CN106916906A - A kind of Primer composition and its kit for detecting infectious diarrhea pathogen - Google Patents
A kind of Primer composition and its kit for detecting infectious diarrhea pathogen Download PDFInfo
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- CN106916906A CN106916906A CN201710305672.7A CN201710305672A CN106916906A CN 106916906 A CN106916906 A CN 106916906A CN 201710305672 A CN201710305672 A CN 201710305672A CN 106916906 A CN106916906 A CN 106916906A
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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
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
The present invention relates to a kind of Primer composition for detecting infectious diarrhea pathogen, it includes at least one set in rotavirus primer sets, EAd primer sets, norovirus primer sets, salmonella primer sets, Shigella primer sets and campylobacter jejuni primer sets；A kind of kit comprising the Primer composition is further related to, its micro-fluidic chip, isothermal amplification reactions liquid, constant-temperature amplification enzyme solutions, negative control thing for also including coating primer；Further relate to a kind of detection method using above-mentioned Primer composition, including the coating of Primer composition, measuring samples nucleic acid extraction, LAMP reactions and result interpretation.The kit that the present invention is provided can quickly and accurately detect infectious diarrhea pathogen in 1 hour, also significant for the treatment of quick auxiliary direction and medication；The detection of the multi objective can be used for regional epidemiological survey and epidemic situation monitoring, to study popularity of the infectious diarrhea in China.
Description
Technical field
The invention belongs to nucleic acid amplification technologies field, and in particular to a kind of primer for detecting infectious diarrhea pathogen
Composition, and the kit and its method that detect infectious diarrhea pathogen based on micro-fluidic chip system.
Background technology
Infectious diarrhea is the most common class disease in the whole world, and the children that the annual whole world is estimated to be 3,200,000≤5 years old die from
Diarrhoea, accounts for the 24.8% of children's cause of the death of the same age；In developing country, annual every child morbidity 1-12 times, developed country is annual every
Child morbidity 1-5 times, it can be seen that, infectious diarrhea is still when the important disease of previous class.Infectious diarrhea refers to various urgency
Property, chronic bacterium, virus, fungi, parasitic infection cause the diarrhoea caused by intestinal inflammatory.We are also except cholera, bacterium
Infectious diarrhea beyond property and amebic dysentery, Typhoid and paratyphoid, referred to as infectious diarrhea, be《Chinese people's republicanism
State's law on the prevention and control of infectious diseases》Specified in Class C infectious disease.
The pathogen of infectious diarrhea has bacterium, virus, parasite, fungi etc..At present, it is main with bacterium and disease in China
Based on poison, virus type has rotavirus, norovirus, adenovirus and Coxsackie virus etc., and bacterium class is mainly salmonella, will
Hayes bacterium, campylobacter jejuni etc.；Tool reported literature, viral infection accounts for infectious diarrhea 90% or so, and bacterium accounts for 10% or so；
It is that rotavirus and (or) adenovirus, norovirus cause to have more than 95% in virus, and bacterium is with salmonella, campylobacter jejuni
Based on Shigella, more than 90% is accounted for.Infectious diarrhea is China's clinically one of most common disease, if patient with severe symptoms is not
Can timely, correctly taking drugs, easy threat to life.However, the disease that routine clinical Analysis of Biochemical cannot be infected patient
Substance makes precise Identification, most of clinical treatments still in the experience medication stage, exist in terms of using senior antibiotic compared with
Big blindness.Clinically the method for the popular identification pathogen for using is mainly by Bacteria Culture detection method at present, but
There is larger defect in Bacteria Culture detection method：Detection time (general 2-3 days or so) long, accuracy rate low (false negative rate and vacation
Positive rate is all higher), pathogen for being difficult to detect by difficult culture etc..
Loop-mediated isothermal amplification technique (Loopmediated isothermal amplification, hereinafter referred to as LAMP
Method) LAMP be 2000 exploitation a kind of constant temperature nucleic acid amplification method, be characterized in for target gene 6 regions design 4 kinds
Special primer, is incubated 30-60 minutes for (60-65 DEG C), you can complete nucleic acid using a kind of strand displacement archaeal dna polymerase in isothermy
Amplified reaction.LAMP has easy, quick, sensitive, special advantage, is particluarly suitable for the application of development at the basic level LAMP kit
Promote.In LAMP technology, primer is the key factor for determining testing result sensitivity and specificity.
The content of the invention
It is an object of the invention to overcome defect of the prior art, there is provided one kind is for detecting infectious diarrhea pathogen
Primer composition and its kit, for rapidly and accurately identifying the species of infectious diarrhea pathogen.
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 Primer composition for detecting infectious diarrhea pathogen, and it includes
Rotavirus primer sets, EAd primer sets, norovirus primer sets, salmonella primer sets, Shigella primer sets
With at least one set in campylobacter jejuni primer sets；
Wherein, the rotavirus primer sets include SEQ ID NO：Rotavirus primers F 3, SEQ ID NO shown in 1：
Rotavirus primer B3, SEQ ID NO shown in 2：Rotavirus primers F IP and SEQ ID NO shown in 3：Colyliform shown in 4
Viral primer BIP；
Wherein, the EAd primer sets include SEQ ID NO：EAd primers F 3, SEQ ID shown in 5
NO：EAd primer B3, SEQ ID NO shown in 6：EAd primers F IP and SEQ ID NO shown in 7：8 institutes
The EAd primer BIP for showing；
Wherein, the norovirus primer sets include SEQ ID NO：Norovirus primers F 3, SEQ ID NO shown in 9：
Norovirus primer B3, SEQ ID NO shown in 10：Norovirus primers F IP and SEQ ID NO shown in 11：Shown in 12
Norovirus primer BIP
Wherein, the salmonella primer sets include SEQ ID NO：Salmonella primers F 3, SEQ ID shown in 13
NO：Salmonella primer B3, SEQ ID NO shown in 14：Salmonella primers F IP and SEQ ID NO shown in 15：Shown in 16
Salmonella primer BIP；
Wherein, the Shigella primer sets include SEQ ID NO：Shigella primers F 3, SEQ ID shown in 17
NO：Shigella primer B3, SEQ ID NO shown in 18：Shigella primers F IP and SEQ ID NO shown in 19：Shown in 20
Shigella primer BIP；
Wherein, the campylobacter jejuni primer sets include SEQ ID NO：Campylobacter jejuni primers F 3, SEQ shown in 21
ID NO：Campylobacter jejuni primer B3, SEQ ID NO shown in 22：Campylobacter jejuni primers F IP and SEQ ID shown in 23
NO：Campylobacter jejuni primer BIP 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 Primer composition further includes internal reference primer sets, and the internal reference primer sets are used to expand people
Hemoglobin-β chains (hemoglobin beta-chain, HBB)；
Wherein, the internal reference primer sets include SEQ ID NO：Internal reference primers F 3, SEQ ID NO shown in 25：26
Shown internal reference primer B3, SEQ ID NO：Internal reference primers F IP and SEQ ID NO shown in 27：Internal reference shown in 28
Primer BIP.
The primer sequence of above-mentioned infectious diarrhea pathogen primer sets and the sequence of internal reference primer sets are as shown in table 1.
The amplimer sequence table of table 1
On the other hand, the present invention provides a kind of kit containing the combination of above-mentioned primer, and it also includes micro-fluidic chip, institute
State micro-fluidic chip and be provided with mutual disconnected 4 reaction detection areas, sample inlet pool that each reaction detection area includes being sequentially communicated,
Distributing reservoir, capillary micro-valve and amplification pond, each reaction detection area have 8 amplification ponds, each in the Primer composition
Primer sets are coated in corresponding amplification pond 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, in each amplification pond, the concentration of primers F 3 is 1~4 μM in each primer sets, and the concentration of primer B3 is
1~4 μM, the concentration of primers F IP is 8~15 μM, and the concentration of primer BIP is 8~15 μM.
Preferably, the kit further includes isothermal amplification reactions liquid, constant-temperature amplification enzyme solutions, negative control thing.
Preferably, the isothermal amplification reactions liquid include 200~250mM Tris-HCl, 300~400mM KCl, 8~
10mM dNTP, 100~120mM MgSO4,1%~5% (W/V) Tween-20,40%~50% (W/V) glycerine, 1~5%
(W/V) PEG8000 and 10~20mM indicator；The constant-temperature amplification enzyme solutions include 10~12U/ μ L archaeal dna polymerases and 20~
30U/ μ L reverse transcriptases.
Preferably, the isothermal amplification reactions liquid includes 250mM Tris-HCl, 300mM KCl, 10mM dNTP, 100mM
MgSO4,5% (W/V) Tween-20,50% (W/V) glycerine, 5% (W/V) PEG8000 and 10mM indicator.
Preferably, the indicator includes HNB, Calcein, cresol red, phenol red, m-cresol purple, bromocresol purple, neutrality
Red, naphtholphthalein, thymol blue.
Preferably, the indicator is dimethyl diaminophenazine chloride, and concentration is 10~20mM, more preferably 10mM.Dimethyl diaminophenazine chloride (Neutral
Red), scientific name " 3- amino -7- methylamino -2- toluphenazines hydrochloride ", is a kind of alkalescent pH indicator, color change interval
It is red under acid condition between pH6.4~8.0 (yellow by red change), is yellow under alkalescence condition；Constant-temperature amplification reacts
Before, because reaction solution is in alkalescence, therefore color is yellow, when there is amplified reaction to occur, with the continuous accumulation of amplified production,
Solution PH environment is changed into acid, therefore color can be changed into red from yellow.
Preferably, the archaeal dna polymerase is Bst archaeal dna polymerases, and the reverse transcriptase is M-MLV reverse transcriptases；It is more excellent
It is the M-MLV reverse transcriptases of 20U/ μ L to elect the Bst archaeal dna polymerases and concentration that concentration is 10U/ μ L as.
Preferably, the negative control thing is the water without RNase.
Finally, the present invention also provide it is a kind of detected using above-mentioned Primer composition infectious diarrhea pathogen for non-
The method of diagnostic purpose, it comprises the following steps：
Step 1) Primer composition coating：Each primer sets in the Primer composition are coated on accordingly respectively
In amplification pond, it is fixed in amplification pond by vacuum drying；
Step 2) measuring samples nucleic acid extraction：The nucleic acid of measuring samples is extracted using paramagnetic particle method；
Step 3) LAMP reactions：Isothermal amplification reactions liquid and archaeal dna polymerase, reverse transcriptase solution are taken, with measuring samples
After nucleic acid is mixed, the sample inlet pool of the micro-fluidic chip of coating primer sets is transferred to, liquid flows into amplification under the action of the centrifugal force
In pond, loop-mediated isothermal amplification reaction is then carried out；
Step 4) result interpretation：The color change expanded after being terminated according to amplification in pond carries out direct naked eyes interpretation or adopts
Color interpretation is carried out with equipment.
Preferably, step 1) described in the coated of Primer composition comprise the following steps that：By the Primer composition
Each primer sets mix with sucrose respectively, are configured to corresponding mixed solution, and the primer sets and sucrose are in the mixed solution
In final concentration be respectively 0.15 μM and 1.0% (mass percent)；Take 1 μ L mixed solutions and click and enter micro-fluidic chip and expand accordingly
Increase in pond, in dry in 37 DEG C of baking ovens, compressing tablet sealer, after punching press is vacuumized, primer sets are coated in corresponding amplification pond；More
Specifically, 8 amplification ponds in each reaction detection area are coated with following primer sets respectively in the micro-fluidic chip：Rotavirus
LMAP primer sets, EAd LAMP primer group, norovirus LAMP primer group, salmonella LAMP primer group, will Hayes
Bacterium LAMP primer group, campylobacter jejuni LAMP primer group, negative controls, internal reference primer sets.
Preferably, step 3) described in LAMP reaction comprise the following steps that：Take 25 μ L isothermal amplification reactions liquids and 10 μ L
Archaeal dna polymerase, 10 μ L reverse transcriptase solution, after being mixed with the nucleic acid of 40 μ L measuring samples, are transferred to coating primer sets miniflow
The sample inlet pool of chip is controlled, in the centrifuge, liquid is flowed into amplification pond under the action of the centrifugal force, in 50 ° of DEG C of isothermal reactions
10 minutes, in 60~63 DEG C of isothermal reactions 50 minutes；Wherein isothermal amplification reactions liquid and archaeal dna polymerase, reverse transcriptase solution is equal
Using the reagent in mentioned reagent box.
Preferably, step 4) described in direct naked eyes interpretation it is specific as follows：If color changes, the sample to be checked
In contain infectious diarrhea pathogen genome nucleic acid；If color does not change, infection is not contained in the sample to be checked
Property diarrhoeal diseases substance genomic nucleic acids.
Preferably, the sample to be tested includes excrement；Accordingly, the sample to be tested nucleic acid includes what is extracted from excrement
DNA or RNA.
Primer sets of the present invention or primer sets composition or kit are being detected or are aiding in detection infectious diarrhea cause of disease
Non-diagnostic purpose application in body falls within protection scope of the present invention.
In the present invention, at least one during the infectious diarrhea pathogen can be as follows：Rotavirus, enteron aisle adenopathy
Poison, norovirus, salmonella, Shigella and campylobacter jejuni.
Compared with prior art, the invention has the advantages that：
The kit that the present invention is provided provides high-throughout detection method, can quickly and accurately detect Common infectious abdomen
Pathogenic infection is rushed down, for clinic, 6 kinds of testing results of pathogen index can be obtained in 1 hour, be not only faster than mesh
The preceding real time fluorescence quantifying PCR method for more generally using, and also have for the treatment of quick auxiliary direction and medication important
Meaning；Meanwhile, the detection of multi objective can be used for regional epidemiological survey and epidemic situation monitoring, to study infectious abdomen
Rush down the popularity in China.
Brief description of the drawings
Fig. 1 is the structural representation of the micro-fluidic chip that the present invention is used；
Fig. 2 and Fig. 3 is the coating schematic diagram of primer sets in the micro-fluidic chip of one embodiment of the invention；
Fig. 4 and Fig. 5 are 6 kinds of testing result figures of the micro-fluidic chip of infectious diarrhea pathogen sample；
Reference is in figure：
11st, reaction detection area；12nd, sample inlet pool；13rd, distributing reservoir；14th, pond is expanded；15th, capillary micro-valve.
A~H：Reaction detection area；1~8：It is coated with the amplification pond of primer sets.
Hereinafter, the amplification pond 1 of reaction detection area A represents that the amplification pond 2 of reaction detection area A is represented with A-2 with A-1,
The like.
Specific embodiment
The invention provides a kind of Primer composition and its kit for detecting infectious diarrhea pathogen and detection
Method, wherein the infectious diarrhea pathogen includes rotavirus, EAd, norovirus, salmonella, will Hayes
At least one in bacterium, campylobacter jejuni, the Primer composition includes the corresponding primer sets of above-mentioned infectious diarrhea pathogen
In at least one set.
Rotavirus, EAd, norovirus, enterovirus EV 71, enterovirus CA16, gland that the present invention is used
Viral 3 types are clinical sample, take from the attached pediatric hospital of Fudan University in Shanghai；Salmonella derives from Chinese medicine bacterium preservation
Administrative center (CMCC), bacterial strain number is CMCC50115；Shigella derives from Chinese medicine bacterium preservation administrative center
(CMCC), bacterial strain number is CMCC51592；Campylobacter jejuni derives from Chinese industrial Microbiological Culture Collection administrative center
(CICC), bacterial strain number is CICC22936；Comma bacillus derives from American Type Culture Collection (ATCC), and bacterial strain number is
ATCC27070；Vibrio alginolyticus derives from Chinese industrial Microbiological Culture Collection administrative center (CICC), and bacterial strain number is
CICC21664；ETEC derives from Chinese medicine bacterium preservation administrative center (CMCC), and bacterial strain number is CMCC44103；
The internal reference that the present invention is used is the pseudovirus of the amplified fragments containing HBB mesh.
With reference to the accompanying drawings and examples, specific embodiment of the invention is further described.Following examples are only
For clearly illustrating technical scheme, and 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
Area 11, each reaction detection area 11 includes the sample inlet pool 12, distributing reservoir 13, capillary micro-valve 15 and the amplification pond 16 that are sequentially communicated,
Sample inlet pool 12 is connected by a curved channel with distributing reservoir 13, and sample holes are provided with sample inlet pool, and distributing reservoir 13 is logical by an arc
Road is connected with waste liquid pool and steam vent；Each reaction detection area 11 is equipped with 8 amplification ponds 16.
Wherein the major function of sample inlet pool 12 is loading reaction solution；The Main Function of distributing reservoir 13 is to evenly distribute reaction solution
Into amplification pond；Amplification pond 15 implements loop-mediated isothermal amplification reaction (LAMP)；Capillary micro-valve 14 mainly utilizes capillary
Pipe controls the retardation of liquid the motion of fluid in micro-fluidic chip.
As shown in figures 2-3, the present invention uses 2 micro-fluidic chips, carries out the detection of infectious diarrhea pathogen.A be containing
There is the reaction detection area of rotavirus nucleic acid；B is the reaction detection area containing EAd nucleic acid；C is to contain norovirus
The reaction detection area of nucleic acid；D is the reaction detection area containing salmonella nucleic acid；E is the reaction inspection containing Shigella nucleic acid
Survey area；F is the reaction detection area containing campylobacter jejuni nucleic acid；G is to contain six kinds of reaction detection areas of pathogen nucleic acid；H is
The reaction detection area of negative sample nucleic acid；Wrapped respectively in amplification pond 1,2,3,4,5,6,8 in above-mentioned reaction detection area A~H
By rotavirus primer sets, EAd primer sets, norovirus primer sets, salmonella primer sets, Shigella primer
Group, campylobacter jejuni primer sets, internal reference primer sets；Deposited without RNA in amplification pond 7 in above-mentioned reaction detection area A~F
The negative control of enzyme water.
Embodiment two
The present embodiment is the Primer composition and its reagent for detecting infectious diarrhea pathogen of the present invention
Box.
At least one in 6 primer sets of the Primer composition of the present invention including independent packaging.The infectious abdomen
Rushing down pathogen includes at least one in following six kinds of pathogen：Rotavirus, EAd, norovirus, salmonella,
Shigella, campylobacter jejuni.Corresponding 6 primer sets of above-mentioned pathogen are respectively rotavirus primer sets, EAd
Primer sets, norovirus primer sets, salmonella primer sets, Shigella primer sets and campylobacter jejuni primer sets, the primer
Combination also includes internal reference primer sets, and the internal reference primer sets are used to expand human hemoglobin-β chains.Above-mentioned its respectively draws
Each primer sequence of thing group refers to table 1.
The present invention relates to a kind of kit containing above-mentioned Primer composition, it also includes the coating as described in embodiment one
The micro-fluidic chip of above-mentioned primer sets, isothermal amplification reactions liquid, constant-temperature amplification enzyme solutions and negative control thing.In coating primer sets
Micro-fluidic chip in, the mol ratio of 4 kinds of primers in each primer sets is as follows：The primer of band " F3 ", 3 μM of names in 3 μM of titles
The primer of band " B3 " in title, the primer of band " FIP " in 12 μM of titles, the primer of band " BIP " in 12 μM of titles.Perseverance in kit
The volume of isothermal amplification reaction liquid is 25 μ L, contains 0.5 μ L 250mmol Tris-HCl, 0.5 μ L 300mmol KCl, 15 μ L
10mM dNTP, 0.5 μ L 120mM MgSO4,0.25 μ L 1% (W/V) Tween-20, (W/V) glycerine of 0.5 μ L 50% and 0.25
μ L 1% (W/V) PEG8000,1.0 μ L 10mM dimethyl diaminophenazine chlorides, 25 μ L are supplied with without RNase water.Constant-temperature amplification enzyme in kit
Solution includes 10 μ L 10U/ μ L archaeal dna polymerases, 10 μ L 20U/ μ L reverse transcriptase solution.
Above-mentioned Bst archaeal dna polymerases be NEB Products, Tris-HCl, KCl, MgSO4, Tween-20, glycerine,
PEG8000, dimethyl diaminophenazine chloride are Sigma Products, and dNTP is Takara Products, and M-MLV reverse transcriptase is Promega companies
Product.
Embodiment three
The present embodiment is to carry out detecting infectious diarrhea pathogen using the Primer composition and kit implemented described in two
Method, it comprises the following steps：
1. the coating of Primer composition；
As shown in Figures 2 and 3, by rotavirus primer sets, EAd primer sets, norovirus primer sets, sramana
Salmonella primer sets, Shigella primer sets, campylobacter jejuni primer sets, internal reference primer sets mix with sucrose respectively, are configured to
Corresponding mixed solution, the final concentration of each primer sets and sucrose in the mixed solution is respectively 0.15 μM and 1.0% (quality
Percentage)；Take 1 μ L mixed solutions and click and enter micro-fluidic chip and expand accordingly in pond (amplification pond 1,2,3,4,5,6,8), expand pond
Without RNase water, by micro-fluidic chip in dry in 37 DEG C of baking ovens, compressing tablet sealer, after punching press is vacuumized, primer sets are wrapped for 7 storages
By in each amplification pond.
2. measuring samples nucleic acid extraction：The nucleic acid of measuring samples is extracted using paramagnetic particle method；
Rotavirus, enteron aisle adenopathy are extracted with nucleic acid extraction kit (Da'an Gene Company, Zhongshan University)
Poison, norovirus, salmonella, Shigella, campylobacter jejuni, operating procedure carry specification and enter by nucleic acid extraction kit
OK, rotavirus, EAd, norovirus, salmonella, Shigella, campylobacter jejuni nucleic acid samples are obtained；
By rotavirus obtained above, EAd, norovirus, salmonella, Shigella, campylobacter jejuni
Nucleic acid presses 1：1：1：1：1：1 ratio mixes, and obtains multiple positive nucleic acid samples；
Enterovirus EV 71, enteron aisle disease are extracted with nucleic acid extraction kit (Da'an Gene Company, Zhongshan University)
Malicious CA16, adenovirus type III, comma bacillus, vibrio alginolyticus, ETEC, operating procedure are carried by nucleic acid extraction kit
Specification is carried out, by enterovirus EV 71, enterovirus CA16, adenovirus type III, comma bacillus, vibrio alginolyticus, E
Sclerotium acid presses 1：1：1：1：1：1 ratio mixes, and obtains negative nucleic acid samples；
Internal reference nucleic acid is extracted with nucleic acid extraction kit (Da'an Gene Company, Zhongshan University)；
Positive nucleic acid sample concentration obtained above is 103Copy/μ L, negative nucleic acid samples concentration is 106Copy/μ L,
Internal reference nucleic acid concentration is 104TD/μL。
3.LAMP reacts
25 μ L isothermal amplification reactions liquids and constant-temperature amplification enzyme solutions described in 20 μ L are taken, with 30 μ L rotavirus nucleic acids and 10 μ L
Internal reference nucleic acid samples mix, and are injected into the sample inlet pool of reaction detection area A of disk micro-fluidic chip；
25 μ L isothermal amplification reactions liquids and constant-temperature amplification enzyme solutions described in 20 μ L are taken, with 30 μ L EAds nucleic acid and 10
μ L internal references nucleic acid samples mix, and are injected into the sample inlet pool of reaction detection area B of disk micro-fluidic chip；
Take 25 μ L isothermal amplification reactions liquids and constant-temperature amplification enzyme solutions described in 20 μ L, with 30 μ L vibrio parahemolyticus nucleic acid and
10 μ L internal references nucleic acid samples mix, and are injected into the sample inlet pool of reaction detection area C of disk micro-fluidic chip；
25 μ L isothermal amplification reactions liquids and constant-temperature amplification enzyme solutions described in 20 μ L are taken, with 30 μ L salmonellas nucleic acid and 10 μ L
Internal reference nucleic acid samples mix, and are injected into the sample inlet pool of reaction detection area D of disk micro-fluidic chip；
25 μ L isothermal amplification reactions liquids and constant-temperature amplification enzyme solutions described in 20 μ L are taken, with 30 μ L Shigellas nucleic acid and 10 μ L
Internal reference nucleic acid samples mix, and are injected into the sample inlet pool of reaction detection area E of disk micro-fluidic chip；
25 μ L isothermal amplification reactions liquids and constant-temperature amplification enzyme solutions described in 20 μ L are taken, with 30 μ L campylobacter jejunis nucleic acid and 10
μ L internal references nucleic acid samples mix, and are injected into the sample inlet pool of reaction detection area F of disk micro-fluidic chip；
25 μ L isothermal amplification reactions liquids and constant-temperature amplification enzyme solutions described in 20 μ L are taken, with the multiple positive nucleic acid of 30 μ L and 10 μ L
Internal reference nucleic acid samples mix, and are injected into the sample inlet pool of reaction detection area G of disk micro-fluidic chip；
Take 25 μ L isothermal amplification reactions liquids and constant-temperature amplification enzyme solutions described in 20 μ L, with 30 μ L mixing negative sample nucleic acid and
10 μ L internal references nucleic acid samples mix, and are injected into the sample inlet pool of reaction detection area H of disk micro-fluidic chip；
The sample inlet pool pad pasting of above-mentioned micro-fluidic chip is closed, is placed in a centrifuge, 3000rpm centrifugations 1min；50 DEG C of perseverances
Incubator reacts 10min；63 DEG C of insulating boxs react 50min.
4. result interpretation；
As shown in Figure 4 and Figure 5, its LAMP reaction result is as follows：It is embedded with rotavirus primer sets, is injected into rotavirus
The amplification pond A-1 and G-1 colors of nucleic acid samples are pink, illustrate to contain rotavirus nucleic acid in two amplification ponds of A-1 and G-1
And there are isothermal amplification reactions；It is embedded with EAd primer sets, is injected into the amplification pond B-2 of EAd nucleic acid samples
It is pink with G-2 colors, illustrates to contain EAd nucleic acid in two amplification ponds of B-2 and G-2, and it is anti-that constant-temperature amplification occurs
Should；It is pink to be embedded with norovirus primer sets, be injected into the amplification pond C-3 and G-3 colors of norovirus nucleic acid samples, is said
Bright C-3 and G-3 contain norovirus nucleic acid and generation isothermal amplification reactions in two amplification ponds；It is embedded with salmonella primer
Group, to be injected into the amplification pond D-4 and G-4 colors of salmonella nucleic acid samples be pink, illustrate that D-4 and G-4 two expand ponds
In containing salmonella nucleic acid and occur isothermal amplification reactions；It is embedded with Shigella primer sets, is injected into Shigella nucleic acid
The amplification pond E-5 and G-5 colors of sample are pink, illustrate to contain Shigella nucleic acid and hair in two amplification ponds of E-5 and G-5
Raw isothermal amplification reactions；It is embedded with campylobacter jejuni primer sets, is injected into the amplification pond F-6 and G- of campylobacter jejuni nucleic acid samples
6 colors are pink, illustrate to contain campylobacter jejuni nucleic acid and generation isothermal amplification reactions in two amplification ponds of F-6 and G-6；Bag
It is embedded with internal reference primer sets, is injected into amplification pond A-8, B-8, C-8, D-8, E-8, F-8, G-8, H-8 color of internal reference nucleic acid
It is pink, illustrates nucleic acid and hair containing internal reference in eight amplification ponds of A-8, B-8, C-8, D-8, E-8, F-8, G-8, H-8
Raw isothermal amplification reactions；And other amplification pond colors be it is faint yellow, illustrate without generation isothermal amplification reactions.
Also the color interpretation in micro-fluidic chip amplification pond can be carried out using relevant device.
The above results show that the kit and its application method of detection infectious diarrhea of the present invention can be accurate
Detection obtained rotavirus, EAd, norovirus, salmonella, Shigella, 6 kinds of campylobacter jejuni in 1 hour
The testing result of pathogen index, effectively increases detection efficiency,.
Specific embodiment of the invention has been 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, any equivalent modifications carried out to the practicality 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 Primer composition and its kit for detecting infectious diarrhea pathogen
<160> 28
<170> PatentIn version 3.5
<210> 1
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223>Rotavirus primers F 3
<400> 1
cactatagat tggaaatcca gat 23
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Rotavirus primer B3
<400> 2
tggacgaaat aactgatcct 20
<210> 3
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223>Rotavirus primers F IP
<400> 3
actttccttg ccttaataac ccaatttgaa agacgatttg agtcg 45
<210> 4
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223>Rotavirus primer BIP
<400> 4
ttcagaatgt tatttcgcaa caacatttga ttgtaaatca cgctcta 47
<210> 5
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223>EAd primers F 3
<400> 5
gctggatcgg agacaggt 18
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>EAd primer B3
<400> 6
tgtcaccacc attgtagcac 20
<210> 7
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223>EAd primers F IP
<400> 7
aactaagggc gagggttgcg gtcagctcgt gtcgtgaga 39
<210> 8
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223>EAd primer BIP
<400> 8
cagttgggca ctctaagggg acagggccat gaggacttga 40
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Norovirus primers F 3
<400> 9
gccctgacaa aactgaagga 20
<210> 10
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223>Norovirus primer B3
<400> 10
cccccagtag ggacatca 18
<210> 11
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223>Norovirus primers F IP
<400> 11
ttccaaacca accagctggg tcccccttgt catctccgaa ga 42
<210> 12
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223>Norovirus primer BIP
<400> 12
ctggactagg ggtcccaacc atggtctttg ggagtgtgga at 42
<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Salmonella primers F 3
<400> 13
ggatgactcg ccatggtatg 20
<210> 14
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223>Salmonella primer B3
<400> 14
ttgttcaaca gctgcgtca 19
<210> 15
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223>Salmonella primers F IP
<400> 15
ctgggcgaca agaccatcac catttgtcct ccgccctgt 39
<210> 16
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223>Salmonella primer BIP
<400> 16
tccccgcatt gttgattgcg atccgcccca tattatccgt a 41
<210> 17
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223>Shigella primers F 3
<400> 17
tcttcgccgg actaccac 18
<210> 18
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223>Shigella primer B3
<400> 18
cgacctgttc acggaatcc 19
<210> 19
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223>Shigella primers F IP
<400> 19
aaccatgctg tcacggcatc agacttctcc atgagtgacg ga 42
<210> 20
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223>Shigella primer BIP
<400> 20
acatgaagag cacgccaaca ccgcagagac ggtatcggaa ag 42
<210> 21
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223>Campylobacter jejuni primers F 3
<400> 21
gcagataaat cgccattcg 19
<210> 22
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223>Campylobacter jejuni primer B3
<400> 22
taagaacgcc cactgaga 18
<210> 23
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223>Campylobacter jejuni primers F IP
<400> 23
cttgccacag actgcgtcag tacttcttat ctggatttaa tgtcg 45
<210> 24
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223>Campylobacter jejuni primer BIP
<400> 24
cgatgttacg gtttgttact gtgatcatcc agtgttgtac gaa 43
<210> 25
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223>Internal reference primers F 3
<400> 25
gcatggtgca cctgactc 18
<210> 26
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Internal reference primer B3
<400> 26
tcaccttagg gttgcccata 20
<210> 27
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223>Internal reference primers F IP
<400> 27
tcaccaccaa cttcatccac gtctgaggag aagtctgccg t 41
<210> 28
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223>Internal reference primer BIP
<400> 28
gctgctggtg gtctaccctt gtcaggggtg gacagatcc 39
Claims (10)
1. a kind of Primer composition for detecting infectious diarrhea pathogen, it is characterised in that including rotavirus primer sets,
EAd primer sets, norovirus primer sets, salmonella primer sets, Shigella primer sets and campylobacter jejuni primer
At least one set in group；
Wherein, the rotavirus primer sets include SEQ ID NO：Rotavirus primers F 3, SEQ ID NO shown in 1：2 institutes
Rotavirus primer B3, SEQ ID NO for showing：Rotavirus primers F IP and SEQ ID NO shown in 3：Colyliform disease shown in 4
Malicious primer BIP；
Wherein, the EAd primer sets include SEQ ID NO：EAd primers F 3, SEQ ID NO shown in 5：
EAd primer B3, SEQ ID NO shown in 6：EAd primers F IP and SEQ ID NO shown in 7：Shown in 8
EAd primer BIP；
Wherein, the norovirus primer sets include SEQ ID NO：Norovirus primers F 3, SEQ ID NO shown in 9：10 institutes
Norovirus primer B3, SEQ ID NO for showing：Norovirus primers F IP and SEQ ID NO shown in 11：Promise shown in 12 is such as
Viral primer BIP；
Wherein, the salmonella primer sets include SEQ ID NO：Salmonella primers F 3, SEQ ID NO shown in 13：14
Shown salmonella primer B3, SEQ ID NO：Salmonella primers F IP and SEQ ID NO shown in 15：Sand shown in 16
Door Salmonella primer BIP；
Wherein, the Shigella primer sets include SEQ ID NO：Shigella primers F 3, SEQ ID NO shown in 17：18
Shown Shigella primer B3, SEQ ID NO：Shigella primers F IP and SEQ ID NO shown in 19：Will shown in 20
Hayes bacterium primer BIP；
Wherein, the campylobacter jejuni primer sets include SEQ ID NO：Campylobacter jejuni primers F 3, SEQ ID shown in 21
NO：Campylobacter jejuni primer B3, SEQ ID NO shown in 22：Campylobacter jejuni primers F IP and SEQ ID NO shown in 23：24
Shown campylobacter jejuni primer BIP.
2. the Primer composition for detecting infectious diarrhea pathogen according to claim 1, it is characterised in that enter
Step includes internal reference primer sets, and the internal reference primer sets are used to expand human hemoglobin-β chains；
Wherein, the internal reference primer sets include SEQ ID NO：Internal reference primers F 3, SEQ ID NO shown in 25：Shown in 26
Internal reference primer B3, SEQ ID NO：Internal reference primers F IP and SEQ ID NO shown in 27：Internal reference primer shown in 28
BIP。
3. a kind of kit of the Primer composition containing described in claim 1 or 2, it is characterised in that also including micro-fluidic core
Piece, the micro-fluidic chip is provided with mutual disconnected 4 reaction detection areas, and each reaction detection area includes that what is be sequentially communicated enters
Sample pond, distributing reservoir, capillary micro-valve and amplification pond, each reaction detection area have 8 amplification ponds, in the Primer composition
Each primer sets are coated in corresponding amplification pond respectively.
4. kit according to claim 3, it is characterised in that in each amplification pond, primers F 3 in each primer sets
Concentration be 1~4 μM, the concentration of primer B3 is 1~4 μM, and the concentration of primers F IP is 8~15 μM, the concentration of primer BIP for 8~
15μM。
5. kit according to claim 3, it is characterised in that the kit further includes isothermal amplification reactions
Liquid, constant-temperature amplification enzyme solutions, negative control thing.
6. kit according to claim 5, it is characterised in that the isothermal amplification reactions liquid includes 200~250mM
Tris-HCl, 300~400mM KCl, 8~10mM dNTP, 100~120mM MgSO4,1%~5% (W/V) Tween-20,
40%~50% (W/V) glycerine, 1~5% (W/V) PEG8000 and 10~20mM indicator；The constant-temperature amplification enzyme solutions include
10~12U/ μ L archaeal dna polymerases and 20~30U/ μ L reverse transcriptases.
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, described
Reverse transcriptase is that concentration is M-MLV reverse transcriptases.
9. a kind of Primer composition using described in claim 1 or 2 is come the method that detects infectious diarrhea pathogen, its feature
It is to comprise the following steps：
Step 1) Primer composition coating：Each primer sets in the Primer composition are coated on corresponding amplification respectively
In pond, it is fixed in amplification pond by vacuum drying；
Step 2) measuring samples nucleic acid extraction：The nucleic acid of measuring samples is extracted using paramagnetic particle method；
Step 3) LAMP reactions：Take isothermal amplification reactions liquid and archaeal dna polymerase, reverse transcriptase solution, the nucleic acid with measuring samples
After mixing, the sample inlet pool of the micro-fluidic chip of coating primer sets is transferred to, liquid is flowed into amplification pond under the action of the centrifugal force,
Then loop-mediated isothermal amplification reaction is carried out；
Step 4) result interpretation：The color change in pond is expanded after terminating according to amplification to be carried out direct naked eyes interpretation or uses 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 sucrose respectively, corresponding mixed solution is configured to, it is described
The final concentration of primer sets and sucrose in the mixed solution is respectively 0.15 μM and 1.0wt%；The mixed solution for taking 1 μ L is clicked and entered
Micro-fluidic chip is expanded in pond accordingly, and in dry in 37 DEG C of baking ovens, compressing tablet sealer, after punching press is vacuumized, primer sets are coated
In corresponding amplification pond.
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| CN107629951A (en) * | 2017-09-29 | 2018-01-26 | 深圳国际旅行卫生保健中心 | Micro-fluidic gene detecting chip |
| CN108187769A (en) * | 2018-01-15 | 2018-06-22 | 西南石油大学 | A kind of rotatable certain angle and the matched integrated form microcosmic oil drive chip of mold |
| CN108531630A (en) * | 2018-06-06 | 2018-09-14 | 上海速创诊断产品有限公司 | The streptococcic primer sets of detection B races include the primer liquid and kit of the primer sets and its application |
| CN108754032A (en) * | 2018-07-19 | 2018-11-06 | 上海速创诊断产品有限公司 | It is a kind of with the isothermal nucleic acid amplification system of high specific and its application |
| CN110592250A (en) * | 2019-11-01 | 2019-12-20 | 中国人民解放军总医院 | LAMP primer combination for detecting intestinal pathogenic bacteria and application thereof |
| CN111057799A (en) * | 2019-11-22 | 2020-04-24 | 浙江大学 | Method for rapidly detecting pinnate mottle virus of sweet potato by using micro-fluidic chip and used primer |
| WO2022121742A1 (en) * | 2020-12-07 | 2022-06-16 | 中国科学院脑科学与智能技术卓越创新中心 | Single cell transcriptome sequencing method |
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| CN107629951A (en) * | 2017-09-29 | 2018-01-26 | 深圳国际旅行卫生保健中心 | Micro-fluidic gene detecting chip |
| CN107629951B (en) * | 2017-09-29 | 2021-03-02 | 深圳国际旅行卫生保健中心 | Micro-fluidic gene detection chip |
| CN108187769A (en) * | 2018-01-15 | 2018-06-22 | 西南石油大学 | A kind of rotatable certain angle and the matched integrated form microcosmic oil drive chip of mold |
| CN108531630A (en) * | 2018-06-06 | 2018-09-14 | 上海速创诊断产品有限公司 | The streptococcic primer sets of detection B races include the primer liquid and kit of the primer sets and its application |
| CN108754032A (en) * | 2018-07-19 | 2018-11-06 | 上海速创诊断产品有限公司 | It is a kind of with the isothermal nucleic acid amplification system of high specific and its application |
| CN108754032B (en) * | 2018-07-19 | 2022-02-18 | 上海速创诊断产品有限公司 | Isothermal nucleic acid amplification system with high specificity and application thereof |
| CN110592250A (en) * | 2019-11-01 | 2019-12-20 | 中国人民解放军总医院 | LAMP primer combination for detecting intestinal pathogenic bacteria and application thereof |
| CN111057799A (en) * | 2019-11-22 | 2020-04-24 | 浙江大学 | Method for rapidly detecting pinnate mottle virus of sweet potato by using micro-fluidic chip and used primer |
| WO2022121742A1 (en) * | 2020-12-07 | 2022-06-16 | 中国科学院脑科学与智能技术卓越创新中心 | Single cell transcriptome sequencing method |
| WO2024077197A1 (en) * | 2022-10-05 | 2024-04-11 | Life Technologies Corporation | Multiplex qpcr panel for gastrointestinal pathogens |
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