CN102329866A - 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 comprises a 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 one type can pass 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); (biovarlymphogranulomavenereum, LGV), the latter two are relevant with human diseases for trachoma biotype (biovar trachoma) and lymphogranuloma venereum biotype.Use indirect microimmunofluorescence test, the trachoma biotype is divided A.B.Ba.C.D.Da.E.F.G.H.I.Ia.J.K14 serotype again, and the LGV biotype has L1.L2.L2a.L34 serotype again.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 of seeing at most in the venereal disease on the one hand, is that it serious persistent infection phenomenon can occur on the other hand.The highest the scorching male urethra infection with 50% of 70% woman uterus is arranged is asymptomatic carrier, and because of no conscious sympton, these people are a high-risk potential contagium socially.There is the scholar to report,, has 50%～70% chance can obtain choamydiae infection through the baby of the positive mother's birth canal birth of chlamydozoan.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 LPS 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 takes place infects that not have the women that CT infects high 3～5 times, and treatment CT can reduce the propagation of HIV.
As a kind of common parasitic microbe of adult's urogenital tract, CT can cause the multiple disease of human infection.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 handling through cicloheximide (cycloheximide).Cultivation needs 48～72h, observes inclusion body with iodine staining or Giemsa staining.
Like DIF. enzyme immunoassays (EIA) etc. also are to make sample with uterine neck or urethral swab.DIF is directly to measure chlamydial LPS or major outer membrane proteantigen, specificity 96%～100%, susceptibility 80%～85% through the fluorescent mark specific antibody.EIA directly measures the chlamydozoan boivin 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 aforesaid method is when being used for urine examination, and susceptibility has only 40%～50%, therefore is not suitable for examination.
The DNA cloning technology comprises polymerase chain reaction (PCR) and ligase chain reaction (LCR), can directly detect the nucleotide sequence of chlamydia trachomatis plasmid in the sample 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 development of molecular biology; The amplification test of transcriptive intermediate, enzyme amplify methods such as immunoreation, 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; For clinical provide do not have wound, sensitive, detection means fast, 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 conventional P CR technology earlier the CT target gene to be increased; Combine ELISA method and the specific probe that is fixed on enzyme plate to hybridize amplified production again; Adopt the titration that develops the color of ELISA method quantitative afterwards to carry out CT-DNA; The advantage of this method is need not acquire extra fluorescent PCR appearance, but this method complex operation is prone to cause operational pollution.The product that Roche company releases the earliest is the analysing amplified product of electrophoretic method; Thereafter; On reagent technical, significant improvement has been arranged, it is apparent that the improvement of the method that product is detected most, after its electrophoretic method reagent, has formally released the PCR test kit based on the ELISA principle.At present, many companies are arranged at the CTPCR test kit of development based on the fluorescence energy transfer principle again, so far, the detection that round pcr is used for chlamydia trachomatis has developed into the very sophisticated 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 the auxiliary diagnosis and the curative effect monitoring of chlamydia trachomatis infection.
For realizing above-mentioned purpose, technical scheme of the present invention is the PCR fluorescent quantitation quick detection kit that a kind of chlamydia trachomatis is provided, and comprises the PCR reaction solution, and said PCR reaction solution contains primer and fluorescent probe; Said 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 further setting of the present invention, said test kit also comprises archaeal dna polymerase, positive quality control product, weak positive quality control product, negative quality control product, positive quantitatively reference article, DNA extraction liquid.
As further setting of the present invention; Introduce an artificial constructed internal reference system and be used to avoid the detected result false negative; The internal reference system comprises confidential reference items probe, primer and confidential reference items DNA, and said 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 further setting of the present invention, said internal reference system is an Arabidopis thaliana internal reference system.
As further setting of the present invention, said positive quality control product concentration is 10 times of gradients 10
6Copies/mL-10
7Copies/mL, said weak positive quality control product concentration is 10 times of gradients 10
3Copies/mL-10
4Copies/mL.
As further setting of the present invention, said archaeal dna polymerase comprises tap enzyme, UNG enzyme.
Another technical scheme of the present invention is the PCR fluorescent quantitation method for quick that a kind of chlamydia trachomatis is provided; 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, said fluorescent probe 5 ' end flag F AM fluorophor, 3 ' end mark TAMRA quenching group.
CT fluorescent PCR detection by quantitative test kit 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 big improvement has been arranged all, improves, and shows that it possesses following performance:
(1) accuracy: detect the internal control reference article Z1-Z10 of enterprise sample, the full yin and yang attribute coincidence rate of result is 100%.
(2) specificity: it is all negative to detect the internal control reference article T1-T10 of enterprise sample results.
(3) sensitivity: the minimum detectability of ability stable detection CT-DNA is 5.0 * 10
2Copies/mL.
(4) precision: detect the internal control reference article 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 article detect figure as a result;
Fig. 2: precision reference article detect figure as a result;
Fig. 3: positive accuracy Z1-Z5 reference article detect figure as a result;
The negative accuracy reference of Fig. 4: Z6-Z10 article detect figure as a result;
Fig. 5: specificity T 1-T10 reference article detect figure as a result;
The negative accuracy internal reference of Fig. 6: Z6-Z10 reference article detect figure as a result
Fig. 7: specificity T 1-T10 internal reference reference article detect figure as a result;
Fig. 8: positive control, critical positive control, negative control reference article detect figure as a result in the test kit;
Fig. 9: standard substance 1e4-1e7 reference article detect figure as a result in the test kit;
Figure 10: typical curve 1e4-1e7 reference article detect figure as a result.
Embodiment
This test kit adopts polymerase chain reaction (PCR) and fluorescence labeling probe technology, the peculiar sequence of chlamydia trachomatis 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 all primers preferably of sensitivity, specificity and selectivity at last.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; Design 20 GC content at random 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, it is minimum therefrom to select self secondary structure; The lower primer of cross-dimerization body Δ G, Blast confirms that the no kind attribute of label primer exists with no similar nucleotide sequence on the NCBI website then.
The composition and the configuration of embodiment 1 test kit
1. main raw material(s) is originated and the preparation method
The CT primer and the probe sequence of this test kit are following:
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′。
Said test kit also comprises archaeal dna polymerase, strong positive quality control product, weak positive quality control product. negative quality control product, positive quantitatively reference article, 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 resorcinolphthalein.-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 resorcinolphthalein.-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: these article have dna polymerase activity, do not have 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.According to supplier's quality standard is that HPLC is pure, and no DNase and RNase pollute.-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 appearance.
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
With dATP, dUTP, dGTP, the dCTP of the 100mM that buys, according to 1: 1.5: 1: 1 (volume ratio) mixed, and 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 centrifugal 1min of synthetic primer 13000rpm, add an amount of pure water (calculating) primer is dissolved into 100 μ M according to resultant quantity, get the 0.5mL centrifuge tube, be distributed into the primer mother liquor of every pipe 100 μ L.Need to add an amount of pure water before producing and be mixed with 10 μ M use liquid.
(3) probe
With the centrifugal 1min of synthetic probe 13000rpm, add an amount of pure water (calculating) probe is dissolved into 100 μ M according to resultant quantity, get the 0.5mL centrifuge tube, be distributed into the probe mother liquor of every pipe 100 μ L.Need to add an amount of pure water before producing 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 like table 1.
Table 1:CT-PCR reaction solution prescription
| Sequence number | Component | Initial concentration | Quantity |
| 1 | Ultrapure water | / | 14.95 |
| 2 | 10×buffer | / | |
| 3 | Mg 2+ | 25mM | 10ul |
| 4 | dN(U)TP | 10Mm、15mM | 1.0 |
| 5 | CT-primer?F | 10uM | 2.5 |
| 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 | 0.5ul |
| 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 ℃, treat the packing of product after, send quality inspection portion to be carried out to the article 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 ℃, treat the packing of product after, send quality inspection portion to carry out quality inspection.
4.CT DNA extraction liquid is produced
CT DNA extraction liquid: use pure water preparation final concentration to be 10mMTris-HCl, 25mMNaOH, 1% Triton-100 (v/v), 1% (v/v) NP-40,0.1mM EDTA.5% (w/v) chelex-100 solution according to turnout; Be sub-packed in the frozen pipe of 2mL with the 1mL/ pipe; Labelled, for use.
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 treated to send quality inspection portion 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 | 10ml |
| 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, room temperature cooling 2-8 ℃ of storage in back.
The preparation of DNA extraction liquid: add NaOH, Chelex 100,1mol/L Tris-Cl (pH8.3), Triton X100, NP 40 and 0.5mol/L EDTA (pH8.0) successively, 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, subsequent use.
2. after confirming that the reaction tubes that need 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, divide to 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 subsequent use that lucifuge is put 4 ℃ of refrigerators.
3. quality control product and quantitative reference article are transferred to the sample preparation district, be put in 4 ℃ of refrigerators 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 the about 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 SPSS earlier, insert the about 2cm of urethra place's rotation with sterile swab again and obtain the secretory product sample, put back to airtight censorship in the pipe.
2.3 female genital tract sample: earlier wipe secretory product too much in the reproductive tract, aseptic terylene swab be positioned over reproductive tract low level 1/3 place with SPSS, along the reproductive tract wall gently rotation obtain secretory product, put back to airtight censorship in the pipe.
3. sample is preserved and censorship: sample can be inspected by ready samples at once, should not surpass 6 days 4-25 ℃ of preservation, and on-liquid property 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 handled:
1.1. in the swab pipe, add the 1ml sterile saline, fully vibration shakes up, and extracts swab;
1.2. the whole liquid after 1.1 processing are transferred in the 1.5ml centrifuge tube (more like secretory product, as only to get 200ul), centrifugal 5 minutes of 10000rpm;
1.3. remove supernatant, in deposition, add the 1ml sterile saline, abundant mixing, centrifugal 5 minutes of 10000rpm;
1.4. remove supernatant, in deposition, 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 handled 10 minutes;
1.5.10 centrifugal 5 minutes of 000rpm, supernatant 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 through PCR method in the chlamydia trachomatis of being bought by ATCC (American Type Culture Collecti), and to be ultraviolet spectrophotometer carry out quantitatively the back gradient dilution to positive reference article desired concn to the plasmid suspension of high density to scaling method.
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 through PCR method in the chlamydia trachomatis of being bought by ATCC (American Type Culture Collecti), and to be ultraviolet spectrophotometer carry out quantitatively the back gradient dilution to positive reference article desired concn to the plasmid suspension of high density to scaling method.
The positive quantitatively reference article of CT; System adopts the plasmid that contains the CT goal gene; Purpose fragment source is to obtain through PCR method in the chlamydia trachomatis of being bought by ATCC (American Type Culture Collecti), and to be ultraviolet spectrophotometer carry out quantitatively the back gradient dilution to positive reference article desired concn to the plasmid suspension of high density to scaling method.Positive quantitatively reference article 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 handled 10 minutes;
Go to 4 ℃ and leave standstill 10-20min, centrifugal 5 minutes of 10 000rpm, supernatant 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 the reference article are handled: instantaneous centrifugal, subsequent use.
The d.PCR reaction
1. application of sample (sample preparation district or sample application zone)
In ready PCR reaction solution pipe, add the testing sample of handling well respectively, negative quality control product sample, positive quality control product, weak positive quality control product sample supernatant 5 μ l, or directly add quantitative reference article 5 μ l, the tight pipe lid of lid back is instantaneous centrifugal.
2.PCR amplification (detection zone)
Ready PCR reaction tubes is positioned on the PCR appearance, and editor's 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 enactment
1.ABI 7500 baselines (baseline) are set: get 2 and arrive preceding 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), promptly the negative quality control product of Ct=30 perhaps " Undet. " be as the criterion.
2.STRATAGENE the Mx3000P baseline is set: select " being fit to baseline (Adaptive baseline) " to establish periodic fluorescent signal.Threshold value (threshold) setting principle is with the vertex of threshold line just above normal negative quality control product amplification curve (random noise line), promptly the negative quality control product of Ct=30 perhaps " No Ct " be 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 regarded as this experiment:
1. negative quality control product: CT (FAM) Ct value=30 or " No Ct " be " Undet. " (ABI 7500) (Mx3000P) perhaps, internal reference (HEX) Ct value＜30, and logarithmic growth curve is preferably arranged.
2. positive quality control product: CT (FAM) has logarithmic growth curve preferably.Quantitatively reference value is at 1.0 * 106copies/ml-1.0 * 107copies/ml.
3. weak positive quality control product: CT (FAM) has logarithmic growth curve preferably.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) be " Undet. " (ABI 7500) perhaps; And internal reference (HEX) channel C t value＜30, and logarithmic growth curve is preferably arranged, then the dna content of CT is less than detecting lower limit.
2.CT Ct value＜40, and logarithmic growth curve is preferably arranged, then by 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 like need and to redeterminate.
3.CT Ct value=40 or " No Ct " be " Undet. " (ABI 7500) (Mx3000P) perhaps; And internal reference (HEX) Ct value=40 or " NoCt " (Mx3000P) perhaps " Undet. " (ABI7500), it is invalid then to reflect, needs to detect again.
3 test kit Performance Evaluations of embodiment
Positive quantitatively reference article 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 with CT have on common disease substance and the taxonomy of identical infection site with the nearer chlamydozoan of CT as specificity reference article, adopt the reference culture of deactivation, reference culture available from Chinese pharmaceutical biological product evaluation or U.S. ATCC.10 specific specificity reference article are followed successively by: T1 Candida albicans, T2 streptococcus aureus, T3B family suis, T4 chlamydia psittaci, T5 CPN, T6 Ureaplasma urealyticum, T7 mycoplasma genitalium, T8 mycoplasma hominis, T9 intestinal bacteria, T10 staphylococcus epidermidis.These 10 specific specificity reference article are detected, and all specificity reference article detect the result should be all negative, false positive must not occur.
With the positive quantitatively reference article of CT as precision reference article 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 test kit products instruction.The result should be: withinrun precision and betweenrun precision all should satisfy CV value≤10%.
CT sensitivity reference article (5.0 * 10
4Copies/mL), be made up of the plasmid that contains the CT goal gene, to be ultraviolet spectrophotometer carry out quantitatively the plasmid suspension of high density scaling method.
Use the DNA diluent to be: S1:5.0 * 10 with above-mentioned sample gradient dilution
4Copies/mL, S2:5.0 * 10
3Copies/mL, S3:5.0 * 10
2Copies/mL, S4:5.0 * 10
1Copies/mL is as sensitivity reference article.
Use sensitivity reference article to detect lowest detection amount S3:5.0 * 10
2Copies/mL should all detect.
Accuracy reference article Z1-Z5 confirms as male, the clinical clinical sample isolated strains of deactivation for being detected by clinical " gold standard ".Accuracy reference article Z6-Z10 is detected by clinical " gold standard " to confirm as feminine gender, the clinical clinical sample isolated strains of deactivation.
Use accuracy reference article to detect, the yin and yang attribute coincidence rate is 100%.
The composition of table 4 reference article
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 the CT standard substance both at home and abroad at present, so the present invention has prepared a cover job and has been used for quality control with reference to article.Starting material and finished product all use this cover work to carry out the performance calibrating with reference to article, set up perfect quality control standard in conjunction with other indexs such as outward appearance calibrating etc.
Through this product accuracy, specificity, repeatability, stability, sensitivity and precision are studied, show that it possesses following performance: see also:
Fig. 1: sensitivity reference article detect figure as a result;
Fig. 2: precision reference article detect figure as a result
Fig. 3: positive accuracy Z1-Z5 reference article detect figure as a result;
The negative accuracy reference of Fig. 4: Z6-Z10 article detect figure as a result;
Fig. 5: specificity T 1-T10 reference article detect figure as a result;
The negative accuracy internal reference of Fig. 6: Z6-Z10 reference article detect figure as a result
Fig. 7: specificity T 1-T10 internal reference reference article detect figure as a result;
Fig. 8: positive control, critical positive control, negative control reference article detect figure as a result in the test kit;
Fig. 9: standard substance 1e4-1e7 reference article detect figure as a result in the test kit;
Figure 10: typical curve 1e4-1e7 reference article detect figure as a result.
(1) accuracy: detect the internal control reference article Z1-Z10 of enterprise sample, the full yin and yang attribute coincidence rate of result is 100%.
(2) specificity: it is all negative to detect the internal control reference article T1-T10 of enterprise sample results.
(3) sensitivity: the minimum detectability of ability stable detection CT-DNA is 5.0 * 10
2Copies/mL.
(4) precision: detect the internal control reference article 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 is merely 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 be done; 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 (8)
1. the PCR fluorescent quantitation quick detection kit of a chlamydia trachomatis comprises the PCR reaction solution, it is characterized in that, said PCR reaction solution contains primer and fluorescent probe; Said 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 '.
2. press the PCR fluorescent quantitation quick detection kit of the described chlamydia trachomatis of claim 1; It is characterized in that said test kit also comprises archaeal dna polymerase, positive quality control product, weak positive quality control product, negative quality control product, positive quantitatively reference article, DNA extraction liquid.
3. press the PCR fluorescent quantitation quick detection kit of the described chlamydia trachomatis of claim 1; It is characterized in that; Introduced an artificial constructed internal reference system and be used to avoid the detected result false negative, the internal reference system comprises confidential reference items probe, primer and confidential reference items DNA.
4. press the PCR fluorescent quantitation quick detection kit of the described chlamydia trachomatis of claim 3; Said internal reference system is an Arabidopis thaliana internal reference system, and said 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.
5. by the PCR fluorescent quantitation quick detection kit of the described chlamydia trachomatis of claim 1, it is characterized in that said positive quality control product concentration is 10 times of gradients 10
6Copies/mL-10
7Copies/mL, said weak positive quality control product concentration is 10 times of gradients 10
3Copies/mL-10
4Copies/mL.
6. by the PCR fluorescent quantitation quick detection kit of the described chlamydia trachomatis of claim 1, it is characterized in that said archaeal dna polymerase comprises tap enzyme, UNG enzyme.
7. the PCR fluorescent quantitation method for quick of a chlamydia trachomatis is characterized in that 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.
8. by the PCR fluorescent quantitation method for quick of the described chlamydia trachomatis of claim 7, it is characterized in that said fluorescent probe 5 ' end flag F AM fluorophor, 3 ' end mark TAMRA quenching group.
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| 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 |
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| CN102329866B (en) | 2013-09-18 |
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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 |