ahead of time.
In addition, the lipopolysaccharides of CT can be induced the generation of TNF-α, stimulates phagocytic cell active, thereby causes tissue injury.CT is the cofactor that HIV propagates, and the women who infects CT HIV occurs infects than high 3～5 times without the women of CT infection, and treatment CT can reduce the propagation of HIV.
As a kind of common parasitic microbe of adult's urogenital tract, CT can cause human infection's various diseases.At present there has been several different methods can utilize certain laboratory condition that CT is made in time, accurately diagnosis.
Doing the chlamydozoan cultivation with uterine neck or urethral swab is to detect chlamydial gold standard, and specificity can reach 100%, and susceptibility is 80%～90%.The most frequently used method is that sample is inoculated in the McCoy cell of processing through cicloheximide (cycloheximide).Cultivating needs 48～72h, observes inclusion body with iodine staining or Giemsa staining.
Such as direct immunofluorescence. enzyme immunoassays (EIA) etc. also are to make sample with uterine neck or urethral swab.Direct immunofluorescence is directly to measure chlamydial lipopolysaccharides or major outer membrane proteantigen, specificity 96%～100%, susceptibility 80%～85% by the fluorescent mark specific antibody.EIA directly measures chlamydial LPS antigen, with other Gram-negative bacteria boivin antigen in the sample cross reaction is arranged, and susceptibility is equivalent to 60%～80% of culture method, specificity 96%～100%.If when aforesaid method was used for urine examination, susceptibility only had 40%～50%, therefore be not suitable for examination.
The DNA cloning technology comprises polymerase chain reaction (PCR) and ligase chain reaction (LCR), can the direct-detection sample in the nucleotide sequence of chlamydia trachomatis plasmid or outer membrane protein.What the RNA TRAP increased is the chlamydozoan ribosome-RNA(rRNA).Can take urine sample to make sample, susceptibility 94%～100%, specificity 98%～100% can be used for the examination test.The gold standard status of cell cultures is replaced by PCR and LCR method at present.
Along with molecular biological development, the amplification test of transcriptive intermediate, enzyme amplify the methods such as immune response, heminested PCR-microvoid plate hybrid method, nest-type PRC, real-time fluorescence PCR detection by quantitative and are all using, the accuracy and the susceptibility that detect have been improved, provide without wound, sensitive, detection means fast for clinical, can be used for the examination test.
At present, the domestic research and development production company that mainly is engaged in the CT detection of nucleic acids adopts fluorescence PCR method to carry out the CT-DNA detection by quantitative more, and this method is easy and simple to handle, and is highly sensitive, and the result is convenient to analyze.Magnificent biotech firm adopts the normal PCR technology first the CT target gene to be increased, again amplified production is hybridized in conjunction with ELISA method and the specific probe that is fixed on enzyme plate, adopt afterwards the titration that develops the color of ELISA method quantitative to carry out CT-DNA, the advantage of this method is not need to acquire extra fluorescent PCR instrument, but this method complex operation easily causes operational pollution.The product that Roche company releases the earliest is the analysing amplified product of electrophoretic method, thereafter, reagent technical significant improvement has been arranged, it is apparent that the improvement of the method that product is detected most, has formally released the PCR test kit based on the ELISA principle after its electrophoretic method reagent.At present, many companies are arranged again at the CTPCR test kit of development based on the fluorescence energy transfer principle, so far, the detection that round pcr is used for chlamydia trachomatis has developed into the very ripe stage.
Summary of the invention
The object of the invention provides a kind of test kit that is applicable to the rapid detection of chlamydia trachomatis in the clinical sample, can be used for auxiliary diagnosis and the curative effect monitoring of chlamydia trachomatis infection.
For achieving the above object, technical scheme of the present invention comprises the PCR reaction solution for the PCR fluorescent quantitation quick detection kit of a kind of chlamydia trachomatis is provided, and described PCR reaction solution contains primer and fluorescent probe; Described primer is divided into upstream primer and downstream primer;
Upstream primer: 5 '-CTGTACATTAGGAGCCACCA-3 ';
Downstream primer: 5 '-CAACAGATTGATCAAAGCTC-3 ';
Fluorescent probe: 5 '-FAM-GATATCTTAAAGGAAATTCAGCATCTTTCAA-TAMRA-3 '.
As the further setting of the present invention, described test kit also comprises archaeal dna polymerase, positive quality control product, weak positive quality control product, negative quality control product, positive quantitatively reference product, DNA extraction liquid.
As the further setting of the present invention, introduce an artificial constructed internal reference system and be used for avoiding the detected result false negative, the internal reference system comprises confidential reference items probe, primer and confidential reference items DNA, and described internal reference system comprises the upstream primer sequence: SEQ ID NO.4, downstream primer sequence: SEQ ID NO.5 and probe sequence: SEQ ID NO.6.
As the further setting of the present invention, described internal reference system is Arabidopis thaliana internal reference system.
As the further setting of the present invention, described positive quality control product concentration is 10 times of gradients 10
6Copies/mL-10
7Copies/mL, described weak positive quality control product concentration is 10 times of gradients 10
3Copies/mL-10
4Copies/mL.
As the further setting of the present invention, described archaeal dna polymerase comprises tap enzyme, UNG enzyme.
Another technical scheme of the present invention is for providing the PCR fluorescent quantitation method for quick of a kind of chlamydia trachomatis, wherein the pcr amplification the primer is: upstream primer sequence such as SEQ ID NO.1, downstream primer sequence such as SEQ ID NO.2, fluorescent probe sequence such as SEQ ID NO.3.
As the further setting of aforesaid method, described fluorescent probe 5 ' end flag F AM fluorophor, 3 ' end mark TAMRA quenching group.
CT fluorescent PCR immue quantitative detection reagent box of the present invention and method have been utilized Taqman core technology platform and Arabidopis thaliana internal reference system, and can effectively remove the inhibition that suppresses pcr amplification, and it is quantitatively more accurate to make.Compare with the simple PCR detection by quantitative in laboratory, this product accuracy is high, high specificity, and in repeatability, stability, sensitivity and precision very large improvement has been arranged, and improves, and shows that it possesses following performance:
(1) accuracy: detect the internal control reference product Z1-Z10 of enterprise sample, the full yin and yang attribute coincidence rate of result is 100%.
(2) specificity: detect the internal control reference product T1-T10 of enterprise sample results all negative.
(3) sensitivity: the minimum detectability of energy stable detection CT-DNA is 5.0 * 10
2Copies/mL.
(4) precision: detect the internal control reference product S1-S4 of enterprise, each sample repeats 5 times, calculates batch interpolation and the equal CV value of difference between batch≤10%.
(5) stability: keeping life is 6 months, and product performance are stable before the deadline.
Description of drawings
Fig. 1: sensitivity reference product detected result figure;
Fig. 2: precision reference product detected result figure;
Fig. 3: positive accuracy Z1-Z5 reference product detected result figure;
The negative accuracy reference product of Fig. 4: Z6-Z10 detected result figure;
Fig. 5: specificity T 1-T10 reference product detected result figure;
The negative accuracy internal reference of Fig. 6: Z6-Z10 reference product detected result figure
Fig. 7: specificity T 1-T10 internal reference reference product detected result figure;
Fig. 8: positive control, critical positive control, negative control reference product detected result figure in the test kit;
Fig. 9: test kit Plays product 1e4-1e7 reference product detected result figure;
Figure 10: typical curve 1e4-1e7 reference product detected result figure.
Embodiment
This test kit adopts polymerase chain reaction (PCR) and fluorescence labeling probe technology, chlamydia trachomatis characteristic sequences in the rapid detection clinical sample, thereby the existence of judgement chlamydia trachomatis.
We design manyly to primer altogether to conserved sequence, and have carried out detailed comparison, have selected at last all primers preferably of sensitivity, specificity and selectivity.We have designed the general dicyclo probe that detects the CT gene in the primer amplification fragment, use the FAM mark, and probe is carried out comprehensive Quality Control and preliminary experiment.
Needed universal tag sequence in two amplification systems, we are when design, 20 GC content of Random Design are about 55%, four kinds of base stochastic distribution of ATGC, the oligonucleotide sequence of base quantity between 18-22bp, use software Primer 5.0 analyze the primer self structure and and sudden change special primer and dicyclo probe between the cross-dimerization body form situation, therefrom select self secondary structure minimum, the lower primer of cross-dimerization body Δ G, then Blast determines existing without the kind attribute with without similar nucleotide sequence of Tag primer on the NCBI website.
Composition and the configuration of embodiment 1 test kit
1. main raw material(s) is originated and the preparation method
CT primer and the probe sequence of this test kit are as follows:
Upstream primer: 5 '-CTGTACATTAGGAGCCACCA-3 ';
Downstream primer: 5 '-CAACAGATTGATCAAAGCTC-3 ';
Fluorescent probe:
5′-FAM-GATATCTTAAAGGAAATTCAGCATCTTTCAA-TAMRA-3′。
The internal reference of this test kit (Suc) primer upstream primer sequence: SEQ ID NO.4, downstream primer sequence: SEQ ID NO.5 and probe sequence: SEQ ID NO.6, specific as follows:
Upstream primer: 5 '-TCATCGTCGCTGGAGCTGGTT-3 ';
Downstream primer: 5 '-CGGCGGTTTGTCAAGCTGAT-3 ';
Fluorescent probe:
5′-HEX-CTTCTTATAGTCACTGCACTAAACTGGAT-TAMRA-3′。
Described test kit also comprises archaeal dna polymerase, strong positive quality control product, weak positive quality control product. negative quality control product, positive quantitatively reference product, DNA extraction liquid.
1.1. primer
Be used for the primer of PCR reaction, ultraviolet detection is A260nm as a result: A280nm 〉=1.5 can be considered qualified primer.-20 ℃ of preservations.
1.2. probe
The CT probe: at oligonucleotide 5 ' end flag F AM, 3 ' end mark BHQ, ultraviolet detection is A260nm as a result: there is absorption peak A280nm 〉=1.5 at the excitation wavelength 520nm place of FAM fluorescein.-20 ℃ of preservations.
SUC II probe: at oligonucleotide 5 ' end mark HEX, 3 ' end mark BHQ, ultraviolet detection is A260nm as a result: there is absorption peak A280nm 〉=1.5 at the excitation wavelength 555nm place of HEX fluorescein.-20 ℃ of preservations.
1.3.DNA polysaccharase (containing 10 * PCR buffer.MgCl2)
Concentration 5U/ μ l contains 10 * PCR Buffer.25mmol/LMgCl
2, according to supplier's quality standard: this product has dna polymerase activity, without 3 ' → 5 ' exonuclease activity and endonuclease activity; The tool thermostability, 94 ℃ of insulations still kept 50% activity after 1 hour.-20 ℃ of preservations.
1.4.dN(U)TP
Comprise: dATP, dCTP, dGTP, dUTP.Be that HPLC is pure according to supplier's quality standard, pollute without DNase and RNase.-20 ℃ of preservations.
1.5.UNG enzyme
Use FermentasIntenational INC. (MBI company) product.Check its technical parameter: SDS-PAGE and detect, purity＞95% has the characteristic in dUTP site in the degrade specifically DNA chain, and 95 ℃ can be with its deactivation after 10 minutes.The quality inspection of UNG enzyme and primer carry out simultaneously, and the detection performance meets primer raw material quality standard and can be used for products production.
1.6. pure water
Use the pure water of Tai Pu company preparation, the limpid inclusion-free of visual inspection is measured its resistivity value more than 1.0M Ω/cm through DDB-305 type electric conductivity instrument.
The quality inspection of pure water and primer quality inspection are carried out simultaneously, and Performance Detection meets primer raw material quality standard can be used for products production.
2.PCR the preparation of reaction solution
2.1 prepare before the dosing
(1)dN(U)TP
DATP, dUTP, dGTP, dCTP with the 100mM that buys, according to 1: 1.5: 1: 1 (volume ratio) mixed, each constituent concentration of mixed solution is respectively: 10mM.15mM.10mM.10mM gets the 0.5mL centrifuge tube, is distributed into the mother liquor of every pipe 100 μ L dN (U) TP.
(3) primer
With the synthetic centrifugal 1min of primer 13000rpm, add an amount of pure water (calculating according to resultant quantity) primer is dissolved into 100 μ M, get the 0.5mL centrifuge tube, be distributed into the primer mother liquor of every pipe 100 μ L.Produce the front an amount of pure water of adding that needs and be mixed with 10 μ M use liquid.
(3) probe
With the synthetic centrifugal 1min of probe 13000rpm, add an amount of pure water (calculating according to resultant quantity) probe is dissolved into 100 μ M, get the 0.5mL centrifuge tube, be distributed into the probe mother liquor of every pipe 100 μ L.Produce the front an amount of pure water of adding that needs and be mixed with 10 μ M use liquid.
2.2.PCR reaction solution dosing
1. be PCR reaction solution prescription of the present invention such as table 1.
Table 1:CT-PCR reaction solution prescription
| Sequence number | Component | Initial concentration | Quantity |
| 1 | Ultrapure water | / | 14.95ul |
| 2 | 10×buffer | / | 9ul |
| 3 | Mg 2+ | 25mM | 10ul |
| 4 | dN(U)TP | 10Mm、15mM | 1.0ul |
| 5 | CT-primer F | 10uM | 2.5ul |
| 6 | CT-primer R | 10uM | 2.5ul |
| 7 | CT-probe | 10uM | 0.25 |
| 8 | Suc II-primer F | 10uM | 0.5ul |
| 9 | Suc II-primer R | 10uM | 0.5 |
| 10 | Suc II-probe | 10uM | 0.3ul |
| 11 | Methane amide | / | 2.5ul |
2.PCR reaction solution packing
Above-mentioned PCR reaction solution is sub-packed in the centrifuge tube of 1.5mL by every pipe 1056 μ L, performs sign, preserve in the refrigerator below-18 ℃, after the packing of product, send quality inspection section to carry out the finished product quality inspection.
3.DNA polysaccharase system configurations
Taq archaeal dna polymerase (5U/ μ L) and the UNG enzyme (1U/ μ L) bought mix according to 1: 1 (volume ratio), add 1 μ L10 in per 10 μ L mixed solutions
5Be packed as 24 μ L/ pipe behind the copies/mLsuc II plasmid mixing, perform sign, preserve in the refrigerator below-18 ℃, after the packing of product, send quality inspection section to carry out quality inspection.
4.CT DNA extraction liquid is produced
CT DNA extraction liquid: preparing final concentration according to turnout with pure water is 10mMTris-HCl, 25mMNaOH, 1% Triton-100 (v/v), 1% (v/v) NP-40,0.1mM EDTA.5% (w/v) chelex-100 solution, be sub-packed in the 2mL cryopreservation tube with the 1mL/ pipe, labelled, stand-by.
Every production 1000ml CT DNA extraction liquid, prepare with the sterilization pure water according to following table: with the packing of 1.2mL/ pipe, labelled, 2-8 ℃ of storage send quality inspection section to carry out quality inspection after the packing of product.
Table 2:CT DNA extraction liquid formula
| The component composition | Every 1000ml |
| NaOH | 1g |
| 1mol/L Tris-Cl pH 8.3 | |
| NP | |
| 40 | 10ml |
| Triton X 100 | 10ml |
| Chelex 100 | 20g |
| 0.5mol/L EDTA pH 8.0 | 0.2ml |
| The sterilization purified water | Be settled to 1000ml |
The preparation of 1mol/L Tris-HCl (pH 8.3): electronic balance takes by weighing Tris alkali, with the purified water dissolving, places solution and makes it be cooled to room temperature.Drip dense HCl, regulate pH with accurate pH meter.
0.5mol/L the preparation of EDTA (pH8.0): electronic balance takes by weighing EDTA-Na22H2O, with the purified water dissolving, takes by weighing NaOH and adds, and regulates pH, constant volume with accurate pH meter. sterilization, 2-8 ℃ of storage after the room temperature cooling.
The preparation of DNA extraction liquid: add successively NaOH, Chelex 100,1mol/L Tris-Cl (pH8.3), Triton X100, NP 40 and 0.5mol/L EDTA (pH8.0), 2-8 ℃ of storage behind the constant volume.
The use of embodiment 2 test kits
A. reagent is prepared (reagent area in preparation)
1. take out DNA extraction liquid, for subsequent use.
2. after determining that the reaction tubes that need to carry out is counted n (sample number+feminine gender+positive quality control product+weak positive quality control product), take out the CT-PCR reaction solution and draw n * 44 μ l CT-PCR reaction solutions, n * 1 μ l archaeal dna polymerase system, add a centrifuge tube and the mixing that vibrates, instantaneous centrifugal after, minute be filled to n PCR reaction tubes, every pipe 45 μ l, transfer to sample application zone behind the lid upper tube cap, it is for subsequent use that lucifuge is put 4 ℃ of refrigerators.
3. quality control product and quantitative reference product are transferred to the sample preparation district, be put in 4 ℃ of refrigerators for subsequent use.
B. sample process (sample process district)
The sample requirement
1. be suitable for sample type: reproductive tract. urethral secretions swab etc.
2. sample collection:
2.1 male urethra sample: get urethral secretions or stretch into approximately 2-4cm of urethra with tiny swab, rotation obtains secretory product gently, puts back to airtight censorship in the pipe.
2.2 female urethra sample: clean urethral orifice with stroke-physiological saline solution first, again with sterile swab insert urethra approximately the rotation of 2cm place obtain the secretory product sample, put back to airtight censorship in the pipe.
2.3 female genital tract sample: wipe secretory product too much in the reproductive tract with stroke-physiological saline solution first, aseptic terylene swab be positioned over reproductive tract low level 1/3 place, along the reproductive tract wall gently rotation obtain secretory product, put back to airtight censorship in the pipe.
3. Sample preservation and censorship: sample is censorship at once, should not surpass 6 days 4-25 ℃ of preservation, and the on-liquid sample can be preserved 6 months at-20 ℃, should use ice bag during the long-distance censorship of sample.
1. sample to be tested is processed:
1.1. add the 1ml sterile saline in the swab pipe, fully vibration shakes up, and extracts swab;
1.2. the whole liquid rotatings after 1.1 processing are moved on in the 1.5ml centrifuge tube (more such as secretory product, as only to get 200ul), centrifugal 5 minutes of 10000rpm;
1.3. remove supernatant, in precipitation, add the 1ml sterile saline, abundant mixing, centrifugal 5 minutes of 10000rpm;
1.4. remove supernatant, in precipitation, add the abundant mixings of 50 μ lDNA extracting solutions (extracting solution includes water-fast material, fully draws behind the mixing), 100 ℃ of constant temperature were processed 10 minutes;
1.5.10 centrifugal 5 minutes of 000rpm, supernatant liquor are used for the PCR reaction.
C, quality control product sample process
CT positive quality control product (1.0 * 10
7Copies/ml), system adopts the plasmid that contains the CT goal gene, purpose fragment source is to obtain by PCR method in the chlamydia trachomatis of being bought by ATCC (American Type Culture Collecti), scaling method after to be ultraviolet spectrophotometer carry out quantitatively to the plasmid suspension of high density gradient dilution to positive reference product desired concn.
The weak positive quality control product (1 * 10 of CT
4Copies/ml), system adopts the plasmid that contains the CT goal gene, purpose fragment source is to obtain by PCR method in the chlamydia trachomatis of being bought by ATCC (American Type Culture Collecti), scaling method after to be ultraviolet spectrophotometer carry out quantitatively to the plasmid suspension of high density gradient dilution to positive reference product desired concn.
The positive quantitatively reference product of CT, system adopts the plasmid that contains the CT goal gene, purpose fragment source is to obtain by PCR method in the chlamydia trachomatis of being bought by ATCC (American Type Culture Collecti), scaling method after to be ultraviolet spectrophotometer carry out quantitatively to the plasmid suspension of high density gradient dilution to positive reference product desired concn.Positive quantitatively reference product DNA concentration is respectively P1:1.0 * 10
7Copie s/ml, P2:1.0 * 10
6Copies/ml, P3:1.0 * 10
5Copies/ml, P4:1.0 * 10
4Copies/ml.
1. take out negative quality control product, instantaneous centrifugal, get 50 μ l in the 1.5ml centrifuge tube, add 50 μ l DNA extraction liquid, abundant mixing, 100 ℃ of constant temperature were processed 10 minutes;
Go to 4 ℃ and leave standstill 10-20min, centrifugal 5 minutes of 10 000rpm, supernatant liquor are used for the PCR reaction.
2. positive quality control product sample process: (with negative quality control product).
3. weak positive quality control product sample process: (with negative quality control product).
4. quantitatively reference product is processed: instantaneous centrifugal, for subsequent use.
The d.PCR reaction
1. application of sample (sample preparation district or sample application zone)
In ready PCR reaction solution pipe, add respectively the testing sample of handling well, negative quality control product sample, positive quality control product, weak positive quality control product sample supernatant liquor 5 μ l, or directly add quantitative reference product 5 μ l, cover tightly the pipe lid rear instantaneous centrifugal.
2.PCR amplification (detection zone)
Ready PCR reaction tubes is positioned on the PCR instrument, and editing sample information is also pressed tabulation 3 loop parameters and is carried out amplified reaction:
Table 3PCR loop parameter
Comprise in the CT-PCR reaction system: CT detects and internal reference.
3, interpretation of result condition is set
1.ABI 7500 baselines (baseline) are set: get 2 and arrive front 3 cycle values of sample threshold value (threshold) as baseline.Threshold value (threshold) setting principle is with the vertex of threshold line just above normal negative quality control product amplification curve (random noise line), and namely the negative quality control product of Ct=30 or " Undet. " are as the criterion.
2.STRATAGENE the Mx3000P baseline is set: select " being fit to baseline (Adaptive baseline) " fluorescent signal when setting.Threshold value (threshold) setting principle is with the vertex of threshold line just above normal negative quality control product amplification curve (random noise line), and namely the negative quality control product of Ct=30 or " No Ct " are as the criterion.
Quality control standard
This test kit is cloudy. and positive quality control product should meet the following conditions simultaneously, otherwise it is invalid to be considered as this experiment:
Negative quality control product: CT (FAM) Ct value=30 or " No Ct " (Mx3000P) or " Undet. " (ABI 7500), internal reference (HEX) Ct value＜30, and preferably Increasing Curve of Logarithm is arranged.
2. positive quality control product: CT (FAM) has preferably Increasing Curve of Logarithm.Quantitatively reference value is at 1.0 * 106copies/ml-1.0 * 107copies/ml.
3. weak positive quality control product: CT (FAM) has preferably Increasing Curve of Logarithm.Quantitatively reference value is 1.0 * 10
3Copies/ml-1.0 * 10
4Copies/ml.
The result judges
1.CT Ct value=40 or " No Ct " (Mx 3000P) or " Undet. " (ABI 7500); And internal reference (HEX) channel C t value＜30, and preferably Increasing Curve of Logarithm is arranged, then the dna content of CT is less than detecting lower limit.
2.CT Ct value＜40, and preferably Increasing Curve of Logarithm is arranged, then by the following method interpretation:
2.1. sample C＜5.00E+002, the dna content of CT＜5.0 * 102copies/ml then, then the CT dna content of sample is that this quantitative data of C copies/ml is only for reference.
2.2. sample 5.00E+002≤C≤5.00E+008, the then dna content of CT=C gene copy.
2.3. the dna content of sample CT＞5.0 * 108copies/ml quantitatively can be diluted to sample in the linearity range such as need and to redeterminate.
3.CT Ct value=40 or " No Ct " are (Mx3000P) or " Undet. " (ABI 7500); And internal reference (HEX) Ct value=40 or " NoCt " (Mx3000P) or " Undet. " (ABI7500), it is invalid then to reflect, needs again to detect.
3 test kit Performance Evaluations of embodiment
Positive quantitatively reference product DNA concentration is respectively P1:1.0 * 10
7Copies/ml, P2:1.0 * 10
6Copies/ml, P3:1.0 * 10
5Copies/ml, P4:1.0 * 10
4Copies/ml.
Select to have on the common disease substance of identical infection site and the taxonomy with the nearer chlamydozoan of CT as the specificity reference product with CT, adopt the reference culture of deactivation, reference culture available from Chinese pharmaceutical biological product identify or U.S. ATCC.10 species specificity reference products are followed successively by: T1 Candida albicans, T2 streptococcus aureus, T3B family suis, T4 chlamydia psittaci, T5 Chlamydia pneumoniae, T6 Ureaplasma urealyticum, T7 mycoplasma genitalium, T8 mycoplasma hominis, T9 intestinal bacteria, T10 staphylococcus epidermidis.This 10 species specificity reference product is detected, and all specificity reference product detected results should be all negative, false positive must not occur.
With the positive quantitatively reference product of CT as precision reference product P1:1.0 * 10
7Copies/ml, P2:1.0 * 10
6Copies/ml, P3:1.0 * 10
5Copies/ml, P4:1.0 * 10
4Copies/ml detects, and repeats 5 times, and concrete operations are carried out according to this reagent kit product working instructions.The result should be: withinrun precision and betweenrun precision all should satisfy CV value≤10%.
CT sensitivity reference product (5.0 * 10
4Copies/mL), be comprised of the plasmid that contains the CT goal gene, to be ultraviolet spectrophotometer carry out quantitatively the plasmid suspension of high density scaling method.
With the DNA diluent with above-mentioned sample gradient dilution be: S1:5.0 * 10
4Copies/mL, S2:5.0 * 10
3Copies/mL, S3:5.0 * 10
2Copies/mL, S4:5.0 * 10
1Copies/mL is as the sensitivity reference product.
Use the sensitivity reference product to detect detection limit S3:5.0 * 10
2Copies/mL should all detect.
Accuracy reference product Z1-Z5 is detected by clinical " gold standard " to be defined as the positive, the clinical clinical sample isolated strains of deactivation.Accuracy reference product Z6-Z10 is detected by clinical " gold standard " to be defined as feminine gender, the clinical clinical sample isolated strains of deactivation.
Use the accuracy reference product to detect, the yin and yang attribute coincidence rate is 100%.
The composition of table 4 reference product
This product quality control system mainly comprises final product quality control and two parts of raw material quality control.Owing to all do not have at present the CT standard substance both at home and abroad, so the present invention having prepared a cover work reference material for quality control.Starting material and finished product all carry out performance detecting with this cover work reference material, in conjunction with the quality control standard of the Erecting and improvings such as other indexs such as outward appearance calibrating.
By this product accuracy, specificity, repeatability, stability, sensitivity and precision are studied, show that it possesses following performance: see also:
Fig. 1: sensitivity reference product detected result figure;
Fig. 2: precision reference product detected result figure
Fig. 3: positive accuracy Z1-Z5 reference product detected result figure;
The negative accuracy reference product of Fig. 4: Z6-Z10 detected result figure;
Fig. 5: specificity T 1-T10 reference product detected result figure;
The negative accuracy internal reference of Fig. 6: Z6-Z10 reference product detected result figure
Fig. 7: specificity T 1-T10 internal reference reference product detected result figure;
Fig. 8: positive control, critical positive control, negative control reference product detected result figure in the test kit;
Fig. 9: test kit Plays product 1e4-1e7 reference product detected result figure;
Figure 10: typical curve 1e4-1e7 reference product detected result figure.
(1) accuracy: detect the internal control reference product Z1-Z10 of enterprise sample, the full yin and yang attribute coincidence rate of result is 100%.
(2) specificity: detect the internal control reference product T1-T10 of enterprise sample results all negative.
(3) sensitivity: the minimum detectability of energy stable detection CT-DNA is 5.0 * 10
2Copies/mL.
(4) precision: detect the internal control reference product S1-S4 of enterprise, each sample repeats 5 times, calculate batch interpolation and batch between equal CV value≤10%.
(5) stability: keeping life is 6 months, and product performance are stable before the deadline.
The above only is embodiments of the invention; be not so limit claim of the present invention; every equivalent structure or equivalent flow process conversion that utilizes specification sheets of the present invention and accompanying drawing content to do; or directly or indirectly be used in other relevant technical fields, all in like manner be included in the scope of patent protection of the present invention.
Sequence table
Claims (1)
1. the PCR fluorescent quantitation quick detection kit of a chlamydia trachomatis comprises the PCR reaction solution, it is characterized in that, described PCR reaction solution contains primer and fluorescent probe; Described primer is divided into upstream primer and downstream primer:
Upstream primer: 5 '-CTGTACATTAGGAGCCACCA-3 ';
Downstream primer: 5 '-CAACAGATTGATCAAAGCTC-3 ';
Fluorescent probe: 5 '-FAM-GATATCTTAAAGGAAATTCAGCATCTTTCAA-TAMRA-3 ';
Described test kit also comprises archaeal dna polymerase system, positive quality control product, weak positive quality control product, negative quality control product, positive quantitatively reference product, DNA extraction liquid, and described archaeal dna polymerase system comprises taq archaeal dna polymerase and UNG enzyme;
Described PCR reaction solution also comprises Arabidopis thaliana internal reference system, and described internal reference system comprises upstream primer: SEQ ID NO.4, downstream primer: SEQ ID NO.5 and probe: SEQ ID NO.6.
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| CN103993085A (en) * | 2014-05-25 | 2014-08-20 | 浙江省医疗器械研究所 | Specific primers, probe and method used for detecting chlamydia trachomatis (CT) |
| CN110157820A (en) * | 2019-04-28 | 2019-08-23 | 深圳市国赛生物技术有限公司 | A kind of primer, probe and kit detecting chlamydia trachomatis |
| CN114317785A (en) * | 2021-12-24 | 2022-04-12 | 苏州中科先进技术研究院有限公司 | Primer probe composition, kit and method for detecting gonococcus |
| CN114369672A (en) * | 2021-12-28 | 2022-04-19 | 苏州中科先进技术研究院有限公司 | Primer probe composition, kit and method for detecting chlamydia trachomatis |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006001972A2 (en) * | 2004-05-28 | 2006-01-05 | Stratagene California | Compositions and methods for the diagnosis of group b streptococcus infection |
| WO2007137650A1 (en) * | 2006-05-29 | 2007-12-06 | Bio-Rad Pasteur | Real-time multiplex detection of three bacterial species responsible for sexually-transmitted diseases |
| CN101305101A (en) * | 2005-11-07 | 2008-11-12 | 西门子医疗保健诊断公司 | Chlamydia trachomatis specific oligonucleotide sequences |
| WO2009035177A1 (en) * | 2007-09-14 | 2009-03-19 | Seegene, Inc. | Genotyping of human papilloma viruses by multiplex amplification and size differentiation |
| CN101671674A (en) * | 2009-03-27 | 2010-03-17 | 厦门艾德生物医药科技有限公司 | Annular primer for amplification of nucleic acid and application thereof |
-
2011
- 2011-09-19 CN CN 201110279330 patent/CN102329866B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006001972A2 (en) * | 2004-05-28 | 2006-01-05 | Stratagene California | Compositions and methods for the diagnosis of group b streptococcus infection |
| CN101305101A (en) * | 2005-11-07 | 2008-11-12 | 西门子医疗保健诊断公司 | Chlamydia trachomatis specific oligonucleotide sequences |
| WO2007137650A1 (en) * | 2006-05-29 | 2007-12-06 | Bio-Rad Pasteur | Real-time multiplex detection of three bacterial species responsible for sexually-transmitted diseases |
| WO2009035177A1 (en) * | 2007-09-14 | 2009-03-19 | Seegene, Inc. | Genotyping of human papilloma viruses by multiplex amplification and size differentiation |
| CN101671674A (en) * | 2009-03-27 | 2010-03-17 | 厦门艾德生物医药科技有限公司 | Annular primer for amplification of nucleic acid and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| Development and Validation of a Rotor-Gene Real-Time PCR Assay for Detection, Identification, and Quantification of Chlamydia trachomatis in a Single Reaction;Hamid Jalal, et al;《Journal of Clinical Microbiology》;20060131;第44卷(第1期);206－213 * |
| Hamid Jalal, et al.Development and Validation of a Rotor-Gene Real-Time PCR Assay for Detection, Identification, and Quantification of Chlamydia trachomatis in a Single Reaction.《Journal of Clinical Microbiology》.2006,第44卷(第1期),206－213. |
| 武建国等.PCR检测的室内质控.《医学实验诊断学进展 卷1》.东南大学出版社,2000,245. * |
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Denomination of invention: A PCR fluorescence quantitative rapid detection kit and method for Chlamydia trachomatis Effective date of registration: 20220112 Granted publication date: 20130918 Pledgee: Yao Qingfeng Pledgor: TRIPLEX INTERNATIONAL BIOSCIENCES (CHINA) Co.,Ltd. Registration number: Y2022350000005 |