CN207828256U - The fluorescent quantificationally PCR detecting kit of coxsackie enterovirus CVA12 - Google Patents
The fluorescent quantificationally PCR detecting kit of coxsackie enterovirus CVA12 Download PDFInfo
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- CN207828256U CN207828256U CN201721799881.3U CN201721799881U CN207828256U CN 207828256 U CN207828256 U CN 207828256U CN 201721799881 U CN201721799881 U CN 201721799881U CN 207828256 U CN207828256 U CN 207828256U
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Abstract
The utility model is related to a kind of fluorescent quantificationally PCR detecting kits of coxsackie enterovirus CVA12, the kit is easy to use, can realize that specific detection, high specificity, sensitivity are high for viral CVA12, it clinically can quickly detect and parting, realize the effect of a calibrating type.The utility model has very strong clinical practice meaning so that clinical treatment is effective much sooner, also has important meaning simultaneously for the prevention and control of hand-foot-and-mouth disease.
Description
Technical field
The utility model is related to the fluorescent quantificationally PCR detecting kits of 12 type enterovirus CVA12 of Coxsack A groups.
Background technology
Hand-foot-and-mouth disease (Hand, foot and mouth disease, HFMD) is by caused by enterovirus infection one
The benign self limiting acute infectious disease of kind, is mainly in 6 years old or less children, it is characterized in that fever, stomatocace, brothers' stern
Portion's macula or maculopapule.Few patients can cause the complication such as myocarditis, pulmonary edema, aseptic cerebrospinal meningitis, encephalitis, cause
Extremely, disability rate is very high, not only influences the life quality of patient, but also occupy and expend great health resource, finance branch
Go out huge.HFMD was once repeatedly popular in many countries and regions, in China, HFMD from 1981 Shanghai occur since, in succession
It is had been reported that on Beijing, Tianjin, Fujian and other places.Since in March, 2008, epidemic situation occurred in Fuyang, China is almost involved
All provinces in continent and area, in China, HFMD number of the infecteds and death toll are in ascendant trend year by year.From 2012, in state
The EV71 and CVA16 of inside and outside many area HFMD is no longer advantage epidemic strain.Other enteroviruses such as 12 type intestines of Coxsack A groups
The proportions such as road virus CVA12 are higher and higher.
Coxsackie virus and EV71 type hand-foot-and-mouth disease clinical symptoms are difficult differentiation, and mostly with row eruption and prevalence, therefore clinically
Be badly in need of it is a kind of can quickly accurately typing detection method, for clinically Individual Diagnosis and treatment provide reliable foundation.
There are viral isolated culture, serological method and common RT-PCR method for the diagnostic method of enterovirus at present.And
Virus culture method is relatively time-consuming and needs dedicated technician, and serological method sensitivity is relatively low, conventional RT-PCR detection
Method is easy to pollute and can only qualitative detection.And fluorescent quantitative PCR technique has the characteristics that sensitivity height, high specificity, convenient quickly,
It is current ideal diagnostic techniques.
When carrying out fluorescence quantitative PCR detection, a PCR reaction generally includes a variety of reaction reagents, such as：Archaeal dna polymerase,
Reaction buffer, sense primer, downstream primer, probe, template and distilled water.It is right to sample, positive control, feminine gender to need
It is carried out at the same time detection according to, positive criteria product, also to do regular-PCR control sometimes, when detection, needs repeatedly to pick and place and draw reagent,
It is very inconvenient.
In general kit, above-mentioned reaction reagent is respectively placed in different containers, is carrying out PCR reactions
When, it needs to take out a certain amount of reaction reagent from different containers, be mixed, then carry out PCR reactions.Due to what is be added
The type of reaction reagent is too many, when adding different reaction reagents, slightly not note that the leakage that will result in reagent adds or repeats
Addition, influences the accuracy of PCR response procedures.In addition, when point taking different reagents, needs constantly to open, closes container,
Also it be easy to cause the secondary pollution of reagent in container.
Utility model content
In order to provide a kind of fluorescence quantitative PCR detection examination of 12 type enterovirus CVA12 of simple, fast Coxsack A groups
Agent box, and in order to avoid the secondary pollution of reagent.The utility model provides following technical scheme：
On the one hand, the utility model provides a kind of quantitative fluorescent PCR inspection of 12 type enterovirus CVA12 of Coxsack A groups
Test agent box, the kit include：Box body 1 and box cover 2, box body 1 is interior to be equipped with several grooves 3, and one is placed in each groove 3
A Reagent Tube group 4；Each Reagent Tube group 4 includes several tube chambers 5 to interconnect；The opening of the tube chamber 5 has
Sealer 6 seals, and the sealer 6 is easily to puncture material or hard material；Premix reaction is provided in the cavity of the tube chamber 5
Liquid, the premix reaction solution includes PCR reaction buffers, PCR primer, label probe and distilled water.
Tube chamber 5 includes PCR reaction buffers, PCR primer, label probe and the distilled water of premix, above-mentioned reaction reagent
It measures by needed for PCR reaction systems, prepares in proportion.So set, when carrying out PCR reactions, can directly draw certain
The premix reaction solution of amount carries out PCR reactions.In view of archaeal dna polymerase needs low-temperature preservation, without premix DNA in tube chamber 5
Polymerase；Furthermore, it is contemplated that properties of samples to be detected is different, also not comprising sample to be tested in tube chamber 5.In practical application
When, the premix reaction solution of tube chamber 5 can be drawn, add archaeal dna polymerase and sample to be tested mixing, can it is very convenient,
The addition of reaction reagent is efficiently carried out, and is not easy to omit or repeat to add.
In one preferred embodiment, the premix reaction solution of 1 reaction system is contained only in each tube chamber 5.So
Setting, on the one hand can ensure that the liquid assimilating inside each tube chamber finishes can discard tube chamber, furthermore it is also possible to need not inhale
Go out the liquid in tube chamber 5, and archaeal dna polymerase and sample to be tested are directly added into each tube chamber 5, utilizes tube chamber 5 direct later
Carry out PCR reactions.
When the sealer 6 is easily to puncture material, preferably masking foil or plastic film, the reagent in drawing tube chamber 5
When, it can directly puncture masking foil；At this time, it is preferred that the amount of reagent in single tube chamber 5 is 1-10 reaction system, works as absorption
After, you can it is convenient, fast to discard a tube chamber 5, and there is no secondary pollution.
In other implementations, when containing only the premix reaction solution of 1 reaction system in each tube chamber 5, sealing
Object 6 is preferably hard material；At this point, opening sealer 6, archaeal dna polymerase and sample to be tested are then added in tube chamber 5, later,
Sealer 6 is covered again, you can directly carry out PCR reactions, it is not necessary to the premixed liquid in tube chamber 5 be sucked out again, reduce operation step
Suddenly.
Further, the arrangement mode of each tube chamber of the Reagent Tube group is：Be arranged in a linear, it is rectangular arrangement or be in
Annular array.Further, the quantity of 1 inner groovy 3 of the box body is 1-20, and the quantity of the tube chamber 5 of each Reagent Tube group 4 is
1-10.Further, the bottom of the tube chamber 5 is in conical or arc-shaped.Further, the edge of the sealer 6 is set
The chimb 7 of extension is set, the material of the chimb 7 is hard material.
Further, it is additionally provided with the container for loading positive in the box body 1, reagent is provided on the box cover 2
Box specification.
The beneficial effects of the utility model are as follows：
The fluorescent quantificationally PCR detecting kit of 12 type enterovirus CVA12 of Coxsack A groups provided by the utility model, one
Secondary absorption can all take reagent or various concentration positive criteria product or different control samples needed for such as one reaction of a kind of reagent
On hand, and it is drawn out respectively, reduces the inconvenience brought because repeatedly picking and placeing Reagent Tube, and be less likely to occur reagent leakage plus
Or add again, improve the accuracy and success rate of experiment, moreover it is possible to avoid the secondary pollution of reagent.In addition, used in the utility model
Special primer and probe can realize specific detection for viral CVA12, and high specificity, sensitivity are high, for clinically may be used
Quickly detection and parting realize the effect of a calibrating type.With very strong clinical practice meaning so that clinical treatment is much sooner
Effectively, also there is important meaning simultaneously for the prevention and control of hand-foot-and-mouth disease.
Description of the drawings
The utility model has following attached drawing：
Fig. 1 is a kind of fluorescent quantificationally PCR detecting kit vertical view of 12 type enterovirus CVA12 of Coxsack A groups.Its
In, 1- box bodys, 2- box covers, 3- grooves, 4- Reagent Tube groups, 5- tube chambers.
Fig. 2 is the side view of the built-in Reagent Tube group 4 of Fig. 1 further grooves 3.Wherein, 4- Reagent Tubes group, 5- tube chambers, 6- sealings
Object, 7- chimbs.
Specific implementation mode
The utility model is described in further detail below in conjunction with attached drawing.
As depicted in figs. 1 and 2, the quantitative fluorescent PCR of the 12 type enterovirus CVA12 of Coxsack A groups described in the present embodiment
Detection kit, including：Box body 1 and box cover 2 are provided with kit specification on box cover 2,3 grooves 3 are equipped in box body 1,
A Reagent Tube group 4 is placed in each groove 3；Each Reagent Tube group 4 includes 7 tube chambers 5 to interconnect；It is described
It is masking foil that the opening of tube chamber 5, which has the sealing of sealer 6, the sealer 6, and in other embodiments, sealer 6 can be with
For plastic film or rigid plastics；Premix reaction solution is provided in the cavity of the tube chamber 5, the premix reaction solution includes 100 μ
L PCR reaction buffers M1,4 μ l sense primers A1,4 μ l downstream primers B1,2 μ l label probes T and 40 μ l ddH2O, it is described
Premix reaction solution is 10 reaction systems.
The sequence of sense primer A1 is：5’-TGCGTAGTCAATAAGCATGGAGTGAG-3’；The sequence of downstream primer B1
For：5’-TATGTAAAGATCTCTAGCTTGCGGCG-3’；Probe T-sequence is：5’-FAM-
CAGGCTTGGCAGGTGTCGTCGTGGTAGAG-BHQ1-3’。
When carrying out fluorescence quantitative PCR detection, the box cover 2 on box body 1 is opened, Reagent Tube group 4 is taken out from groove 3, is used
Hand pinches short side 7, and sealer 6 is directly punctured using liquid transfer gun head, then dispenses the premix reagent in tube chamber 5 to different
In PCR pipe, template to be measured and archaeal dna polymerase are added into each PCR pipe, carries out fluorescence quantitative PCR detection；Reaction condition：37 DEG C,
2min；95 DEG C, 10min；95 DEG C, 10s, 60 DEG C, 45s, 40 cycles.
Fluorescent quantitation is carried out to the cDNA of CVA6, CVA10, CVA16, EV71, CVB3, CVB4, CVB5, CVA2 and CVA12
PCR is detected, the results show that primer A1 and B1 and probe T only have specific amplification to the template CVA12 of itself, is shown designed
Primer A1 and B1 and probe T have good specificity.
Using positive criteria product as template, its sensitivity is detected using the quantitative fluorescent PCR reaction system of foundation.As a result it shows
Show, the Monitoring lower-cut for CVA12 standard items is 2.60 × 102copies/μl。
To 105~101The positive criteria product of copies/ μ l concentration carries out 3 repetitions and detects, Statistics Application soft SA S into
As a result row analysis shows that the Ct value differences of its amplification curve are not different notable, the coefficient of variation is respectively less than 5%, shows that this method has
Preferable repeatability and stability (being shown in Table 1).
Table 1.CVA12 quantitative fluorescent PCR system Repeatability checkings
Acquire 91 parts of clinical samples of Jinan City, Shandong Province children's hospital from 2017.Clinical sample is carried using Trizol methods
RNA is taken, is cDNA through reverse transcription.Quantitative fluorescent PCR and regular-PCR detection are carried out respectively, and the results are shown in Table 2.
2. clinical sample of table detects
| Quantitative fluorescent PCR | Regular-PCR | |
| It is positive | 9 | 6 |
| It is negative | 82 | 85 |
Claims (8)
1. the fluorescent quantificationally PCR detecting kit of coxsackie enterovirus CVA12, which is characterized in that the kit includes：Box
Body (1) and box cover (2), box body (1) is interior to be equipped with several grooves (3), and a Reagent Tube group (4) is placed in each groove (3)；Often
A Reagent Tube group (4) includes several tube chambers (5) to interconnect；
The opening of the tube chamber (5) is provided with sealer (6), and the sealer (6) is easily to puncture material or hard material；
It is provided with premix reaction solution in the cavity of the tube chamber (5), the premix reaction solution includes that PCR reaction buffers, PCR draw
Object, label probe and distilled water.
2. kit according to claim 1, which is characterized in that the sealer (6) is masking foil or plastic film.
3. kit according to claim 1 or 2, which is characterized in that the row of each tube chamber (5) of the Reagent Tube group (4)
Row mode is：It is arranged in a linear, rectangular arrangement or arranged in a ring.
4. kit according to claim 1 or 2, which is characterized in that the quantity of box body (1) inner groovy (3) is 1-
20, the quantity of the tube chamber (5) of each Reagent Tube group (4) is 1-10.
5. kit according to claim 1 or 2, which is characterized in that the bottom of the tube chamber (5) is in cone or circular arc
Shape.
6. kit according to claim 1 or 2, which is characterized in that the convex of extension is arranged in the edge of the sealer (6)
The material on side (7), the chimb (7) is hard material.
7. kit according to claim 1 or 2, which is characterized in that be additionally provided with the positive sample of loading in the box body (1)
The container of product.
8. kit according to claim 1 or 2, which is characterized in that be provided with kit explanation on the box cover (2)
Book.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201721799881.3U CN207828256U (en) | 2017-12-21 | 2017-12-21 | The fluorescent quantificationally PCR detecting kit of coxsackie enterovirus CVA12 |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201721799881.3U CN207828256U (en) | 2017-12-21 | 2017-12-21 | The fluorescent quantificationally PCR detecting kit of coxsackie enterovirus CVA12 |
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| CN207828256U true CN207828256U (en) | 2018-09-07 |
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| CN201721799881.3U Expired - Fee Related CN207828256U (en) | 2017-12-21 | 2017-12-21 | The fluorescent quantificationally PCR detecting kit of coxsackie enterovirus CVA12 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109439529A (en) * | 2018-12-29 | 2019-03-08 | 中国人民解放军军事科学院军事医学研究院 | A kind of reaction tube carrying out multiple nucleic acid amplification |
-
2017
- 2017-12-21 CN CN201721799881.3U patent/CN207828256U/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109439529A (en) * | 2018-12-29 | 2019-03-08 | 中国人民解放军军事科学院军事医学研究院 | A kind of reaction tube carrying out multiple nucleic acid amplification |
| WO2020135873A1 (en) * | 2018-12-29 | 2020-07-02 | 中国人民解放军军事科学院军事医学研究院 | Reaction tube for multiple nucleic acid amplification |
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| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180907 Termination date: 20201221 |