CN101117646A - Primer, probe and method for detecting human urological genital tract causal agent - Google Patents
Primer, probe and method for detecting human urological genital tract causal agent Download PDFInfo
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Abstract
The present invention discloses a guiding object and a probe for testing the human urogenital tract pathogens, and a method for testing the human urogenital tract pathogens using the guiding object and the probe. The present invention can test three different pathogens including Neisseria gonorrhoeae, mycoplasma ureaplasma and chlamydia trachomatis in a same reaction tube simultaneously. The present invention not only can increase the early detection rate of the sexually transmitted diseases, but also can reduce the operating time of the medical personnel, lower the cost, and lighten the economic burden of the patient.
Description
Technical field
The present invention relates to the check of pathogenic agent in people's urogenital tract secretory product, relate in particular to a kind of primer, probe and method that detects human urological genital tract causal agent.
Background technology
Progressively intensification along with China's open degree, add that society goes up that some people is subjected to the influence of " free love " thought and to the ignorant of sexually transmitted disease (STD), new std patient has appearred successively in some open cities and developed area, flow of personnel between the current in addition various places is very frequent, and the generation of venereal disease and propagation are the gesture that grows in intensity.Report venereal disease (except the HIV/AIDS) 809,550 examples as the common accumulative total of 31 provinces in the whole nation (municipality directly under the Central Government, autonomous region) in 2004, just risen 10% last one year.
National venereal disease epidemic situation analysis report showed in 2004, and Diplococcus gonorrhoeae (is called for short gonococcus, NG), Ureaplasma urealyticum (UU), chlamydia trachomatis (CT) become three kinds of main pathogenic agent that cause venereal disease.To partly prescription on individual diagnosis person's detection demonstration of this city, the patient who merges 2 kinds or 3 kinds pathogenic infections increases year by year.Sexually transmitted disease (STD) is very big to the hazardness of human health.Particularly to the young adult, consequence is even more serious, and the meeting that has causes lifelong health problem, comprises infertile, sterile, chronic pain and the danger that increases aids infection, and people's physical and mental health and family social constituted serious threat.Sexually transmitted disease (STD) is carried out early monitoring and diagnosis, is an important component part in the prevention and treatment of venereal diseases work, for improving the venereal disease curative ratio, shortens the course of disease and infective stage, control and elimination contagium, and spreading of disease of prevention property has important effect.
The method of inspection to Diplococcus gonorrhoeae, Ureaplasma urealyticum and chlamydia trachomatis mainly contains plate coating checking, bacterium and cell cultures, serological test and PCR method at present.Plate coating checking is to detect gonococcal main method, but the state of urethral secretions can influence the susceptibility of detection; Bacterium and cell cultures be with specimen inoculation in suitable medium or cell, make its amplification, and further make evaluation.Culture method specificity height can detect the pathogenic agent of living in patient's secretory product, can be in conjunction with drug sensitive test as the judgement of curative effect of medication, and the gold standard that past Chang Zuowei detects.But owing to be subjected to the influence of factors such as specialized technology, reagent, equipment, cycle are long, at present clinically generally do not adopt, only carry out scientific research activity, or as the gold standard of new diagnostic reagent evaluation of methodology in R﹠D institution; Serological test can provide quick diagnosis, once is considered to a kind of ideal diagnostic method, but because susceptibility and specificity are not high enough, the expensive price of import antibody and the experiment that can not be used to sieve; The PCR method is used to infect early stage pathogen diagnosis, becomes the new lover of pathogen detection gradually.The PCR method is simple to operate, consuming time less, highly sensitive, as long as just have a copy to be detected in theory.But as extremely sensitive amplification experiment, misoperation very easily pollutes and false-positive result occurs.
The real-time fluorescence quantitative PCR of Chu Xianing (real-time quantitative PCR) technology has realized the leap of PCR from qualitative to quantitative in recent years, and it has solved the past experimentation substantially because of polluting the false positive that occurs.Real-time fluorescence quantitative PCR is meant in the PCR reaction system and adds fluorophor, utilizes the fluorescent signal accumulation whole PCR process of monitoring in real time, the method for by typical curve unknown template being carried out quantitative analysis at last.In the real-time fluorescence quantitative PCR technology, the selection of specificity fluorescent probe and design are vital.It is that the fluorescent chemicals mark is formed fluorescently-labeled dna probe to specific oligonucleotide, combines with the PCR product is specific by probe, can realize homogeneous phase, real-time, detection by quantitative to product in the PCR process.According to the fluorophor mark with realize the different of resonance energy transfer mode, the used probe of fluorescent PCR can be divided into five big classes, is respectively: Taqman probe, molecular beacon, signal primer, hybridization probe, DNA-RNA-DNA chimeric probe.
In the real-time fluorescence quantitative PCR technology, the development of quantitative PCR instrument is another determinative.The polychrome multi-channel detection is current main flow trend, and hyperchannel refers to detect simultaneously the multiple fluorescence in the sample, makes and can detect multi-template simultaneously in single tube.At present, all has multichannel fluorescent PCR instrument in most of hospital.In addition, for satisfying the demand of polychrome multi-channel detection, developed at present and comprised FAM, TET, VIC, the fluorophor that HEX etc. nearly ten kinds.
But at present nearly all what use clinically all is single inspection PCR product, once can only detect a kind of pathogenic agent, be unfavorable for the detection to polyinfection, and operating process is time-consuming relatively, the cost height.
Summary of the invention
One of the technical problem to be solved in the present invention provides a kind of primer that detects human urological genital tract causal agent.
Two of the technical problem to be solved in the present invention provides a kind of probe that detects human urological genital tract causal agent.
Three of the technical problem to be solved in the present invention provides a kind of method that detects human urological genital tract causal agent.
Four of the technical problem to be solved in the present invention provides a kind of test kit that detects human urological genital tract causal agent.
In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:
In one aspect of the invention, provide a kind of primer that detects human urological genital tract causal agent, comprised at least one pair of of following primer centering:
(1) be used to the to increase primer of Diplococcus gonorrhoeae target nucleic acid sequence is right, and its first primer sequence contains at least 15 continuous nucleotides of SEQ ID NO:1, and its second primer sequence contains at least 15 continuous nucleotides of SEQID NO:2;
(2) be used to the to increase primer of Ureaplasma urealyticum target nucleic acid sequence is right, and its first primer sequence contains at least 15 continuous nucleotides of SEQID NO:3, and its second primer sequence contains at least 15 continuous nucleotides of SEQ ID NO:4;
(3) be used to the to increase primer of chlamydia trachomatis target nucleic acid sequence is right, and its first primer sequence contains at least 15 continuous nucleotides of SEQ ID NO:5, and its second primer sequence contains at least 15 continuous nucleotides of SEQ ID NO:6.
Preferably, described primer is to comprising at least: SEQ ID NO:1, SEQ ID NO:2; And/or SEQ ID NO:3, SEQ ID NO:4; And/or SEQ ID NO:5, SEQ ID NO:6.
In another aspect of this invention, provide a kind of probe that detects human urological genital tract causal agent, comprised following at least a kind of oligonucleotide sequence:
(1) oligonucleotide sequence of the target nucleic acid sequence of Diplococcus gonorrhoeae of amplification being hybridized with above-mentioned primer, this sequence comprises at least 15 continuous nucleotides of SEQ ID NO:7 or its complementary sequence;
(2) oligonucleotide sequence of the target nucleic acid sequence of Ureaplasma urealyticum of amplification being hybridized with above-mentioned primer, this sequence comprises at least 15 continuous nucleotides of SEQ ID NO:8 or its complementary sequence;
(3) oligonucleotide sequence of the target nucleic acid sequence of chlamydia trachomatis of amplification being hybridized with above-mentioned primer, this sequence comprises at least 15 continuous nucleotides of SEQ ID NO:9 or its complementary sequence.
Preferably, described probe comprises a kind of oligonucleotide sequence among SEQ ID NO:7, SEQ ID NO:8 and the SEQ IDNO:9 at least.
In another aspect of this invention, provide a kind of method that detects human urological genital tract causal agent, may further comprise the steps:
(1) extracts sample of nucleic acid;
(2) with at least one pair of above-mentioned primer sequence, the nucleic acid that extracts with step (1) is template, the target nucleic acid sequence of amplification pathogenic agent;
(3) detect amplified production, and analytical results.
Preferably, also comprise in the described step (2): adding can with the target nucleic acid sequence probe sequence of hybridizing of step (2) the primer to amplification.
In another aspect of this invention, also provide a kind of test kit that detects human urological genital tract causal agent, having comprised: at least one pair of primer sequence of the present invention.
Preferably, described test kit also comprises at least a probe sequence of the present invention, and the target nucleic acid sequence of this probe sequence and the amplification of contained primer sequence is hybridized.
More preferably, described test kit comprises at least a combination in following primer and the probe combinations:
(1) SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:7;
(2) SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:8;
(3) SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:9.
In the present invention, term " primer " is meant a kind of oligonucleotide, can be natural also can be synthetic, it can be used as induce dna synthetic starting point under certain condition, can bring out synthetic and nucleic acid chains complementary primer extension product under these conditions, promptly in the presence of four kinds of different triphosphoric acid dezyribonucleosides and a kind of polymerization agent (being archaeal dna polymerase or reversed transcriptive enzyme), in a kind of suitable damping fluid and under suitable temperature, carry out above-mentioned synthetic.Preferred primer is the sub-thread oligodeoxyribonucleotide.The appropriate length of primer depends on this primer design purposes, but generally between 15～25 Nucleotide, short primer molecule needs lower temperature usually, thereby forms fully stable hybridization complex with template.Primer needn't reaction template accurate sequence, but must be fully complementary, with template hybridization and to cause DNA synthetic.
In the present invention, term " probe " is meant the one section single stranded DNA or the RNA molecule of the tape label that can discern specific nucleotide sequence, and it only combines with detected specific nucleotide sequence.Probe location is positioned as close to upstream primer.For guaranteeing binding specificity, the appropriate length of probe is generally between 15～45 Nucleotide.
Owing to adopt technique scheme, the primer of detection human urological genital tract causal agent of the present invention, probe and method, can in a reaction tubes, detect Diplococcus gonorrhoeae (NG), Ureaplasma urealyticum (UU), three kinds of different pathogenic agent of chlamydia trachomatis (CT) simultaneously, not only improve the early detective rate of sexually transmitted disease (STD), and minimizing medical worker's operating time, reduce cost, and alleviate patient's economical load.
Description of drawings
Fig. 1 is the electrophorogram of the present invention Diplococcus gonorrhoeae, Ureaplasma urealyticum and the three kinds of pathogenic agent of chlamydia trachomatis that detect;
Fig. 2 is the quantitative fluorescent PCR figure that the present invention detects Diplococcus gonorrhoeae separately;
Fig. 3 is the quantitative fluorescent PCR figure that the present invention detects Ureaplasma urealyticum separately;
Fig. 4 is the quantitative fluorescent PCR figure that the present invention detects chlamydia trachomatis separately;
Fig. 5 is the quantitative fluorescent PCR figure that the present invention detects Diplococcus gonorrhoeae, Ureaplasma urealyticum and three kinds of pathogenic agent of chlamydia trachomatis simultaneously.
Embodiment
The present invention is further detailed explanation below in conjunction with drawings and Examples.
Following examples only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions among the embodiment, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
1. primer of Diplococcus gonorrhoeae and probe
According to the target nucleic acid sequence (Genbank Acession:M10316) of Diplococcus gonorrhoeae, select suitable zone design primer and probe, designed two groups of primers and probe altogether, through experiment relatively, selected wherein one group, concrete sequence is as follows:
NG-F1:5 '-GCT ACG CAT ACC CGC GTT GC-3 ' (SEQ ID NO:1 is positioned at target sequence 3141-3160bp)
NG-R1:5 '-CGA AGA CCT TCG AGC AGA CA-3 ' (SEQ ID NO:2 is positioned at target sequence 3531-3512bp)
NG-F1, the long 391bp (see figure 1) of the amplified production of NG-R1 is corresponding to the cryptic plasmid district of target sequence.
Hybridization probe:
NG-P1:FAM-5 '-CTG TTT AAG TCG TCC AGC TCG TTC-3 '-BHQ1 (SEQ ID NO:7 is positioned at target sequence 3251-3275bp)
After testing, the sensitivity of NG-F1, NG-R1, NG-P1 detection can reach the horizontal (see figure 2) of E3.
2. the primer of Ureaplasma urealyticum and probe
According to the target nucleic acid sequence (Genbank Acession:X51315) of Ureaplasma urealyticum, select suitable zone design primer and probe, designed two groups of primers and probe altogether, through experiment relatively, selected wherein one group, concrete sequence is as follows:
UU-F1:5 '-CAG GTA AAT TAG TAC CAG-3 ' (SEQ ID NO:3 is positioned at target sequence 543-560bp)
UU-R1:5 '-ACG ACG TCC ATA AGC AAC T-3 ' (SEQ ID NO:4 is positioned at target sequence 757-739bp)
UU-F1, the long 215bp (see figure 1) of the amplified production of UU-R1 is positioned at urine enzyme gene regions.
UU-P1:HEX-5 '-CCG TCC TAT CCA AGT TGG ATC ACA-3 '-BHQ1 (SEQ ID NO:8 is positioned at target sequence 643-666bp)
After testing, the sensitivity of UU-F1, UU-R1, UU-P1 detection can reach the horizontal (see figure 3) of E3.
3. primer of chlamydia trachomatis and probe
According to the target nucleic acid sequence (Genbank Acession:CP000052) of chlamydia trachomatis, select suitable zone design primer and probe, designed two groups of primers and probe altogether, through experiment relatively, selected wherein one group, concrete sequence is as follows:
CT-F1:5 '-CTA GGC GTT TGT ACT CCG TCA-3 ' (SEQ ID NO:5 is positioned at target sequence 604-624bp)
CT-R1:5 '-TCC TCA GAA GTT TAT GCA CT-3 ' (SEQ ID NO:6 is positioned at target sequence 803-784bp)
CT-F1, the long 200bp (see figure 1) of the amplified production of CT-R1 is corresponding to the cryptic plasmid district of target sequence.
CT-P1:ROX-5 '-GAG AGA ACG TGC GGG CGA TTT GC-3 '-BHQ2 (SEQID NO:9 is positioned at target sequence 688-711bp)
After testing, CT-F1, CT-R1, the sensitivity that CT-P1 detects can reach the horizontal (see figure 4) of E3.
4. primer synthesizes and purification process
It is synthetic that synthetic method is that solid phase tris phosphite method is carried out chemical dna, and purification process is a polyacrylamide gel electrophoresis, and primer uses that ultraviolet spectrophotometer detects, the amount of resetting before use after dilution, guarantees its A260/A280＞1.7.
5. probe synthesizes and purification process
The synthetic two-step approach that adopts of probe, the first step is connected in 5 of DNA ' end with the tris phosphite method with Reporter.Second step was to adopt the Postlink method Quencher that NHS activates esterification to be connected up (Reporter can use FAM, HEX, TET etc.; Quencher adopts TAMRA, BHQ1, BHQ2 etc. can modify in the middle of 3 ' end or DNA).Unstable in alkaline environment because of considering fluorescence dye, the synthon of employing is Fastamidite, to guarantee the stability of fluorescence.
The probe purification process: employing be HPLC purifying on the PAGE gel electrophoresis basis, the first step is before Postlink connects Quencher, with PAGE most impurity is removed, particularly may remove with the impurity that Quencher is connected to form by product, this helps the raising of Quencher joint efficiency and the purifying work of back; Second step was under certain condition other impurity to be removed with the PAGE electrophoresis after having connected Quencher again, to obtain more highly purified product; The 3rd step promptly crossed dHPLC and carries out purifying, will check it to go out peak position and purity for product behind the purifying, used ultraviolet spectrophotometer quantitative under the 260nm wavelength again.
After dilution, the use ultraviolet spectrophotometer detects probe before use, the amount of resetting, and guarantees its A260/A280＞1.7.
6. other materials
Purified water meets " the main raw and auxiliary material quality control standard of Chinese biological goods " requirement, the Hot-Start enzyme by excusing from death biotech firm provide, dNTP (dATP: dCTP: dGTP: dUTP=25mM: 25mM: 25mM: 25mM), the UNG enzyme provides by Pu Luomaige company.All chemical reagent are analytical pure or the above rank of analytical pure, and all reagent all are up to the standards.
7. reference substance
(1) negative control product: purified water.Alternate test is up to the standards.
(2) positive reference substance: the NG of concentration known, UU, CT pathogenic micro-organism bacterium liquid, the NG of concentration known, UU, CT pathogenic micro-organism plasmid DNA solution.
8. detection method
The present invention uses multiplex PCR fluorescence principle, in same reaction tubes, detect gonococcus, Ureaplasma urealyticum, chlamydia trachomatis simultaneously, in same reaction tubes, add separately primer and probe, because 5 of three kinds of probes ' end is the report fluorescence of mark different wave length respectively, when the PCR reaction is carried out, different report fluorophors discharges fluorescence, and the fluorometric assay instrument reads the fluorescent signal of three kinds of wavelength respectively and analyzes, and the detection of three kinds of pathogenic agent is once finished in the experiment.
Condition to reaction is optimized, and final reaction system and the reaction conditions of determining is as follows:
(1) multiple fluorescence PCR reaction system (selecting 40 μ L reaction systems for use)
Contain in the 40 μ L reaction systems: 2.5 μ L, 10 * PCR reaction buffer (Mg2+free), 3mmol/L Mg2+, 0.125mmol/L dNTP, 0.25 μ mol/L NG-F, 0.25 μ mol/L NG-R, 0.125 μ mol/L NG-P, 0.25 μ mol/L UU-F, 0.25 μ mol/L UU-R, 0.156 μ mol/LUU-P, 0.25 μ mol/L CT-F, 0.25 μ mol/L CT-R, 0.3125 μ mol/L CT-P, Hot-Start polysaccharase 2U, UNG enzyme 0.2U, the template amount is 3 μ L, make up water to 40 μ L.
(2) multiple fluorescence PCR amplification condition
The pcr amplification condition is defined as 42 ℃ 5 minutes, 95 ℃ pre-sex change in 15 minutes; 95 ℃ 15 seconds, 60 ℃ 60 seconds; 37 ℃ of constant temperature.
The PCR instrument that adopts is ABI PRISM
7000 PCR fluorescence detectors, selected detection fluorescence is FAM, HEX, the ROX passage, and when PCR circulation second goes on foot 60 ℃, collect fluorescent signal.Instrument detecting channel selecting passage 1,2,4; Other is a default value.Detected result as shown in Figure 5, in Fig. 5, in the curve of three risings topmost a curve represent NG, intermediary second curve is represented UU, the 3rd curve represented CT.
Per sample, the Ct value of positive control, negative control and critical contrast is determined whether to exist in the sample DNA of three kinds of pathogenic agent, thereby is judged patient's pathogenic infection situation.
1. starting material that test kit of the present invention adopted
(1) primer and probe
Primer of the present invention and probe are responsible for synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
(2) other materials
Purified water meets " the main raw and auxiliary material quality control standard of Chinese biological goods " requirement, the Hot-Start enzyme by excusing from death biotech firm provide, dNTP (dATP: dCTP: dGTP: dUTP=25mM: 25mM: 25mM: 25mM), the UNG enzyme provides by Pu Luomaige company.All chemical reagent are analytical pure or the above rank of analytical pure, and all reagent all are up to the standards.
(3) reference substance
1) negative control product
Purified water.Alternate test is up to the standards.
2) positive reference substance
The NG of concentration known, UU, CT pathogenic micro-organism bacterium liquid, the NG of concentration known, UU, CT pathogenic micro-organism plasmid DNA solution.
2. detection method
(1) sample cracking
Sampling 300 μ l are in the 0.5ml centrifuge tube from the centrifuge tube that has the secretory product suspension liquid, and centrifugal 10 minutes of 15000rpm abandons supernatant; Add lysis buffer 50 μ l.In 100 ℃ of heating 10 minutes, centrifugal 5 minutes of 15000rpm got 3 μ l supernatants and carries out the PCR reaction.
Be noted that the sample after the cracking should be kept at-20 ℃, have a question, should take out duplicate detection as the result, just discardable after clear and definite result has been arranged.Before each repeated experiments, all to will take behind the cracking specimen centrifuge (13000rpm, 5 minutes).
(2) preparation of PCR reaction reagent
1) preparation of primer and probe
Primer, probe are the 50D/ pipe, will be housed the centrifugal several seconds of 1.5ml centrifuge tube 13000rpm of primer or probe, make primer dry powder be gathered in the pipe end, add 500 μ l purified water, left standstill 10 minutes behind the vibration mixing, probe needs lucifuge to leave standstill, and makes it abundant dissolving, the centrifugal several seconds of 13000rpm.
2) preparation of 100 person-portions, three joint inspection PCR reaction solutions
Get 4ml 10 * buffer, 0.2ml 25mmol/L dNTP, 0.2ml 50 μ mol/L NG-F, 0.2ml 50 μ mol/L NG-R, 0.1ml 50 μ mol/L NG-P, 0.2ml 50 μ mol/L UU-F, 0.2ml 50 μ mol/L UU-R, 0.125ml 50 μ mol/L UU-P, 0.2ml 50 μ mol/L CT-F, 0.2ml 50 μ mol/L CT-R, 0.25ml 50 μ mol/L CT-P, be settled to 35ml with purified water, fully mixing.
3) Quality Control of reaction solution
Detect negative internal control product, positive internal control product with reaction solution to be checked, sensitivity internal control product, detected result meets: the CT value of negative internal control product is greater than 38 or do not have the CT value, positive internal control product CT≤28, sensitivity internal control product CT=29～33 then are qualified.
(3) preparation and the Quality Control of Hot-start polysaccharase and UNG enzyme mixed solution (containing 0.5U/ μ l Hot-star polysaccharase and 0.05U/ μ l UNG enzyme)
Get 2500U Hot-start polysaccharase, the mixing of 250U UNG enzyme respectively, be equipped with the 5ml enzyme solution.Alternate test is qualified.
(4) preparation of lysis buffer
Get 1M Tris-HCl (pH8.0) solution 10ml, 0.5M EDTA (pH8.0) 2.0ml, 5M NaCl 20ml, 10%Trition X-100 100ml adds purified water and is settled to 1000ml, fully autoclaving behind the mixing.Alternate test is qualified.
(5) preparation of reference substance
1) preparation of positive reference substance and Quality Control
Get NG, the UU, the CT pathogenic micro-organism plasmid DNA solution that contain concentration known,, measure positive reference substance CT≤28 with enterprise's internal control product with the TE dilution.
2) preparation of critical reference substance
Get NG, UU, the CT pathogenic micro-organism bacterium liquid of concentration known, with 10 times of purified water dilutions.Measure critical reference substance CT＜36 with enterprise's internal control product.
(6) work in-process calibrating
With enterprise's internal control product calibrating.
1) specificity calibrating
Get 8 parts of clinical definites and be all negative sample of NG, UU, CT as the performing PCR test of going forward side by side of negative (specificity) reference material, operation is carried out in strict accordance with the test kit specification sheets---and negative match-rate should be 100%, for qualified.
2) accuracy calibrating
Get clinical definite and be NG, UU, CT and be each 3 parts of male samples as the accuracy reference material, carry out the PCR test, operation is carried out in strict accordance with the test kit specification sheets---and positive coincidence rate answers 100%, for qualified.
3) sensitivity calibrating
With the clinical samples of high titre (107copies/ml concentration with relevant examination criteria product demarcation) with 10 times gradient stepwise dilution to 103copies/ml, get 103copies/ml, 104copies/ml, 105copies/ml, 106copies/ml as enterprise's internal control susceptibility reference material or the 103copies/ml of quantitative standards strain, 104copies/ml, 105copies/ml, 106copies/ml as the susceptibility reference material.Get the Diplococcus gonorrhoeae of 103copies/ml titre, Ureaplasma urealyticum, chlamydia trachomatis mixes, and as the critical reference product of three check reagent, detected result CT meets CT=32～36.
4) precision calibrating
With 1 part of critical contrast, repeat to do 10 times, the CT value variation coefficient must not be greater than 15%.
(7) packing, labeling, packing
1) packing
With micropipet ready reagent branch is packed in the centrifuge tube of cleaning, the tight pipe lid of lid, loading amount is as shown in table 1:
Table 1
| Composition | Sign amount (μ l) | The actual loading amount (μ l) of dividing |
| The PCR reaction solution | 840 | 900 |
| Lysis buffer | 1200 | 1200 |
| Hot-start polysaccharase+UNG enzyme | 48 | 50 |
| |
200 | 200 |
| |
200 | 200 |
| |
20 | 20 |
2) labeling
With the label that is up to the standards, be attached on the corresponding centrifuge tube.
3) packing
According to the composition of test kit, corresponding centrifuge tube is inserted on the box liner, in the box of packing into, put into specification sheets, seal.The test kit assembling requires as shown in table 2 below:
Table 2
| Composition | Quantity (pipe) |
| The |
1 |
| |
1 |
| Hot-start polysaccharase+UNG enzyme |
1 |
| |
1 |
| |
1 |
| |
1 |
3. test kit finished product calibrating
(1) outward appearance calibrating
Calibration method: range estimation.
Standard verification: kit package is perfect, and with specification sheets; Each reagent component is complete, reagent branch loading amount meets composition requirement.
(2) specificity calibrating, accuracy calibrating, sensitivity calibrating, precision calibrating
Examine and determine with work in-process.
4. preserve and validity period
Under-20 ℃ of preservation conditions, examine and determine certainly qualified from validity period be 6 months.
5. test kit operation instruction
Test kit of the present invention adopts nucleic acid amplification technologies combined with fluorescent label probe hybridizing method that the DNA of NG, UU, CT is carried out qualitative detection.
Using method:
(1) reagent is prepared
Press the quantity of sample and required contrast quantity preparation reaction system, be stored in 4 ℃.
(2) sample cracking
Sampling 300 μ l are in the 0.5ml centrifuge tube from the centrifuge tube that has the secretory product suspension liquid, and centrifugal 10 minutes of 15000rpm abandons supernatant; Add lysis buffer 50 μ l.In 100 ℃ of heating 10 minutes, centrifugal 5 minutes of 15000rpm got 3 μ l supernatants and carries out the PCR reaction.
(3) application of sample
In ready PCR reaction solution, add cracking sample supernatant liquor or calibration product respectively.
(4) PCR reaction
Normally exposed installation is decided program, increases on the fluorescent PCR instrument.
Preserve:
In-20 ℃ of preservations, use before the deadline.
Sequence table
＜110〉Shenyou Jianhai Bio-Technology Co., Ltd., Shanghai
＜120〉primer, probe and the method for detection human urological genital tract causal agent
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Claims (9)
1. a primer that detects human urological genital tract causal agent is characterized in that, comprises at least one pair of of following primer centering:
(1) be used to the to increase primer of Diplococcus gonorrhoeae target nucleic acid sequence is right, and its first primer sequence contains at least 15 continuous nucleotides of SEQ ID NO:1, and its second primer sequence contains at least 15 continuous nucleotides of SEQID NO:2;
(2) be used to the to increase primer of Ureaplasma urealyticum target nucleic acid sequence is right, and its first primer sequence contains at least 15 continuous nucleotides of SEQ ID NO:3, and its second primer sequence contains at least 15 continuous nucleotides of SEQ ID NO:4;
(3) be used to the to increase primer of chlamydia trachomatis target nucleic acid sequence is right, and its first primer sequence contains at least 15 continuous nucleotides of SEQ ID NO:5, and its second primer sequence contains at least 15 continuous nucleotides of SEQ ID NO:6.
2. primer as claimed in claim 1 is characterized in that, described primer is to comprising at least: SEQ ID NO:1, SEQ ID NO:2; And/or SEQ ID NO:3, SEQ ID NO:4; And/or SEQ ID NO:5, SEQ ID NO:6.
3. a probe that detects human urological genital tract causal agent is characterized in that, this probe comprises following at least a kind of oligonucleotide sequence:
(1) oligonucleotide sequence of the target nucleic acid sequence of Diplococcus gonorrhoeae of amplification being hybridized with the described primer of claim 1, this sequence comprises at least 15 continuous nucleotides of SEQ ID NO:7 or its complementary sequence;
(2) oligonucleotide sequence of the target nucleic acid sequence of Ureaplasma urealyticum of amplification being hybridized with the described primer of claim 1, this sequence comprises at least 15 continuous nucleotides of SEQ ID NO:8 or its complementary sequence;
(3) oligonucleotide sequence of the target nucleic acid sequence of chlamydia trachomatis of amplification being hybridized with the described primer of claim 1, this sequence comprises at least 15 continuous nucleotides of SEQ ID NO:9 or its complementary sequence.
4. probe as claimed in claim 3 is characterized in that, described probe comprises a kind of oligonucleotide sequence among SEQ IDNO:7, SEQ ID NO:8 and the SEQID NO:9 at least.
5. a method that detects human urological genital tract causal agent is characterized in that, may further comprise the steps:
(1) extracts sample of nucleic acid;
(2) with the described primer sequence of at least one pair of claim 1, the nucleic acid that extracts with step (1) is template, the target nucleic acid sequence of amplification pathogenic agent;
(3) detect amplified production, and analytical results.
6. method as claimed in claim 5 is characterized in that, described step also comprises in (2): adding can with the target nucleic acid sequence probe sequence of hybridizing of step (2) the primer to amplification.
7. a test kit that detects human urological genital tract causal agent is characterized in that, comprising: the described primer sequence of at least one pair of claim 1.
8. test kit as claimed in claim 7 is characterized in that, also comprises the described probe sequence of at least a claim 3, and the target nucleic acid sequence of this probe sequence and the amplification of contained primer sequence is hybridized.
9. test kit as claimed in claim 8 is characterized in that, comprises at least a combination in following primer and the probe combinations:
(1) SEQ ID NO:1, SEQID NO:2 and SEQ ID NO:7;
(2) SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:8;
(3) SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA200710093933XA CN101117646A (en) | 2007-07-06 | 2007-07-06 | Primer, probe and method for detecting human urological genital tract causal agent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA200710093933XA CN101117646A (en) | 2007-07-06 | 2007-07-06 | Primer, probe and method for detecting human urological genital tract causal agent |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101117646A true CN101117646A (en) | 2008-02-06 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA200710093933XA Pending CN101117646A (en) | 2007-07-06 | 2007-07-06 | Primer, probe and method for detecting human urological genital tract causal agent |
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| Country | Link |
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| CN (1) | CN101117646A (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102094072A (en) * | 2009-12-11 | 2011-06-15 | 上海裕隆临床检验中心有限公司 | Fluorescent polymerase chain reaction (PCR) kit for detecting ureaplasma urealyticum infection by SYBR Green method |
| CN102094073A (en) * | 2009-12-11 | 2011-06-15 | 上海裕隆临床检验中心有限公司 | Fluorescent polymerase chain reaction (PCR) kit for detecting chlamydia trachomatis infection by SYBR Green method |
| CN101824471B (en) * | 2009-12-14 | 2012-12-12 | 北京鑫诺美迪基因检测技术有限公司 | Composition and kit for simultaneously detecting mycoplasma urealytium, mycoplasma hominis and mycoplasma genitalium |
| CN107937580A (en) * | 2017-12-26 | 2018-04-20 | 湖南圣湘生物科技有限公司 | The application method of the primer and probe of urogenital tract microorganism detection, kit and kit |
| CN109825615A (en) * | 2019-01-09 | 2019-05-31 | 中生方政生物技术股份有限公司 | Primer, probe, kit and the detection method of chlamydia trachomatis, gonococcus and urea substance Multiple detection |
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| CN113215321A (en) * | 2021-05-26 | 2021-08-06 | 北京毅新博创生物科技有限公司 | Detection product for detecting genital tract pathogen by MALDI-TOF MS and application |
| WO2022228187A1 (en) * | 2021-04-26 | 2022-11-03 | 西南医科大学附属医院 | Kit for detecting ureaplasma urealyticum, chlamydia trachomatis and neisseria gonorrhoeae and method therefor |
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| CN102094072A (en) * | 2009-12-11 | 2011-06-15 | 上海裕隆临床检验中心有限公司 | Fluorescent polymerase chain reaction (PCR) kit for detecting ureaplasma urealyticum infection by SYBR Green method |
| CN102094073A (en) * | 2009-12-11 | 2011-06-15 | 上海裕隆临床检验中心有限公司 | Fluorescent polymerase chain reaction (PCR) kit for detecting chlamydia trachomatis infection by SYBR Green method |
| CN101824471B (en) * | 2009-12-14 | 2012-12-12 | 北京鑫诺美迪基因检测技术有限公司 | Composition and kit for simultaneously detecting mycoplasma urealytium, mycoplasma hominis and mycoplasma genitalium |
| CN107937580A (en) * | 2017-12-26 | 2018-04-20 | 湖南圣湘生物科技有限公司 | The application method of the primer and probe of urogenital tract microorganism detection, kit and kit |
| CN109825615A (en) * | 2019-01-09 | 2019-05-31 | 中生方政生物技术股份有限公司 | Primer, probe, kit and the detection method of chlamydia trachomatis, gonococcus and urea substance Multiple detection |
| CN112301137A (en) * | 2020-02-06 | 2021-02-02 | 广州普世利华科技有限公司 | RDA method and kit for rapidly detecting ureaplasma urealyticum |
| CN112301137B (en) * | 2020-02-06 | 2024-03-22 | 广州普世利华科技有限公司 | RDA method and kit for rapidly detecting ureaplasma urealyticum |
| WO2022228187A1 (en) * | 2021-04-26 | 2022-11-03 | 西南医科大学附属医院 | Kit for detecting ureaplasma urealyticum, chlamydia trachomatis and neisseria gonorrhoeae and method therefor |
| CN113215321A (en) * | 2021-05-26 | 2021-08-06 | 北京毅新博创生物科技有限公司 | Detection product for detecting genital tract pathogen by MALDI-TOF MS and application |
| CN113215321B (en) * | 2021-05-26 | 2022-09-30 | 北京毅新博创生物科技有限公司 | Detection product for detecting genital tract pathogen by MALDI-TOF MS and application |
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