CN110157820A - A kind of primer, probe and kit detecting chlamydia trachomatis - Google Patents
A kind of primer, probe and kit detecting chlamydia trachomatis Download PDFInfo
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- CN110157820A CN110157820A CN201910359593.3A CN201910359593A CN110157820A CN 110157820 A CN110157820 A CN 110157820A CN 201910359593 A CN201910359593 A CN 201910359593A CN 110157820 A CN110157820 A CN 110157820A
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- primer
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- chlamydia trachomatis
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Abstract
The present invention discloses a kind of primer, probe and kit for detecting chlamydia trachomatis, is related to nucleic acid detection technique field.Fluorescent PCR of the primer for chlamydia trachomatis nucleic acid detects, and the primer includes upstream primer and downstream primer, wherein the nucleotide sequence of the upstream primer is as shown in SEQ ID No:1, and the nucleotide sequence of the downstream primer is as shown in SEQ ID No:2；The nucleotide sequence of the probe is as shown in SEQ ID No:3；The kit includes nucleic acid extraction liquid, PCR reaction solution, archaeal dna polymerase, positive control and negative control, wherein the PCR reaction solution includes reaction buffer, dNTPs, MgCl2, the primer and the probe.The present invention is intended to provide a kind of fluorescent PCR kit for detecting chlamydia trachomatis nucleic acid, has the advantages that specific good, high sensitivity.
Description
Technical field
The present invention relates to nucleic acid detection technique field, in particular to a kind of primer, probe and examination for detecting chlamydia trachomatis
Agent box.
Background technique
Chlamydia trachomatis (CT) is Grain-negative pathogen, is a kind of with unique growth cycle, stringent cytozoicus
Prokaryotic microorganism, it can cause urogenital infections, be one of domestic and international most common sexual reverse.Mesh
It has 15 serotypes for preceding discovery, and different serotype can cause different diseases.Wherein A, B, Ba and the Chlamydia strain energy of c-type
Cause trachoma, L1, L2, L3 type can cause lymphogranuloma venereum, and D-K type then causes non-gonococcal urethritis.CT pathogen
Infection lacks specific symptoms often, easily formation Latent infection, brings difficulty, while Yi Yuqi when Chlamydia propagation to clinical diagnosis
Its microbial pathogens mixed infection, causes the complexity of diagnosing and treating.Therefore, it establishes early stage, fast and accurately detect CT
The method of infection is of great significance.
Fluorescence PCR assay can directly detect the nucleotide sequence of chlamydia trachomatis plasmid or outer membrane protein in sample, tool
There are high accuracy, high specific and high sensitivity.It is many research and practice have shown that, fluorescence used in Fluorescence PCR assay
Probe improves the specificity and sensitivity of detection, and detection CT DNA will be quick compared with pathogen culture method and immunology diagnosis method
Sense, and accurate detection can also be made to symptomless infection person, chlamydia trachomatis (CT) can be infected and make early diagnosis；It is logical
Crossing optimization nucleic acid extraction step makes operation easier rapidly, is suitble to processing great amount of samples.
Have the kit based on real-time fluorescent PCR technology detection CT DNA both at home and abroad at present to be applied in clinical detection,
But part kit is due to sample processing method, primer and probe design site, reaction reagent tolerance is different and causes difference
The false negative of degree causes detection efficiency low, specific not high.
Summary of the invention
The main object of the present invention is to propose a kind of primer, probe and kit for detecting chlamydia trachomatis, it is desirable to provide
A kind of fluorescent PCR kit detecting chlamydia trachomatis nucleic acid has the advantages that specific good, high sensitivity.
In order to achieve the object, the present invention proposes a kind of primer for detecting chlamydia trachomatis, it to be used for chlamydia trachomatis nucleic acid
Fluorescent PCR detection, the primer includes upstream primer and downstream primer, wherein the nucleotide sequence of the upstream primer is such as
Shown in SEQ IDNo:1, the nucleotide sequence of the downstream primer is as shown in SEQ ID No:2.
Further, the invention also provides a kind of probe for cooperating the primer to use, the nucleotides sequences of the probe
Column are as shown in SEQ IDNo:3.
Optionally, 3 ' end label BHQ1 quenching groups of the probe, 5 ' end flag F AM fluorophors of the probe.
In addition, the fluorescent PCR for chlamydia trachomatis nucleic acid detects, the examination the invention also provides a kind of kit
Agent box includes nucleic acid extraction liquid, PCR reaction solution, archaeal dna polymerase, positive control and negative control, wherein the PCR reaction solution
Including reaction buffer, dNTPs, MgCl2, the primer and the probe.
Optionally, the nucleic acid extraction liquid includes nucleic acid extraction liquid I and nucleic acid extraction liquid II, wherein the nucleic acid extraction
Liquid I includes 20%~30% polyethylene glycol and 3M~5M sodium chloride, and the nucleic acid extraction liquid II includes 0.1%~0.5% tween-
20 and 0.1%~0.5% lauryl sodium sulfate.
Optionally, in the kit, the reaction system of PCR reaction is prepared by following volume ratio: 35.5 parts of PCR are anti-
Answer liquid, 0.5 part of archaeal dna polymerase and 4 parts of templates, wherein the template be the positive control, the negative control or
Measuring samples DNA.
Optionally, the PCR reaction solution includes: 1 × reaction buffer, 0.1 μM of dNTPs, 0.5 μM of MgCl2, 0.15 μM draw
Object is to, 0.15 μM of probe and 2.5U archaeal dna polymerase.
In technical solution provided by the invention, by 7 kinds of different serotypes chlamydia trachomatises such as D, E, H, I, J, K, L2
Genome sequence analyzed, it is determined that its highly conserved sequence selects the trpR gene of chlamydia trachomatis as target region,
Devise the primer and probe of specificity for the highly conserved sequence, and there may be homology sequence, can by sexual transmission
The no cross reactions such as other pathogens have specificity well.Compare the fluorescent PCR detection of existing chlamydia trachomatis nucleic acid
Kit, it is proposed by the present invention include the kit of the primer and probe can be in a relatively short period of time to genital discharge
CT-DNA in the unknown samples such as object is fast and accurately detected, and has good specificity, high sensitivity, detection efficiency high and inspection
Survey the wide advantage of range.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
Other relevant attached drawings are obtained according to these attached drawings.
Fig. 1 is curve graph when kit proposed by the present invention detection sample is the positive in embodiment 1；
Fig. 2 is curve graph when kit proposed by the present invention detection sample is feminine gender in embodiment 1；
Fig. 3 is the sequencing result figure of No. 7 samples in embodiment 1；
Fig. 4 is the sequencing result figure of No. 8 samples in embodiment 1；
Fig. 5 is the accuracy testing result figure of kit proposed by the present invention in embodiment 2；
Fig. 6 is the specific test result figure of kit proposed by the present invention in embodiment 3；
Fig. 7 is the sensitivity test result figure of kit proposed by the present invention in embodiment 4；
Fig. 8 is the Precision test result figure of kit proposed by the present invention in embodiment 5；
Wherein, in Fig. 1,2,5,6,7,8, abscissa (Cycle) represents CT value, and ordinate (△ Rn) represents fluorescence amplification.
The embodiments will be further described with reference to the accompanying drawings for the realization, the function and the advantages of the object of the present invention.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Have the kit based on real-time fluorescent PCR technology detection CT DNA both at home and abroad at present to be applied in clinical detection,
But part kit is due to sample processing method, primer and probe design site, reaction reagent tolerance is different and causes difference
The false negative of degree causes detection efficiency low, specific not high.
In consideration of it, the present invention proposes a kind of primer, probe and kit for detecting chlamydia trachomatis, it is desirable to provide Yi Zhongjian
The fluorescent PCR kit for surveying chlamydia trachomatis nucleic acid has the advantages that specific good, high sensitivity.
Specifically, the present invention proposes a kind of primer for detecting chlamydia trachomatis, can be used for the glimmering of chlamydia trachomatis nucleic acid
Light PCR detection, the primer includes upstream primer and downstream primer, wherein the nucleotide sequence of the upstream primer such as SEQ
Shown in IDNo:1, the nucleotide sequence of the downstream primer is as shown in SEQ ID No:2.
Further, the invention also provides a kind of probe, the probe cooperates the primer to use.The core of the probe
Nucleotide sequence is as shown in SEQ IDNo:3.
The gene of following 7 kinds of different serotypes chlamydia trachomatises is obtained from GenBank (www.ncbi.nlm.nih.gov)
Sequence: the strain of chlamydia trachomatis D type, the strain of chlamydia trachomatis E type, the strain of chlamydia trachomatis H-type, the strain of chlamydia trachomatis I type, trachoma clothing are former
The strain of body J-type, the strain of chlamydia trachomatis K-type and chlamydia trachomatis L2 type strain.By NCBI and bioanalysis software to above-mentioned 7 kinds not
The gene order of homologous serotype chlamydia trachomatis is analyzed, and determines the highly conserved sequence of chlamydia trachomatis, selects trachoma clothing
The trpR gene of substance is as target sequence, according to primer and the design principle of TaqMan probe, for the highly conserved sequence
The primer and fluorescence probe of specificity are devised, and in the end the 5' flag F AM fluorophor of probe, base is quenched in the end 3' label BHQ1
Group.Accordingly, the nucleotide sequence of the primer and probe of design is as follows:
Upstream primer: 5 '-GCAGGAAGAGATCCTGATA-3 ' (SEQ IDNo:1)
Downstream primer: 5 '-GACTTTTGGATTCGGGATAA-3 ' (SEQ IDNo:2)
Probe: 5 '-FAM-ATCGGAGGTGGCTCCAACGCTATT-BHQ1-3 ' (SEQ IDNo:3).
By above-mentioned design, the specific primer and probe that a set of amplification target sequence is chlamydia trachomatis are obtained, and by giving birth to
The synthesis of work bioengineering (Shanghai) limited liability company, the sterile 1 × TE buffer of synthetic oligonucleotides dry powder
(pH8.0) 100 μM are dissolved to, -20 DEG C of preservations.
In addition, the invention also provides a kind of kits that the fluorescent PCR for chlamydia trachomatis nucleic acid detects.The examination
Agent box includes nucleic acid extraction liquid, PCR reaction solution, archaeal dna polymerase, positive control and negative control, wherein the PCR reaction solution
Including reaction buffer, dNTPs (dATP, dTTP, dGTP, dCTP), MgCl2, the primer and the probe.
In the present embodiment, the primer includes upstream primer and downstream primer, wherein the nucleotides sequence of the upstream primer
Column are as shown in SEQ IDNo:1, and the nucleotide sequence of the downstream primer is as shown in SEQ ID No:2；The nucleotide of the probe
Sequence is as shown in SEQ IDNo:3.
Since above-mentioned primer and probe is that the genome sequence based on 7 kinds of different serotypes chlamydia trachomatises of analysis obtains
Highly conserved sequence and design, therefore, compare the fluorescence PCR detection reagent kit of existing chlamydia trachomatis nucleic acid, this
The kit that invention proposes has higher specificity, sensitivity and detection efficiency, solves existing kit and is easy to appear
False negative, the problem that specificity is low, detection efficiency is low.
In the present embodiment, the positive control is the recombinant plasmid comprising chlamydia trachomatis distinguished sequence, the special sequence
Be classified as the target sequence of the trpR gene of the chlamydia trachomatis expanded by above-mentioned primer and probe, and by raw work bioengineering (on
Sea) limited liability company's synthesis, the dry powder plasmid of synthesis is dissolved to 10 with 1 × TE buffer (pH8.0)4Copies/ μ L, as
Positive control is simultaneously saved in -20 DEG C.
In the present embodiment, the nucleic acid extraction liquid includes nucleic acid extraction liquid I and nucleic acid extraction liquid II.Nucleic acid extraction liquid I packet
Include polyethylene glycol and sodium chloride, wherein the mass fraction of polyethylene glycol is 20%~30%, the molar concentration of sodium chloride be 3M~
5M；Nucleic acid extraction liquid II includes Tween-20 and lauryl sodium sulfate, wherein the mass fraction of Tween-20 be 0.1%~
0.5%, the mass fraction of lauryl sodium sulfate is 0.1%~0.5%.
Wherein, polyethylene glycol can precipitate and be enriched with the pathogen in sample, and nucleic acid extraction liquid II can be with rapid cleavage disease
Somatoblast discharges nucleic acid, and process is without repeatedly centrifugation and abandons liquid.Pathogen by precipitating and with being enriched in sample, simultaneously
It reduces during the extraction process and abandons liquid number, remain the pathogen of low concentration sample to the maximum extent, effectively reduce False-Negative Rate,
Improve the sensitivity and detection accuracy of kit.When specific operation, nucleic acid extraction liquid I is first added into sample liquid, from
After the heart, liquid is discarded supernatant, nucleic acid extraction liquid II is then added, 10 minutes is kept the temperature in 100 DEG C after whirlpool concussion mixes, makes cell
Amplifying nucleic acid is sufficiently discharged, and is centrifuged again after cooling, and supernatant is retained.
In addition, the reaction buffer can be Tris-Cl buffer；Archaeal dna polymerase is thermal starting archaeal dna polymerase；Yin
Property control be no enzyme sterile water.
For the amplification efficiency being optimal, sensitivity and the detection efficiency of kit are improved, is verified through test of many times, obtained
Optimize reaction system.In the present embodiment, in the kit, the reaction system of PCR reaction is prepared by following volume ratio: 35.5
Part described PCR reaction solution, 0.5 part of archaeal dna polymerase and 4 parts of templates, wherein the template is the positive control, institute
State negative control or measuring samples DNA.
Further, the PCR reaction solution is prepared according to the following formulation: 1 × reaction buffer, 0.1 μM of dNTPs, 0.5 μ
MMgCl2, 0.15 μM of primer pair, 0.15 μM of probe and 2.5U archaeal dna polymerase.
It should be noted that kit storage and transport at -20 DEG C.
In addition, the application method of kit of the present invention, comprising the following steps:
Step S10, sample process: taking the swab sample of clinical acquisitions, and 1mL~2mL sterile saline is added, sufficiently shakes
It swings and shakes up rinsing, draw 200 μ L sample rinsing liquids into 1.5mL EP pipe, 50 μ L nucleic acid extraction liquid I, 12000rpm centrifugations are added
It inhales after five minutes and abandons supernatant, 30~50 μ L nucleic acid extraction liquid II are then added, keep the temperature 10 minutes in 100 DEG C after whirlpool concussion mixing,
12000rpm is centrifuged 1 minute after cooling, and supernatant is used for subsequent PCR amplification as template.The negative control of kit is by above-mentioned
Sample processing method carries out.
Wherein, the swab sample type of the clinical acquisitions include genital tract sample, female urethra secretion sample and
Male urethra secretion sample etc..
Step S20, PCR amplification: according to number of awaiting test sample n+3 (sample number n+ negative control+positive control+blank pair
According to), take PCR reaction solution 35.5 μ L × (n+3) in kit, archaeal dna polymerase 0.5 μ L × (n+3) to mix well rear each expansion
Increase reaction tube and dispense 36 μ L, positive control, negative control and the 4 μ L of n sample handled well is separately added into, so that overall reaction
System is 40 μ L, covers lid, is transferred to PCR amplification room, on real-time fluorescence PCR instrument, is expanded according to following reaction condition
Increase reaction:
Step S30, result interpretation: after reaction, instrument automatically save as a result, can use the included software of instrument into
Row automatically analyzes, and initial value, end value and the threshold value that can also manually adjust baseline are analyzed, and then records sample Ct value
With as a result, simultaneously in accordance with the following methods carry out result interpretation.
1) quality-control product
A. negative control and the detection Ct value of blank control should be shown as Undect；
B. the detection Ct value of positive control answers≤36.
Meet two above condition, this time experiment is considered as effectively, otherwise in vain, need to reform.
2) judgement of result
In the case where meeting quality-control product testing requirements:
A. sample is shown as Undet in FAM channel C t value, then is judged as negative；
B. sample is then judged as positive in value≤36 FAM channel C t；
C. sample is in value≤36 FAM channel C t 40≤Ct of value, it is proposed that and it reforms and (nucleic acid extraction sample size can be doubled), if
It reforms result Ct value and is judged as positive less than 40, be otherwise feminine gender.
Technical solution of the present invention is described in further detail below in conjunction with specific embodiments and the drawings, it should be understood that
Following embodiment is only used to explain the present invention, is not intended to limit the present invention.
1 kit Performance Evaluation of embodiment
Kit of the present invention and comparative example kit chlamydia trachomatis in 10 unknown women Cervical scrapes samples
Detection.
Comparative example: chlamydia trachomatis/nucleic acid detection reagent of selection Guangdong Hybribio Biotech Co., Ltd. production
Box (PCR- fluorescence probe method) is used as similar kit comparative example, and detection method is carried out according to the specification of the kit.
The detection method of kit of the present invention carries out as steps described below:
1, sample collection and processing
1) sample collection: then swab is inserted into uterine neck at 1cm~1.5cm, gently oppresses by cleaning cervix opening outer surface
And taken out after rotating swab 15s~20s, obtain columnar epithelial cell sample.Swab should be avoided when sampling encounters vaginal wall.
2) sample process: taking the swab sample of clinical acquisitions, and 1mL~2mL sterile saline is added, fullys shake and shakes up
Rinsing.200 μ L sample rinsing liquids are drawn into 1.5mL EP pipe, 50 μ L DNA extracting solution I, 12000rpm are added centrifugation 5 minutes
It inhales afterwards and abandons supernatant, 30~50 μ L DNA extracting solution II are added, 10 minutes are kept the temperature in 100 DEG C after whirlpool concussion mixing, after cooling
12000rpm is centrifuged 1 minute, and supernatant is used for subsequent PCR amplification as template.The above-mentioned sample process of the negative control of kit
Method carries out.
Wherein, nucleic acid extraction liquid I includes the polyethylene glycol of 30% (w/w) and the sodium chloride of 5M；Nucleic acid extraction liquid II includes
The lauryl sodium sulfate of the polysorbas20 of 0.5% (w/w) and 0.2% (w/w).
2, reagent prepares
According to number of awaiting test sample 10+3, PCR reaction solution 35.5 μ L × (10+3) in kit, 0.5 μ L of archaeal dna polymerase are taken
× (10+3) mixes well rear each amplified reaction pipe and dispenses 36 μ L, be separately added into positive control, negative control and processing
Good 10 samples, 4 μ L, so that overall reaction system is 40 μ L, another reaction tube dispenses 40 μ L, any template conduct is not added
Blank control covers lid and is transferred to PCR amplification room.
3, PCR amplification
1) PCR reaction tube is put into fluorescent PCR instrument, sample names is set；
2) fluorescence channel selects: the selection channel FAM (Reporter:FAM, Quencher:None) is arranged according to following table
PCR reaction condition sets operation PCR amplification program.
4, interpretation of result:
After reaction, instrument automatically saves is automatically analyzed as a result, can use the included software of instrument, can also be with
Initial value, end value and the threshold value for manually adjusting baseline are analyzed, and sample Ct value and result are then recorded.For detecting Ct
The sample of value≤36 is reported as the CT-DNA positive, and S type is presented in sample to be tested amplification curve at this time, as shown in Figure 1；For detection
Show the sample without Ct value, being reported as CT-DNA feminine gender, sample to be tested amplification curve is straight at this time, as shown in Figure 2；For detection
Value≤36 Ct 40≤Ct of value are then detected and (can be doubled nucleic acid extraction sample size) to the part sample, again if reforming result
Ct value is judged as positive still less than value≤36 item 40≤Ct, is otherwise feminine gender.
It records kit and comparative example kit of the present invention and detects sand in above 10 unknown women Cervical scrapes samples
The comparing result of chlamydia oculogenitale is as shown in table 1:
Table 1 detects the success ratio of chlamydia trachomatis in women Cervical scrapes sample
No. 7 inconsistent to testing result and No. 8 sample pcr amplification products send raw work bioengineering (Shanghai) share limited
Company's sequence verification.No. 7 sample sequencing results are as shown in figure 3, No. 8 sample sequencing results are as shown in Figure 4.Resulting core will be sequenced
Nucleotide sequence compares analysis by NCBI, is determined as chlamydia trachomatis positive sample.Illustrate the similar kit for comparing commercialization,
Kit accuracy of the present invention is high, and testing result is objective.
The accuracy testing of 2 kit of embodiment
8 parts of enterprise's positive reference product and 8 parts of enterprise's negative reference product are detected with kit of the present invention, detection method is according to reality
The kit operating procedure of the present invention for applying the offer of example 1 carries out, as a result as shown in Figure 5.
As seen from Figure 5,8 parts of enterprise's positive reference product can determine whether all positives at S type amplification curve；8 parts of enterprises
Industry negative reference product can determine whether all feminine genders in straight amplification curve and without Ct value.Enterprise's positive reference product and negative ginseng
Examining product coincidence rate result coincidence rate is all 100%.Illustrate that kit accuracy of the present invention is high.
The specific test of 3 kit of embodiment
In order to detect the specificity of kit of the present invention, Ureaplasma urealyticum, gonococcus, pneumonia are detected with kit of the present invention
It is mycoplasma, mycoplasma genitalium, HPV 18, human cytomegalovirus, mycoplasma hominis, herpes simplex virus type 2, big
Intestines Escherichia, Friedlander's bacillus, pseudomonas aeruginosa, Shigella dysenteriae.The sheet that detection method is provided according to embodiment 1
Invention kit operating procedure carries out, as a result as shown in Figure 6.
As seen from Figure 6, positive control result is the positive, and above 12 kinds of pathogenic microorganisms are to be all negative.Illustrate this
Invention kit is only capable of specific amplification chlamydia trachomatis, anti-without intersecting with the generation of the nucleic acid of the other bacteriums of intravaginal and virus
It answers, does not occur false positive, the goodness of fit 100%, it was demonstrated that kit specificity of the present invention is good.
The sensitivity test of 4 kit of embodiment
Positive reference product are to be diluted to 10 by 10 times5Copies/μL、104Copies/μL、103Copies/μL、
102The plasmid containing chlamydia trachomatis target gene segment of Copies/ μ L, 20Copies/ μ L pass through raw work bioengineering
The women Cervical scrapes sample that the synthesis of (Shanghai) limited liability company and DNA concentration are about 20Copies/ μ L.Negative reference
Product for no chlamydia trachomatis genetic fragment sterile saline.
Above-mentioned positive and negative reference material and minimum detection limit reference material are detected using kit of the present invention, detection side
Method is carried out according to the kit operating procedure of the present invention that embodiment 1 provides, and testing result is as shown in Figure 7.Referring to Fig. 7, to most
Low detection limit sample carries out definite value, and concentration is 20Copies/ μ L, shows that kit of the present invention has good sensitivity.
The precision test of 5 kit of embodiment
Precision test, every part of sample retest 10 are carried out to two parts of precision reference material J1 and J2 using this kit
Secondary, detection method is carried out according to the kit operating procedure of the present invention that embodiment 1 provides, as a result as shown in Figure 8.Wherein, described
Precision reference material J1 and J2 is confirmed as the positive clinical sample preparation of chlamydia trachomatis and is formed by detecting.
As can be seen from Figure 8, the amplification curve Ct value of two parts of precision repeated samples shows concentration, analyzes through data
Show that the Ct value coefficient of variation (CV%) less than 5%, shows kit of the present invention precision with higher.
In conclusion kit of the present invention has the advantages that specificity is good, accuracy is high, high sensitivity, accuracy are high, and
Compared with existing similar kit, do not occur false negative, there is higher accuracy.
The above is only a preferred embodiment of the present invention, is not intended to limit the scope of the invention, for this field
For technical staff, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any
Modification, equivalent replacement, improvement etc. should all be included within the scope of the present invention.
SEQUENCE LISTING
<110>Shenzhen Goldsite Diagnostics Inc.
<120>a kind of primer, probe and kit for detecting chlamydia trachomatis
<130> 2019.4.25
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213>artificial synthesized: upstream primer
<400> 1
gcaggaagag atcctgata 19
<210> 2
<211> 20
<212> DNA
<213>artificial synthesized: downstream primer
<400> 2
gacttttgga ttcgggataa 20
<210> 3
<211> 24
<212> DNA
<213>artificial synthesized: probe
<400> 3
atcggaggtg gctccaacgc tatt 24
Claims (7)
1. a kind of primer for detecting chlamydia trachomatis, the fluorescent PCR for chlamydia trachomatis nucleic acid is detected, which is characterized in that institute
Stating primer includes upstream primer and downstream primer, wherein the nucleotide sequence of the upstream primer is as shown in SEQ IDNo:1, institute
The nucleotide sequence of downstream primer is stated as shown in SEQ ID No:2.
2. a kind of probe being used cooperatively with primer as described in claim 1, which is characterized in that the nucleotides sequence of the probe
Column are as shown in SEQ IDNo:3.
3. probe as claimed in claim 2, which is characterized in that 3 ' end label BHQ1 quenching groups of the probe, the spy
5 ' end flag F AM fluorophors of needle.
4. a kind of kit, the fluorescent PCR for chlamydia trachomatis nucleic acid is detected, which is characterized in that the kit includes core
Sour extracting solution, PCR reaction solution, archaeal dna polymerase, positive control and negative control, wherein the PCR reaction solution includes that reaction is slow
Fliud flushing, dNTPs, MgCl2, primer as described in claim 1 and probe as claimed in claim 2 or claim 3.
5. kit as claimed in claim 4, which is characterized in that the nucleic acid extraction liquid includes nucleic acid extraction liquid I and nucleic acid
Extracting solution II, wherein the nucleic acid extraction liquid I includes 20%~30% polyethylene glycol and 3M~5M sodium chloride, and the nucleic acid mentions
Taking liquid II includes 0.1%~0.5% Tween-20 and 0.1%~0.5% lauryl sodium sulfate.
6. kit as claimed in claim 4, which is characterized in that in the kit, the reaction system of PCR reaction is by as follows
Volume ratio is prepared: 35.5 parts of PCR reaction solutions, 0.5 part of archaeal dna polymerase and 4 parts of templates, wherein the template is
The positive control, the negative control or measuring samples DNA.
7. kit as claimed in claim 6, which is characterized in that the PCR reaction solution includes: 1 × reaction buffer, 0.1 μ
MdNTPs、0.5μMMgCl2, 0.15 μM of primer pair, 0.15 μM of probe and 2.5U archaeal dna polymerase.
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| CN201910359593.3A CN110157820A (en) | 2019-04-28 | 2019-04-28 | A kind of primer, probe and kit detecting chlamydia trachomatis |
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| CN201910359593.3A CN110157820A (en) | 2019-04-28 | 2019-04-28 | A kind of primer, probe and kit detecting chlamydia trachomatis |
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