CN107099619A - A kind of LAMP primer composition thing and its kit for detecting respiratory pathogen - Google Patents
A kind of LAMP primer composition thing and its kit for detecting respiratory pathogen Download PDFInfo
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- CN107099619A CN107099619A CN201710305673.1A CN201710305673A CN107099619A CN 107099619 A CN107099619 A CN 107099619A CN 201710305673 A CN201710305673 A CN 201710305673A CN 107099619 A CN107099619 A CN 107099619A
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- 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
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- 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
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The present invention relates to a kind of Primer composition for being used to detect respiratory pathogen, it includes at least one set in mycoplasma pneumoniae primer sets, CPN primer sets, first/influenza B virus primer sets, parainfluenza virus primer sets, adenovirus primer sets, Respiratory Syncytial Virus(RSV) primer sets；A kind of kit for including the Primer composition is further related to, it also includes micro-fluidic chip, naked eyes visible indicator that bag drapes over one's shoulders primer；A kind of detection method using above-mentioned Primer composition is further related to, includes coating, measuring samples nucleic acid extraction, LAMP reactions and the visualization result interpretation of Primer composition.The kit and approach application that the present invention is provided carry out visualization judgement on micro-fluidic chip, rapidly and accurately realize the instant detection of 7 kinds of respiratory pathogens, by color change naked eyes result of determination, this makes the present invention easier in actual applications, quick and easily operated, suitable for execute-in-place.
Description
Technical field
The invention belongs to nucleic acid amplification technologies field, and in particular to a kind of LAMP primer for being used to detect respiratory pathogen
Composition, and the kit and its method that detect respiratory pathogen based on micro-fluidic chip system.
Background technology
Respiratory tract infection is a kind of common infectious disease of China, refer to the nasal cavity of pathogenic infection human body, throat, tracheae and
The respiratory systems such as bronchus.The infection of the upper respiratory tract mainly has virus to cause, and refers to nasal cavity, pharynx or throat's inflammation, most commonly anxious
The property infection of the upper respiratory tract, has stronger infectiousness also to cause severe complication.ALRI is by virus, bacterium, Zhi Yuan
The microorganisms such as body, Chlamydia cause, and ALRI includes bronchitis, pneumonia etc., if can clearly cause infection during treatment
Pathogen, then can select effective antibiotic or antiviral therapy.Atypical respiratory pathogens are being breathed now simultaneously
Accounting is increasing in road pathogen, and there is a situation where multiple infection.Therefore quick detection is realized, many pathogen detections are
As trend.
The method for the identification pathogen clinically applied at present is mainly detected by culture, but cultivation detection time
Difference, accuracy is low, while many more difficult cultures of respiratory pathogen.And such disease has similar clinical symptoms and prevalence
Property feature, and there is a situation where multiple infection.
Respiratory tract joint inspection reagent Pneumoslide produces for VIRCELL companies of Spain, and 1 card can detect human serum simultaneously
Middle 9 IgM antibodies of respiratory tract infection, including the type of legionella pneumophilia 1 (LP1), mycoplasma pneumoniae (MP), the hot Richettsia of Q
(COX), CPN (CP), adenovirus (ADV), Respiratory Syncytial Virus(RSV) (RSV), influenza A virus (IFA), B-mode stream
Influenza Virus (IFB), parainfluenza virus (PIV), but result need to use immunofluorescence microscopy, to distinguishing that it is longer that yin and yang attribute needs
Time is differentiated.
Microflow control technique is to realize that micro-volume is reacted with micro-processing technology, to replace conventional molecular biological, chemistry, exempt from
Epidemiology or Pharmaceutical Analysis reaction, and many flux or high pass quantitative response can be realized so that experiment testing cost be greatly reduced, significantly carry
High efficiency.
Loop-mediated isothermal amplification (is directed to target sequence 6 according to the amplification characteristic and specific primer of strand displacement archaeal dna polymerase
4 specific primers of individual region design), ensure primer under isothermal conditions smoothly and template using strand displacement archaeal dna polymerase
With reference to and carry out amplified reaction, have the specificity being equal and sensitivity with PCR, may be implemented under constant temperature
Continuous rapid amplifying, while can also add ring primer, amplification efficiency is improved, better than PCR.
Pertinent respiratory pathogen detection technology is mainly IIF, colloidal gold method, ELISA and
Fluorescent PCR method.Wherein, IIF result of determination is needed by fluorescence microscope, and result of determination needs detection
Personnel are experienced；Although collaurum detection is quick, sensitivity is not high, false negative result easily occurs.Enzyme linked immunological is examined
The specificity of survey and sensitivity is not high, and operating process is cumbersome, time and effort consuming.Fluorescent PCR method being capable of accurate detection pathogen
Nucleic acid, but most 3 of multiple fluorescence PCR Product checking target spot, detection time is long, and detection architecture and operating process are more complicated,
It is difficult to carry out Site Detection, it is necessary to the problems such as professional operates particular detection instrument.
Many researchs are combined loop-mediated isothermal amplification with micro-fluidic chip at present, to pathogen nucleic acid, tumor marker base
Cause, resistance site etc. are used for quickly detecting, but research needs to judge result by other instruments above.
Therefore, the technical difficult point faced at present be to research and develop it is a can while detect various respiratory road pathogen,
And it is easy to the system of result of determination.
The content of the invention
It is an object of the invention to overcome defect of the prior art to be used to detect respiratory pathogen there is provided a kind of
LAMP primer composition thing and its kit and method, it uses micro-fluidic chip to realize the higher atypia breathing of 7 kinds of infection rates
Road pathogen (mycoplasma pneumoniae (MP), CPN (CP), adenovirus (ADV), Respiratory Syncytial Virus(RSV) (RSV), first/second
Type influenza virus (IFA＆IFB, altogether inspection), parainfluenza virus (PIV)) parallel parsing, and synchronous naked eyes colour developing judges that raising is exhaled
The detection flux and operability of the related pathogen in road are inhaled, cost is reduced, shortens detection time.
To achieve the above object, the present invention is adopted the following technical scheme that：
On the one hand, the present invention provides a kind of LAMP primer composition thing for being used to detect respiratory pathogen, and it includes pneumonia
Mycoplasma primer sets, CPN primer sets, first/influenza B virus primer sets, parainfluenza virus primer sets, adenovirus are drawn
At least one set in thing group, Respiratory Syncytial Virus(RSV) primer sets；
Wherein, the mycoplasma pneumoniae primer sets include SEQ ID NO：Mycoplasma pneumoniae outer primer 1, SEQ shown in 1
ID NO：Mycoplasma pneumoniae outer primer 2, SEQ ID NO shown in 2：Mycoplasma pneumoniae inner primer 1 and SEQ ID NO shown in 3：
Mycoplasma pneumoniae inner primer 2 shown in 4；
Wherein, the CPN primer sets include SEQ ID NO：CPN outer primer 1, SEQ shown in 5
ID NO：CPN outer primer 2, SEQ ID NO shown in 6：CPN inner primer 1 and SEQ ID NO shown in 7：
CPN inner primer 2 shown in 8；
Wherein, the first/influenza B virus primer sets include SEQ ID NO：Outside first/influenza B virus shown in 9
Primer 1, SEQ ID NO：First/influenza B virus outer primer 2, SEQ ID NO shown in 10：First/influenza B shown in 11
Viral inner primer 1 and SEQ ID NO：First/influenza B virus inner primer 2 shown in 12；
Wherein, the parainfluenza virus primer sets include SEQ ID NO：Parainfluenza virus outer primer 1, SEQ shown in 13
ID NO：Parainfluenza virus outer primer 2, SEQ ID NO shown in 14：Parainfluenza virus inner primer 1 and SEQ ID shown in 15
NO：Parainfluenza virus inner primer 2 shown in 16；
Wherein, the adenovirus primer sets primer sets include SEQ ID NO：Adenovirus primer sets outer primer 1 shown in 17,
SEQ ID NO：Adenovirus primer sets outer primer 2, SEQ ID NO shown in 18：The He of adenovirus primer sets inner primer 1 shown in 19
SEQ ID NO：Adenovirus primer sets inner primer 2 shown in 20；
Wherein, the Respiratory Syncytial Virus(RSV) primer sets include SEQ ID NO：Draw outside Respiratory Syncytial Virus(RSV) shown in 21
Thing 1, SEQ ID NO：Respiratory Syncytial Virus(RSV) outer primer 2, SEQ ID NO shown in 22：Respiratory Syncytial Virus(RSV) shown in 23
Inner primer 1 and SEQ ID NO：Respiratory Syncytial Virus(RSV) inner primer 2 shown in 24.
In order to further optimize above-mentioned technical proposal, the technical measures that the present invention is taken also include：
Preferably, the LAMP primer composition thing further comprises Quality Control primer sets, and the Quality Control primer sets include SEQ ID
NO：Quality Control outer primer 1, SEQ ID NO shown in 25：Quality Control outer primer 2, SEQ ID NO shown in 26：In Quality Control shown in 27
Primer 1 and SEQ ID NO：Quality Control inner primer 2 shown in 28.
The primer sequence of above-mentioned respiratory pathogen primer sets and the sequence of Quality Control primer sets are as follows：
The amplimer sequence table of table 1
On the other hand, the present invention provides a kind of kit containing above-mentioned LAMP primer composition, and it also includes micro-fluidic core
Piece, the micro-fluidic chip is provided with mutual disconnected 4 reaction detection parts, and each reaction detection part includes being sequentially communicated
Liquid storage area, trough, ball valve and reacting hole, the liquid storage area sets well, and each reaction detection part has 8 reactions
Each primer sets in hole, the Primer composition are coated in corresponding reacting hole respectively.
In order to further optimize above-mentioned technical proposal, the technical measures that the present invention is taken also include：
Preferably, the micro-fluidic chip is disc micro-fluidic chip.
Preferably, the concentration of outer primer 1 is 50 μM in each primer sets, and the concentration of outer primer 2 is 50 μM, inner primer 1
Concentration is 50 μM, and the concentration of inner primer 2 is 50 μM；Wherein, in the kit, the outer primer 1, outer primer 2, inner primer 1,
The volume ratio of inner primer 2 is 1:1:4~8:4~8.
Preferably, the kit further comprises reaction solution, archaeal dna polymerase, indicator, negative control thing.
Preferably, the reaction solution reacts including 10~15mM dNTPs, 10 × Isothermal Amplification
Buffer solution, 100~200m mM MgSO4 aqueous solution；Wherein, dNTPs, reaction buffer, the MgSO4 aqueous solution in the reaction solution
Volume ratio be 7~9:4~6:2.
Preferably, the reaction solution includes 12mMdNTPs, 10 × Isothermal Amplification reaction buffering
Liquid, the 150mM MgSO4 aqueous solution；Wherein, dNTPs, reaction buffer, the volume ratio of the MgSO4 aqueous solution are 7 in the reaction solution
~9:4~6:2, more preferably 8：5：2.
Preferably, the archaeal dna polymerase is Bst archaeal dna polymerases, and concentration is 8U/ μ l.
Preferably, the indicator includes HNB, Calcein, cresol red, phenol red, m-cresol purple, bromocresol purple, neutrality
Red, naphtholphthalein, thymol blue, its concentration are 50~200 μM.
Preferably, the indicator is cresol red, and final concentration of 50~200 μM of reaction system, more preferably reaction system are whole
Concentration is 100 μM,
Preferably, the negative control thing is water.
Finally, the present invention also provide it is a kind of detected using above-mentioned Primer composition respiratory pathogen for non-diagnostic
Purpose method, it comprises the following steps：
Step 1) Primer composition coating：Each primer sets in the Primer composition are coated on accordingly respectively
In reacting hole, it is fixed on by vacuum drying in reacting hole；
Step 2) measuring samples nucleic acid extraction：The nucleic acid of measuring samples is extracted using paramagnetic particle method；
Step 3) LAMP reactions：Extract reaction solution with archaeal dna polymerase, indicator, after being mixed with the nucleic acid of measuring samples, turn
The liquid storage area of the micro-fluidic chip of coating primer sets is moved to, liquid is flowed into reacting hole under the action of the centrifugal force, then carries out ring
Mediated isothermal amplification reacts；
Step 4) result interpretation：Terminate the color change in rear reacting hole according to amplification to carry out direct naked eyes interpretation or adopt
Color interpretation is carried out with equipment.
Preferably, step 1) described in the coated of Primer composition comprise the following steps that：Step 1) described in primer sets
The coated of compound comprises the following steps that：Each primer sets of the Primer composition are mixed with agarose respectively, are configured to
Corresponding mixed solution, takes 1 μ L mixed solution to click and enter in the corresponding reacting hole of micro-fluidic chip, is dried in clean super-clean bench
Afterwards, vacuumize after drying 2 hours, primer sets are coated in corresponding reacting hole；Wherein, agarose in the mixed solution
Final concentration of 0.1% (mass percent), the mixed solution contains 4~8 μM of inner primers 1,4~8 μM of 2,1 μM of inner primers and drawn outside
Thing 1,1 μM of outer primer 2；More specifically, 8 reacting holes of each reaction detection part are coated with respectively in the micro-fluidic chip
Following primer sets：Mycoplasma pneumoniae primer sets, CPN primer sets, first/influenza B virus primer sets, parainfluenza virus
Primer sets, adenovirus primer sets, Respiratory Syncytial Virus(RSV) primer sets, Quality Control primer sets, negative controls.
Preferably, step 3) described in LAMP reaction comprise the following steps that：Take 30 μ l reaction solutions, 4 μ lDNA polymerases molten
Liquid, 5 μ l 2mM cresol reds, the nucleic acid of 20 μ l measuring samples, add water and complement to 100 μ l reaction mixture, and vortex concussion is equal
Take 80 μ l reaction mixtures to inject in each well after even to seal after membrana oralis, the Quick spin 1200rpm/ in centrifuge
Min 15s, 3200rpm/min 30s, sample introduction is to reacting hole under the action of the centrifugal force, in 50 DEG C of isothermal reaction 20min, at 60 DEG C
Lower isothermal reaction 60min.
Preferably, step 4) described in direct naked eyes interpretation it is specific as follows：If color changes, the sample to be checked
In contain respiratory pathogen genomic nucleic acids；If color does not change, respiratory diseases are not contained in the sample to be checked
Chlamydia genes group nucleic acid.
Preferably, step 4) described in equipment include constant temperature micro-fluidic chip nucleic acid augmentative instrument, it is detectable corresponding to expand
Increase curve and ct values.
Preferably, in step 1) before, include the cleaning step of micro-fluidic chip, it is specific as follows：Micro-fluidic chip is adopted
With plasma cleaning, micro-fluidic chip surface is rinsed with plasma cleaner, is air-dried with inert gas.
Preferably, in step 1) coating Primer composition micro-fluidic chip be packaged using pressure-sensitive bonding film.The pressure
Quick bonding film adhesive strength, it is resistant to conventional heating temperature, does not have notable harmful effect to the reaction carried out.
The method for preparing following any products falls within protection scope of the present invention：
A. it is described detection respiratory pathogen ring mediated isothermal amplification Primer composition；
B. it is described detection respiratory pathogen micro-fluidic chip；
The following of the isothermal duplication primer set composition of described detection respiratory pathogen any are used for non-diagnostic
The application of purpose falls within protection scope of the present invention：
A. the primer set composition of the isothermal duplication of the detection respiratory pathogen is preparing the detection respiratory tract
Application in the micro-fluidic chip of pathogen；
B. the primer set composition of the isothermal duplication of the detection respiratory pathogen is preparing the detection respiratory tract
The application of the reagent or kit of pathogen；
C. the primer set composition of the isothermal duplication of the detection respiratory pathogen is preparing the detection respiratory tract
Application in the system of pathogen.
The present invention also protects a kind of method of the detection respiratory pathogen for non-diagnostic purpose, including a or b：
A. the nucleic acid of sample to be tested is extracted, with the primer set composition of the isothermal duplication of the detection respiratory pathogen
Isothermal duplication is carried out, amplified production is detected, determines whether sample to be tested contains corresponding primer sets in the primer set composition
Corresponding respiratory pathogen pathogen；
B. the nucleic acid of respiratory pathogen to be measured is extracted, with the isothermal duplication primer set of the detection respiratory pathogen
Isothermal duplication is carried out, amplified production is detected, it is accordingly to draw in the primer set composition to determine respiratory pathogen to be measured
The corresponding respiratory pathogen of thing group.
In the present invention, the respiratory pathogen can be at least one of following：Mycoplasma pneumoniae, pneumonia clothing are former
Body, first/influenza B virus, parainfluenza virus, adenovirus and Respiratory Syncytial Virus(RSV).
Compared with prior art, the invention has the advantages that：
Kit and its application method that the present invention is used, add naked eyes visible indicator, and the indicator can be in reaction
It is preceding to add, amplification is not influenceed, can be with Indicator Reaction result；The micro-fluidic chip that the present invention is used has 32 reacting holes, as long as
In chip production, embedding primer sets can carry out 32 cores on the chip of same design in advance in different reacting holes
Acid reaction, realizes the detection mode of multisample multi objective, while setting Quality Control primer sets in the chips, can reach quality control
It is required that；
The present invention can rapidly and accurately realize 7 kinds by being used in the method for carrying out visualizing judgement on micro-fluidic chip
The instant detection of atypical respiratory pathogens, not only realizes to each small size well and matches somebody with somebody liquid, it is to avoid operating personnel are difficult to distinguish
The problem of whether being loaded successfully recognized, while by color change naked eyes result of determination so as to reach visual requirement, this makes this
Invention it is easier in actual applications, quick and easily operation, suitable for execute-in-place, its by LAMP isothermal duplications with
Micro-fluidic chip macroscopic is combined, and the detection of multiple respiratory pathogen can be achieved, for rapidly and accurately identifying
The species of respiratory pathogen.
The present invention's will be appreciated that takes into account micro-fluidic chip detection performance and operability, can both combine supporting
Instrument, can also be according to naked-eye observation final result, therefore the system is adapted to wanting for high throughput automated or easy operation
Ask.
Brief description of the drawings
The structural representation for the micro-fluidic chip that Fig. 1 uses for the present invention；
Fig. 2 for one embodiment of the invention micro-fluidic chip in primer sets coating schematic diagram；
Fig. 3 is the testing result figure of the micro-fluidic chip of respiratory pathogen sample；
Reference is in figure：
1st, egative film；2nd, reaction detection part；3rd, through hole；4th, sealer；21st, well；22nd, liquid storage area；23rd, trough；24、
Reacting hole；25th, ball valve；26th, waste liquid tank；27th, steam vent；28th, the first curved channel；29th, the second curved channel.
1#~4#：Reaction detection part；A~H：It is coated with the reacting hole of primer sets.
Hereinafter, reaction detection part 1# reacting hole A is represented with 1#-A, reaction detection part 1# reacting hole B with
1#-B represents, the like.
Embodiment
The invention provides a kind of Primer composition and its kit and application method for being used to detect respiratory pathogen,
Wherein described respiratory pathogen includes mycoplasma pneumoniae, CPN, first/influenza B virus, parainfluenza virus, adenopathy
At least one of poison and Respiratory Syncytial Virus(RSV), the Primer composition is included in the corresponding primer sets of above-mentioned respiratory pathogen
At least one set.
With reference to the accompanying drawings and examples, the embodiment to the present invention is further described.Following examples are only
For clearly illustrating technical scheme, and it can not be limited the scope of the invention with this.
Embodiment one
Present embodiment describes micro-fluidic chip of the present invention.
As shown in figure 1, the micro-fluidic chip that the present invention is used is disc micro-fluidic chip, it includes 4 reaction detections
Part 2, each reaction detection part 2 includes well 21, liquid storage area 22, trough 23, reacting hole 24, ball valve 25, waste liquid tank
26th, steam vent 27, the first curved channel 28 and the second curved channel 29, the above-mentioned curved channel 29 of first curved channel 28 and second
On egative film 1, the first curved channel 28 connection trough 23 and steam vent 27, the second curved channel 29 connection well 21,
Liquid storage area 22 and trough 23, above-mentioned ball valve 25 are located between trough 23 and reacting hole 2, and each reaction detection part has 8
Individual reacting hole.
Above-mentioned micro-fluidic chip carries out sample introduction centrifugation by different wells, and trough is communicated by ball valve with liquid storage area,
Centrifuged by the low speed (1000rpm) of centrifuge, the liquid of liquid storage area is flowed into trough successively under the action of the centrifugal force；It is reserved
Area is communicated by ball valve with reacting hole, then is centrifuged by (3000rpm) at a high speed, and the liquid of trough is under the action of the centrifugal force successively
Flow into reacting hole, surplus liquid is flowed into waste liquid tank；Reacted at a certain temperature in reacting hole, heating enters reaction rank
Duan Shi, ball valve prevents the liquid in well to be back in trough and then spread to reacting hole around, while ensureing reacting hole
In liquid be full up state, it is ensured that reaction carry out and prevent pollution.
As shown in Fig. 2 the present invention carries out the detection of respiratory pathogen using micro-fluidic chip.Wherein 1# is to contain pneumonia
The reaction detection part of mycoplasma nucleic acid；2# is the reaction detection part containing CPN nucleic acid；3# is to contain first/B-mode
The reaction detection part of influenza nucleic acids；4# is the reaction detection part containing parainfluenza virus nucleic acid；In above-mentioned reaction inspection
Survey in reacting hole A, B, C, D, E, F, G in part 1#~4# and be coated with mycoplasma pneumoniae primer sets, CPN primer respectively
Group, first/influenza B virus primer sets, parainfluenza virus primer sets, adenovirus primer sets, Respiratory Syncytial Virus(RSV) primer sets,
Quality Control primer sets；It is the negative control of water in reacting hole H in above-mentioned reaction detection part 1#~4#.Also another 1 can be set up
Micro-fluidic chip, set respectively 5#~8# adenoviral nucleic acid, Respiratory Syncytial Virus(RSV) nucleic acid, containing six kinds of pathogen nucleic acids,
The reaction detection part of negative sample nucleic acid, its each reacting hole coating material and upper identical (not shown)；
Embodiment two
The present embodiment is the LAMP primer composition thing and its reagent of the present invention for being used to detect respiratory pathogen
Box.
LAMMP Primer compositions of the present invention include at least one in 6 pathogen primer sets.The respiratory diseases
Substance includes at least one of following six kinds of pathogen：Mycoplasma pneumoniae, CPN, first/influenza B virus, sidestream
Influenza Virus, adenovirus, Respiratory Syncytial Virus(RSV).Corresponding 6 primer sets of above-mentioned pathogen be respectively mycoplasma pneumoniae primer sets,
CPN primer sets, first/influenza B virus primer sets, parainfluenza virus primer sets, adenovirus primer sets, respiratory tract are closed
Cellular virus primer sets, primer combination also includes Quality Control primer sets (negative Quality Control, positive quality control).Its above-mentioned each primer sets it is each
Primer sequence refers to table 1.
The present invention relates to a kind of kit containing above-mentioned LAMP primer composition thing, it also includes as described in Example 1
It is coated with micro-fluidic chip, reaction solution, archaeal dna polymerase, indicator and the negative control thing of above-mentioned Primer composition.In coating primer
In the micro-fluidic chip of composition, the concentration of 4 kinds of primers in each primer sets is as follows：The concentration of outer primer 1 is 50 μM, is drawn outside
The concentration of thing 2 is 50 μM, and the concentration of inner primer 1 is 50 μM, and the concentration of inner primer 2 is 50 μM；Wherein, the outer primer 1, draw outside
Thing 2, inner primer 1, the volume ratio of inner primer 2 are 1:1:4~8:4~8.In the kit, the volume of reaction solution is 30 μ l, including
12mM dNTPs, 10 × Isothermal Amplification reaction buffers, the 150mM MgSO4 aqueous solution, wherein
DNTPs, reaction buffer, the volume ratio of the MgSO4 aqueous solution are 8：5：2；The volume of archaeal dna polymerase is 4 μ l, and it is that concentration is
8U/ μ l Bst archaeal dna polymerases；The volume of indicator is 5 μ l, and it is 2mM cresol reds.
Embodiment three
The present embodiment is to carry out detection respiratory tract pathogen using the Primer composition and kit implemented described in two
The method of non-diagnostic purpose, it comprises the following steps：
1. the cleaning step of micro-fluidic chip；
Micro-fluidic chip uses plasma cleaning, rinses micro-fluidic chip surface with plasma cleaner, uses inert gas
Air-dry.
2. the coating of Primer composition；
As shown in Figures 2 and 3, mycoplasma pneumoniae primer sets, CPN primer sets, first/influenza B virus are drawn
Thing group, parainfluenza virus primer sets, adenovirus primer sets, Respiratory Syncytial Virus(RSV) primer sets, Quality Control primer sets respectively with agar
Sugar mixing, is configured to corresponding mixed solution, in above-mentioned mixed liquor, contains 4~8 μM of inner primers 1,4~8 μM of μ of inner primer 2,1
Final concentration of 0.1% (mass percent) of agarose in M outer primers 1,1 μM of outer primer 2, the mixed solution；1 μ L are taken to mix
Solution is clicked and entered in the corresponding reacting hole of micro-fluidic chip (reacting hole A, B, C, D, E, F, G), reacting hole H storage water, will be micro-fluidic
Chip is after drying after being dried in clean super-clean bench, is vacuumized 2 hours, and primer sets are coated in corresponding reacting hole, so
The micro-fluidic chip is packaged using pressure-sensitive bonding film afterwards.
3. measuring samples nucleic acid extraction：The nucleic acid of measuring samples is extracted using paramagnetic particle method；
With paramagnetic particle method nucleic acid extraction kit extract mycoplasma pneumoniae (MP), CPN (CP), adenovirus (ADV),
Respiratory Syncytial Virus(RSV) (RSV), first/influenza B virus (IFB), parainfluenza virus (PIV) nucleic acid, operating procedure are as follows：
The purifying of 3.1 nucleic acid extractions prepares
A) lysate is please used if any precipitation after 56 DEG C of warm bath are completely dissolved to precipitation.
B) before use, plus 24mL absolute ethyl alcohols mixed, and carry out mark and (ensure that bottle cap is tightened, prevent second into washing lotion three
Alcohol volatilizees).
C) after proteinase mixture is fully mixed, dispensed according to every μ L of pipe 20 into each centrifuge tube.
D) according to target amount in 10 μ L of every pipe packing into each centrifuge tube.
3.2 nucleic acid extraction purification steps
A) to it is above-mentioned divided 200 μ l clinical samples and yin and yang attribute added in protease and interior target centrifuge tube compare, gently
It is light to mix；Then 400 μ l lysates are added, vortex oscillation 10s, 56 DEG C are incubated 10 minutes.
B) and then coprecipitated dose of 300 μ l nucleic acid and 2 μ l magnetic beads are added, turn upside down centrifuge tube 10 times, makes magnetic bead distribution in pipe
Uniformly.
C) centrifuge tube 10 minutes is stood, during which turned upside down centrifuge tube 5 times every two minutes, makes magnetic bead distribution in pipe equal
It is even.
D) centrifuge tube is placed on centrifuge, 8000g centrifugation 1min, and liquid in pipe is sucked with pipettor.
E) 500 μ l washing lotions one are added, tube wall magnetic bead, vortex oscillation 10s, until its distribution are softly broken up with pipettor gun head
Uniformly.
F) centrifuge tube is placed on centrifuge, 8000g centrifugation 1min suck liquid in pipe with pipettor.
G) 500 μ l washing lotions two are added, tube wall magnetic bead, vortex oscillation 10s, until its distribution are softly broken up with pipettor gun head
Uniformly.
H) centrifuge tube is placed on centrifuge, 8000g centrifugation 1min suck liquid in pipe with pipettor.
I) 500 μ l washing lotions three are added, tube wall magnetic bead, vortex oscillation 10s, until its distribution are softly broken up with pipettor gun head
Uniformly.
J) centrifuge tube is placed on centrifuge, 8000g centrifugation 1min suck liquid in pipe with pipettor.
K) centrifuge tube is placed on centrifuge, 8000g centrifugation 30s suck residual liquid in pipe with pipettor.
L) lid, drying at room temperature 2 minutes are opened.
M) 60 μ l eluents are added, tube wall magnetic bead is broken up with pipettor gun head, until it is evenly distributed.56 DEG C are incubated 3 points
Clock.
N) centrifuge tube is placed on centrifuge, 10000g centrifugations 1min.60 μ l supernatants being collected into are drawn to one
In the clean centrifuge tube of nuclease free, nucleic acid solution as after purification is used for constant temperature amplified fluorescence.
4.LAMP reacts
Taking 30 μ l constant-temperature amplifications buffer solutions, (dNTPs containing 12mM, 10 × Isothermal Amplification reaction are slow
Fliud flushing, the 150mM MgSO4 aqueous solution, three's volume ratio are 8：5：2), 4 μ l constant-temperature amplifications enzyme solutions (archaeal dna polymerase containing Bst,
Concentration be 8U/ μ l), 5 μ l 2mM cresol reds, the nucleic acid of 20 μ l measuring samples, add water and complement to 100 μ l reaction mixture,
Take 80 μ l reaction mixtures to inject in each well after vortex concussion is uniform to seal after membrana oralis, the Quick spin in centrifuge
1200rpm/min 15s, 3200rpm/min 30s, reaction mixture sample introduction is to reacting hole under the action of the centrifugal force；
Chip after sample-adding is put into necessary instrument micro-fluidic chip nucleic acids instrument, completes amplified reaction, 50 DEG C
20min；60℃60min；Or micro-fluidic chip is put into constent temperature heater, 50 DEG C of 20min；It is anti-that 60 DEG C of 60min complete amplification
Should.
Wherein, the nucleic acid of reaction detection part 1# measuring samples contains mycoplasma pneumoniae nucleic acid；Reaction detection part 2#
The nucleic acid of measuring samples contain CPN nucleic acid；The nucleic acid of reaction detection part 3# measuring samples contains first/B-mode
Influenza nucleic acids；The nucleic acid of reaction detection part 4# measuring samples contains the reaction detection part of parainfluenza virus nucleic acid.
5. result interpretation；
5.1 carry out interpretation using equipment：There is S-shaped in the positive reacting hole of necessary instrument micro-fluidic chip nucleic acids instrument
Amplification curve, negative reaction hole does not have amplification curve, if detecting certain reacting hole amplification curve occurs, the reacting hole is corresponding
The Testing index of the sample is the positive, i.e., contain respiratory pathogen nucleic acid and generation isothermal amplification reactions in reacting hole.
5.2 carry out interpretation using macroscopicization：It is yellow in the positive reacting hole of chip, negative reaction hole is red,
If detecting certain reacting hole yellow occurs, the Testing index of the corresponding sample of the reacting hole is the positive, i.e., described to be measured
The color of sample is yellow, then contains the genome of corresponding respiratory pathogen in the sample to be tested liquid, if described treat test sample
The color of product is red, then the genome of corresponding respiratory pathogen is not contained in the sample to be tested liquid.
As shown in figure 3, the throat swab sample of corresponding type is carried out after nucleic acid extraction, as template, it is added to micro-fluidic
The corresponding reaction detection part 1# (mycoplasma pneumoniae) of chip, reaction detection part 2# (CPN), reaction detection part
3# (influenza B virus), reaction detection part 4# (parainfluenza virus) carry out isothermal duplication, as a result as follows：Reaction detection part
1# reacting hole is macroscopic testing result of the mycoplasma pneumoniae clinical sample in reaction tank, and 1#-A, 1#-G are yellow, its
He is red；Reaction detection part 2# reacting hole is that mycoplasma pneumoniae clinical sample detects knot in the macroscopicization of reaction tank
Really, 2#-B, 2#-G are yellow, and other are red；Reaction detection part 3# reacting hole is that influenza B virus clinical sample exists
The macroscopic testing result of reaction tank, 3#-C, 3#-G are yellow, and other are red；Reaction detection part 4# reacting hole
It is macroscopic testing result of the parainfluenza clinical sample in reaction tank, 4#-D, 4#-G are yellow, and other are red.
The above results show that the kit and its method of detection respiratory pathogen of the present invention are micro- by being used in
The method that visualization judgement is carried out on fluidic chip, can be achieved 7 kinds of atypical respiratory pathogens (mycoplasma pneumoniae, pneumonia clothing
Substance, adenovirus, Respiratory Syncytial Virus(RSV), first/influenza B virus, parainfluenza virus) instant detection, not only realize to each
Small size well matches somebody with somebody liquid, it is to avoid operating personnel are difficult the problem of whether identification is loaded successfully, while passing through color change naked eyes
Result of determination is so as to reach visual requirement, and this makes the present invention easier in actual applications, quick and easily grasped
Make, suitable for execute-in-place.
The specific embodiment of the present invention is described in detail above, but it is only used as example, and the present invention is not intended to limit
In particular embodiments described above.To those skilled in the art, it is any to the practicality carry out equivalent modifications and replace
In generation, is also all among scope of the invention.Therefore, the equalization made without departing from the spirit and scope of the invention is converted and repaiied
Change, all should be contained within the scope of the invention.
SEQUENCE LISTING
<110>Shanghai Su Chuan diagnostic products Co., Ltd
<120>A kind of LAMP primer composition thing and its kit for detecting respiratory pathogen
<160> 28
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223>Mycoplasma pneumoniae outer primer 1
<400> 1
gcaagtccaa ctcaaggcg 19
<210> 2
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223>Mycoplasma pneumoniae outer primer 2
<400> 2
tcaatggcgg tacggttg 18
<210> 3
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223>Mycoplasma pneumoniae inner primer 1
<400> 3
agtcgttaac cacggcgttc agcgattttg gtactgcccc t 41
<210> 4
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223>Mycoplasma pneumoniae inner primer 2
<400> 4
gcaaattcac aacgaccccg cgcataaggc gcatcgtaca g 41
<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223>CPN outer primer 1
<400> 5
aatgaactac caaacgtttc t 21
<210> 6
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223>CPN outer primer 2
<400> 6
tgtttacaga gaattgcgat ac 22
<210> 7
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223>CPN inner primer 1
<400> 7
ttcccataag gctccacgag ggagttgttg aactttacac a 41
<210> 8
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223>CPN inner primer 2
<400> 8
cggttgtgca actttgggag gttacagatc acattaagtt cttca 45
<210> 9
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223>First/influenza B virus outer primer 1
<400> 9
gcagaccaat gtgtgtgg 18
<210> 10
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223>First/influenza B virus outer primer 2
<400> 10
ctcctggtgc cttttctg 18
<210> 11
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223>First/influenza B virus inner primer 1
<400> 11
gtgcattata ggaaagcacc cgcgaaagct tcaatactcc ac 42
<210> 12
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223>First/influenza B virus inner primer 2
<400> 12
caggacaaaa atcaggcaac tagcatcgat aacgttttgg gttga 45
<210> 13
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223>Parainfluenza virus outer primer 1
<400> 13
gatgagctca acactggaa 19
<210> 14
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223>Parainfluenza virus outer primer 2
<400> 14
tgattgaggc tccccatc 18
<210> 15
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223>Parainfluenza virus inner primer 1
<400> 15
tggaaagatg tctctgaact gtttgatgaa ctcggagtaa caca 44
<210> 16
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223>Parainfluenza virus inner primer 2
<400> 16
aaccaacagg tggatcagcc ttgatctgct ctatgttcga att 43
<210> 17
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223>Adenovirus outer primer 1
<400> 17
cctcggagta cctgagt 17
<210> 18
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223>Adenovirus outer primer 2
<400> 18
agcgcacttt ataggagta 19
<210> 19
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223>Adenovirus inner primer 1
<400> 19
acggtggggt ttctaaactt gtccgggcct ggtgcaatt 39
<210> 20
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223>Adenovirus inner primer 2
<400> 20
ggctcccacc catgatgtta cgtggtatct tcccgatcca 40
<210> 21
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223>Respiratory Syncytial Virus(RSV) outer primer 1
<400> 21
accaaccaat caatcaacc 19
<210> 22
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223>Respiratory Syncytial Virus(RSV) outer primer 2
<400> 22
tgttagtgat gcgggatc 18
<210> 23
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223>Respiratory Syncytial Virus(RSV) inner primer 1
<400> 23
gccccatctt gttctttttt tgttaattga tcaatcagca ccct 44
<210> 24
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223>Respiratory Syncytial Virus(RSV) inner primer 2
<400> 24
tatggaaaca tacgtgaaca agctcatctt tttctagaac attgtactg 49
<210> 25
<211> 16
<212> DNA
<213> Artificial Sequence
<220>
<223>Quality Control outer primer 1
<400> 25
cgccggccat gtacgt 16
<210> 26
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223>Quality Control outer primer 2
<400> 26
gtagtcggtc aggtcccg 18
<210> 27
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223>Quality Control inner primer 1
<400> 27
tccccagagt ccatgacaat gctccaggct gtgctgtcc 39
<210> 28
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223>Quality Control inner primer 2
<400> 28
tggggtcacc cacacggtgc cagccaggtc caga 34
Claims (10)
1. a kind of LAMP primer composition thing for being used to detect respiratory pathogen, it is characterised in that including mycoplasma pneumoniae primer
Group, CPN primer sets, first/influenza B virus primer sets, parainfluenza virus primer sets, adenovirus primer sets, breathing
At least one set in road syncytial virus primer sets；
Wherein, the mycoplasma pneumoniae primer sets include SEQ ID NO：Mycoplasma pneumoniae outer primer 1, SEQ ID shown in 1
NO：Mycoplasma pneumoniae outer primer 2, SEQ ID NO shown in 2：Mycoplasma pneumoniae inner primer 1 and SEQ ID NO shown in 3：4 institutes
The mycoplasma pneumoniae inner primer 2 shown；
Wherein, the CPN primer sets include SEQ ID NO：CPN outer primer 1, SEQ ID shown in 5
NO：CPN outer primer 2, SEQ ID NO shown in 6：CPN inner primer 1 and SEQ ID NO shown in 7：8 institutes
The CPN inner primer 2 shown；
Wherein, the first/influenza B virus primer sets include SEQ ID NO：First/influenza B virus outer primer shown in 9
1、SEQ ID NO：First/influenza B virus outer primer 2, SEQ ID NO shown in 10：First/influenza B virus shown in 11
Inner primer 1 and SEQ ID NO：First/influenza B virus inner primer 2 shown in 12；
Wherein, the parainfluenza virus primer sets include SEQ ID NO：Parainfluenza virus outer primer 1, SEQ ID shown in 13
NO：Parainfluenza virus outer primer 2, SEQ ID NO shown in 14：Parainfluenza virus inner primer 1 and SEQ ID NO shown in 15：
Parainfluenza virus inner primer 2 shown in 16；
Wherein, the adenovirus primer sets primer sets include SEQ ID NO：Adenovirus primer sets outer primer 1, SEQ shown in 17
ID NO：Adenovirus primer sets outer primer 2, SEQ ID NO shown in 18：Adenovirus primer sets inner primer 1 and SEQ shown in 19
ID NO：Adenovirus primer sets inner primer 2 shown in 20；
Wherein, the Respiratory Syncytial Virus(RSV) primer sets include SEQ ID NO：Respiratory Syncytial Virus(RSV) outer primer 1 shown in 21,
SEQ ID NO：Respiratory Syncytial Virus(RSV) outer primer 2, SEQ ID NO shown in 22：Draw in Respiratory Syncytial Virus(RSV) shown in 23
Thing 1 and SEQ ID NO：Respiratory Syncytial Virus(RSV) inner primer 2 shown in 24.
2. the LAMP primer composition thing according to claim 1 for being used to detect respiratory pathogen, it is characterised in that enter one
Step includes Quality Control primer sets, and the Quality Control primer sets include SEQ ID NO：Quality Control outer primer 1, SEQ ID NO shown in 25：26
Shown Quality Control outer primer 2, SEQ ID NO：Quality Control inner primer 1 and SEQ ID NO shown in 27：Quality Control inner primer shown in 28
2。
3. a kind of kit of the LAMP primer composition thing containing described in claim 1 or 2, it is characterised in that also including miniflow
Chip is controlled, the micro-fluidic chip is provided with mutual disconnected 4 reaction detection parts, and each reaction detection part is included successively
Liquid storage area, trough, ball valve and the reacting hole of connection, the liquid storage area set well, and each reaction detection part has 8
Each primer sets in reacting hole, the Primer composition are coated in corresponding reacting hole respectively.
4. kit according to claim 3, it is characterised in that the concentration of the outer primer 1 in each primer sets is 50 μM,
The concentration of outer primer 2 is 50 μM, and the concentration of inner primer 1 is 50 μM, and the concentration of inner primer 2 is 50 μM；Wherein, the outer primer 1,
Outer primer 2, inner primer 1, the volume ratio of inner primer 2 are 1:1:4~8:4~8.
5. kit according to claim 4, it is characterised in that the kit further comprises reaction solution, DNA polymerizations
Enzyme, indicator, negative control thing.
6. kit according to claim 5, it is characterised in that the reaction solution include 10~15mM dNTPs, 10 ×
Isothermal Amplification reaction buffers, 100~200mM MgSO4 aqueous solution；Wherein, in the reaction solution
DNTPs, reaction buffer, the volume ratio of the MgSO4 aqueous solution are 7~9:4~6:2.
7. kit according to claim 6, it is characterised in that the indicator include HNB, Calcein, cresol red,
Phenol red, m-cresol purple, bromocresol purple, dimethyl diaminophenazine chloride, naphtholphthalein, thymol blue.
8. kit according to claim 6, it is characterised in that the archaeal dna polymerase is Bst archaeal dna polymerases, concentration
For 8U/ μ l.
9. a kind of Primer composition using described in claim 1 or 2 exists the method that detects respiratory pathogen, its feature
In comprising the following steps：
Step 1) Primer composition coating：Each primer sets in the Primer composition are coated on corresponding reaction respectively
In hole, it is fixed on by vacuum drying in reacting hole；
Step 2) measuring samples nucleic acid extraction：The nucleic acid of measuring samples is extracted using paramagnetic particle method；
Step 3) LAMP reactions：Extract reaction solution with archaeal dna polymerase, indicator, after being mixed with the nucleic acid of measuring samples, be transferred to
The liquid storage area of the micro-fluidic chip of primer sets is coated with, liquid is flowed into reacting hole under the action of the centrifugal force, then carries out ring mediation
Isothermal amplification reactions；
Step 4) result interpretation：Terminate the color change in rear reacting hole according to amplification to carry out direct naked eyes interpretation or use to set
It is standby to carry out color interpretation.
10. method according to claim 9, it is characterised in that step 1) described in Primer composition it is coated specific
Step is as follows：Each primer sets of the Primer composition are mixed with agarose respectively, corresponding mixed solution is configured to, takes
1 μ L mixed solution is clicked and entered in the corresponding reacting hole of micro-fluidic chip, after being dried in clean super-clean bench, vacuumizes dry 2 small
Shi Hou, primer sets are coated in corresponding reacting hole；Wherein, in the mixed solution agarose final concentration of 0.1wt%,
The mixed solution contains 4~8 μM of inner primers 1,4~8 μM of inner primers, 2,1 μM of outer primer 1,1 μM of outer primer 2.
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| CN108486262A (en) * | 2018-06-06 | 2018-09-04 | 上海速创诊断产品有限公司 | A kind of LAMP primer composition object, kit and its method of detection mycoplasma pneumoniae |
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| CN112375669A (en) * | 2020-10-30 | 2021-02-19 | 清华大学深圳国际研究生院 | Micro-fluidic chip for extracting, purifying and detecting nucleic acid |
| WO2022124919A1 (en) * | 2020-12-08 | 2022-06-16 | Genomtec S.A. | Set of primers, composition of reagents and method of detecting atypical bacteria |
| CN112941242A (en) * | 2021-04-20 | 2021-06-11 | 美格医学检验所(广州)有限公司 | Primer, probe, kit and application for detecting respiratory syncytial virus |
| CN113073151A (en) * | 2021-05-12 | 2021-07-06 | 广州普世利华科技有限公司 | Primer probe composition for detecting adenovirus HAdV by using enzyme digestion probe report system and kit thereof |
| CN114632558A (en) * | 2021-12-17 | 2022-06-17 | 上海交通大学医学院附属仁济医院 | Microfluidic chip and preparation method and application thereof |
| CN114632558B (en) * | 2021-12-17 | 2023-08-18 | 上海交通大学医学院附属仁济医院 | Microfluidic chip and preparation method and application thereof |
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