CN106544434A - It is intersect the method that amplification detects Listeria monocytogenes with reference to gold nano bio-sensing more - Google Patents
It is intersect the method that amplification detects Listeria monocytogenes with reference to gold nano bio-sensing more Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6804—Nucleic acid analysis using immunogens
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Abstract
The invention discloses a kind of method for intersecting amplification with reference to gold nano bio-sensing detection Listeria monocytogenes more, 5 ' the end labelling biotin of cross primer CP1 or CP2 of the methods described in many cross substitutions amplification, in 5 ' the end labelling hapten of amplimer C1 or C2, methods described can be by gold nano biosensor Visual retrieval for the amplified production of Listeria monocytogenes lmo0733.Methods described is convenient, quick, sensitive, special, is suitable for various nucleotide fragments detections.
Description
Technical field
The invention discloses a kind of Listeria monocytogenes detection method, belongs to microbiological art.
Background technology
Listeria monocytogenes (Listeria monocytogens, LM) are a kind of Gram-positive brevibacterium, have motion energy
Power, facultative intracellular bacterial parasite.Listeria monocytogenes are widely present in nature, are important food-borne Zoonosis cause of diseases
Bacterium, Listeria monocytogenes present in food have danger to the safety of the mankind, and the bacterium can still grow numerous in 4 DEG C of environment
Grow, be one of the main pathogenic fungi that chilled food threatens human health.Listeria monocytogenes can cause mankind's severe infections.Lee
The Susceptible population of this special bacterium disease is mainly hypoimmunity crowd and immunodeficiency crowd, the former include old people, neonate and
Anemia of pregnant woman；The latter is tumor patient, HIV sufferers and organ graft recipient etc..Mankind's listeriosis main clinic symptoms are to lose
Mass formed by blood stasis, meningitiss, anemia of pregnant woman's miscarriage, stillbirth and heat generation gastroenteritiss etc., the bacterium can pass through gut barrier, blood brain barrier and Placenta Hominiss
Barrier.Listeria monocytogenes disease is not high in the infection rate of the mankind, but the sick mortality rate is high and widely paid close attention to, once
Become the great public health problem of American-European countries.The country there is no and cause the report that alimentary toxicosis break out by Listeria monocytogenes, real
Border presence or absence fails to understand that Sporadic cases is all had been reported that in multiple provinces and cities.Therefore, quickly and accurately control to give clinical patient
The Epidemiological study of Listeria monocytogenes is treated, carries out, research and development one save time, the Listeria monocytogenes that laborsaving and specificity is higher
Detection method necessitates.
At present for Listeria monocytogenes isolation identification depends on traditional Zengjing Granule and biochemical identification, the method
Time-consuming about 5 to 7 days, including increasing bacterium twice, selecting to cultivate and follow-up biochemical identification, its inferior position takes time and effort, chemical result
Interpretation depend on the subjective judgment of people, cause result to repeat poor, easily misjudge.With the fast development of nucleic acid diagnostic techniques, one
A little diagnostic techniquess (such as regular-PCR technology, Fluorescence PCR assay) based on PCR are used for quickly examining for Listeria monocytogenes
It is disconnected, but these methods depend on the instrument and equipment of costliness, need follow-up electrophoretic procedures, expensive probe to synthesize, and it is ripe
Experienced operator.For some laboratorys for falling behind cannot be carried out, the utilization of these technologies is limited.Examine with these at present
The PCR method and Real-time PCR method of survey technology diagnosis Listeria monocytogenes, selected genes of interest (such as hly,
PrfA, iap) poor specificity, easily there is false positive amplification.In addition the poor sensitivity of these detection techniques, detection process take
It is longer, it is unfavorable for quick detection and Emergent detection.Therefore, quickly and accurately treat to give clinical patient, food borne pathogenses
Body monitoring and the Epidemiological study of Listeria monocytogenes, research and development one save time, the diagnostic method that laborsaving and specificity is higher,
Identification Listeria monocytogenes that can be quick, sensitive, special necessitate.
In order to overcome the inferior position of PCR amplification techniques, many isothermal amplification technologies are developed in recent years.Isothermal amplification technology compared with
For round pcr, thermal cycling amplification equipment is not relied on, response speed is fast, and sensitivity is good.Therefore, isothermal amplification technology is favourable
In realizing rapid amplifying, easy detection and field diagnostic.Up to the present, the isothermal amplification technology for having developed has more than 10 kinds, should
With the relatively broad constant-temperature amplification (HDA) for having rolling circle amplification (RCA), strand displacement amplification (SDA), unwindase to rely on, ring mediation
Constant-temperature amplification (LAMP), intersection amplification (CPA) etc..However, these constant temperature technologies realize that nucleic acid amplification needs various enzymes while work
Make, depend on the reagent of costliness, complicated operating procedure.Therefore the practicality of these methods, convenience, operability await
Improve, especially in quick diagnosis field and low developed area.In order to overcome the bad of round pcr and existing isothermal amplification technology
Gesture, realizes convenient, quick, sensitive and special amplifying nucleic acid sequence, and in the field such as biology, agricultural, medical science, inventor is in the recent period
A kind of new nucleic acid amplification technologies have been set up, many cross substitution amplification (Multiple Cross Displacement have been named as
Amplification, MCDA), related content is disclosed in CN104946744A, and the patent document is constituted as prior art document
One part of present specification.
MCDA realizes nucleic acid amplification under constant temperature, and only using a kind of constant temperature substituted enzyme, amplification rate is fast, react spirit
Quick, specificity is high.In the present invention, in order that the technology is more extensive, more economical in biological, medicine and health field application, send out
MCDA technologies are combined based on MCDA by a person of good sense with nano biological detection technique, developed dependence MCDA technologies, are realized soon
Speed, the nanobiosensor technology of sensitive detection, the technology are named as the cores for intersecting amplification with reference to gold nano bio-sensing more
Sour diagnostic techniquess (Multiple cross displacement amplification coupled with gold
Nanoparticle-based lateral flow biosensor, MCDA-LFB), and the Technology application is increased into Li Si in single
The detection of special bacterium.
The present invention designs a set of MCDA amplimers for the specific gene lmo0733 of Listeria monocytogenes, it is intended to verify,
Evaluate MCDA-LFB technologies and set up quick, the sensitive and special MCDA-LFB detection bodies for being directed to Listeria monocytogenes pathogen
System.
The content of the invention
Based on foregoing invention purpose, present invention firstly provides a kind of application intersects amplification with reference to gold nano bio-sensing
The method of testing goal gene, the method comprising the steps of：
(1) from 3 ' ends of the genes of interest fragment, the first arbitrary sequence F1s of setting, the second arbitrary sequence P1s, from
The 5 ' of the genes of interest fragment are held, and set the 3rd arbitrary sequence F2, the 4th arbitrary sequence P2, in second arbitrary sequence
5 ' the 5th arbitrary sequences C1 of end setting of P1s, and/or in 3 ' the 6th arbitrary sequences of end setting of the 4th arbitrary sequence P2
C2s, sets the 7th arbitrary sequence D1 and the 8th arbitrary sequence R1 at the 3 ' ends and 5 ' ends of sequence C 1, respectively the 5 ' of sequence C 2s
End and 3 ' ends set the 9th arbitrary sequence D2s and the tenth arbitrary sequence R2s respectively；
(2) displacement primers F 1 is provided, the primers F 1 is containing the sequence complementary with sequence F1s, there is provided cross primer CP1,
The primer CP1 is from 5 ' ends successively containing sequence C 1 and sequence P1 complementary with sequence P1s, there is provided displacement primers F 2, described to draw
Thing contains sequence F2, there is provided cross primer CP2, and the primer CP2 is from 5 ' ends successively containing the complementary sequence C 2 of sequence C 2s and sequence
Row P2, the 5 ' ends for being simultaneously provided in cross primer CP1 or CP2 are marked with the cross primer CP1* or CP2* of biotin；
(3) amplimer is provided, the amplimer includes the amplimer C1 containing sequence C 1, and/or contains and sequence
Row C2s complementary amplimer C2, the amplimer D1 containing sequence D 1, the amplimer R1 containing sequence R1, containing with sequence
Arrange the complementary amplimer D2 of D2s and containing the amplimer R2 complementary with sequence R2s, be simultaneously provided in the amplimer C1
Or the haptenic amplimer C1* or C2* of 5 ' end labellings of C2；
(4) in the presence of chain shift-type polymerase (Bst), melting temperature regulator, primer, application target genetic fragment is made
For template constant-temperature amplification DNA；
(5) using the amplified production of gold nano biosensor detecting step (4).
In a preferred technical scheme, it is ground in the hapten of 5 ' the end institute labellings of the amplimer C1* or C2*
Gaoxin.
It is further preferable that in 5 ' the end labelling biotin of cross primer CP1*, on the amplimer C1*5 ' ends labelling ground
Gaoxin.
In a technical scheme being more highly preferred to, the gold nano biosensor includes a backboard, in the backboard
On set gradually equipped with sample pad, gold standard pad, nitrocellulose filter and adsorptive pads, set gradually on the nitrocellulose filter
Detection line and control line, are coated with the streptomycin parent of golden nanometer particle coupling successively in gold standard pad, detection line and control line region
The bovine serum albumin being coupled with element, anti digoxin antibody and biotin.
In a preferred technical scheme, the constant-temperature amplification is carried out in 60-63 DEG C of environment.
In a technical scheme being more highly preferred to, the constant-temperature amplification is carried out in 61 DEG C of environment.
In a preferred technical scheme, lmo0733 of the genes of interest for Listeria monocytogenes.
Preferably, the sequence such as SEQ ID NO of the displacement primers F 1:Shown in 1, the sequence of the displacement primers F 2 is such as
SEQ ID NO:Shown in 2；The sequence of the cross primer CP1 such as SEQ ID NO:Shown in 3, the 5 ' of the cross primer CP1
End is marked with the sequence such as SEQ ID NO of the cross primer CP1* of biotin:Shown in 4, the sequence of the cross primer CP2
Such as SEQ ID NO:Shown in 5, the sequence such as SEQ ID NO of primer C1:Shown in 6, ground is marked with the 5 ' ends of the primer C1 high
The sequence of pungent primer C1* such as SEQ ID NO:Shown in 7, the sequence such as SEQ ID NO of primer C2:Shown in 8, the sequence of primer D1
Such as SEQ ID NO:Shown in 9, the sequence such as SEQ ID NO of primer D2:Shown in 10, the sequence such as SEQ ID NO of primer R1:11 institutes
Show, the sequence such as SEQ ID NO of primer R2:Shown in 12.
The present invention additionally provides one group for intersecting drawing for amplification Listeria monocytogenes lmo0733 genes on the other hand
Thing sequence, the sequence include：Such as SEQ ID NO:Displacement primers F 1 shown in 1, such as SEQ ID NO:Displacement primer shown in 2
F2, such as SEQ ID NO:Cross primer CP1 shown in 3, such as SEQ ID NO:Cross primer CP2 shown in 5, such as SEQ ID NO:
Amplimer C1 shown in 6, such as SEQ ID NO:Amplimer C2 shown in 8, such as SEQ ID NO:Amplimer shown in 9
D1, such as SEQ ID NO:Amplimer D2 shown in 10, such as SEQ ID NO:Amplimer R1 shown in 11, such as SEQ ID NO:
Amplimer R2 shown in 12, the 5 ' ends for being simultaneously provided in cross primer CP1 or CP2 are marked with the cross primer CP1* of biotin
Or CP2*, and in the haptenic amplimer C1* or C2* of 5 ' end labellings of the amplimer C1 or C2.
In a preferred technical scheme, in 5 ' the end labelling biotin of cross primer CP1*, amplimer C1*'s
5 ' ends are marked with Digoxin.
Many intersection amplifications that the present invention is provided are with reference to gold nano bio-sensing testing goal gene Listeria monocytogenes
The method of lmo0733 has excellent detection sensitivity, its detection range be 10ng～10fg, and have detect faster
Speed, it is only necessary to 20 minutes, you can obtain the amplified production that can supply visualization LFB detections.
With common pathogenic bacterium and conditioned pathogen DNA (Listeria monocytogenes, salmonella, shigella dysenteriae, vibrio cholera, pair
Hemolytic vibrio, Vibrio vulnificus, enterococcus faecalis, staphylococcus aureuses, campylobacter jejuni, Bacillus cereuss, intestinal are pathogenic
Escherichia coli, enterotoxigenic E.Coli, enteroinvasive E.Coli etc.) for template evaluate MCDA-LFB technologies specificity.
The MCDA-LFB technologies that the present invention is provided can accurately differentiate Listeria monocytogenes, illustrate that the specificity of MCDA-LFB methods is good
It is good, also demonstrate that the primer sequence provided by the present invention for MCDA has excellent specificity and expanding effect.
Description of the drawings
Fig. 1 .MCDA-LFB expand principle and gold nano biosensor structure schematic diagram；
The position of Fig. 2 .MCDA-LFB design of primers and direction schematic diagram；
Fig. 3 .MCDA-LFB primers are verified and MCDA-LFB testing result collection of illustrative plates；
Fig. 4. standard MCDA-LFB optimal reaction temperature test result collection of illustrative plates；
Fig. 5 .MCDA-LFB detect the sensitivity results collection of illustrative plates of Listeria monocytogenes；
The optimum reacting time test result collection of illustrative plates of Fig. 6 .MCDA-LFB technologies；
The specific detection of Fig. 7 .MCDA-LFB technologies evaluates collection of illustrative plates
Specific embodiment
Further describe the present invention with reference to specific embodiment, advantages of the present invention and feature will be with description and
It is apparent.But these embodiments are only exemplary, do not constitute any restriction to protection scope of the present invention.
1.MCDA-LFB expands principle
(1) detectable product is built by MCDA amplifications
MCDA reaction systems include 10 primers, recognize 10 regions of target sequence, including 2 intersect inner primer, i.e. CP1
With CP2 (Cross Primer, CP), 2 displacement primer, i.e. F1 and F2,6 amplimers, i.e. D1, C1, R1, D2, C2 and R2.
In order to build detectable product, at 5 ' ends labelling biotin (Biotin), amplimer C1's cross primer CP1 or CP2 or C2 exist
5 ' ends labelling Digoxin (Dig), the primer of new labelling are named as CP1*, CP2*, C1* and C2*.CP1* includes Cl (C1s regions
Complementary seriess) and P1, i.e. 5 '-Cl-P1；CP2 includes C2 (complementary seriess in C2s regions) and P2, i.e. 5 '-C2-P2.Two friendships
Fork primer CP1 and CP2 are the main primers for mediating MCDA amplifications；Displacement primers F 1 and F2 displacements play displacement in MCDA reactions
Effect, replaces cross primer CP1 and CP2；Six amplimer D1, C1, R1, D2, C2* and R2 can speed up MCDA reactions and increase
Plus MCDA product amounts, amplification principle is referring to Figure 1A.
In order to make it easy to understand, CP2* and C2* primers are omitted in amplification schematic diagram.It is under set constant temperature, double
In the dynamic balance state in half dissociation and quasi integration, any one primer is carried out chain DNA to the complementary portions of double-stranded DNA
When base pairing extends, another chain will dissociate, and become single-stranded.First in the presence of Bst archaeal dna polymerases, drawn with CP1*
3 ' ends of thing P1 sections are starting point, are matched with corresponding DNA complementary seriess, start strand displacement DNA synthesis.F1 primers and P1s
Front end F1s sequences are complementary, with 3 ' ends as starting point, by the effect of strand-displacement activity archaeal dna polymerase, displace CP1 first and draw
The DNA of thing synthesis, simultaneously synthesizing itself DNA.The DNA that final F1 primers are synthesized into forms double-strand with template DNA.But
Strand displacement is carried out by F1 primers by the DNA that cross primer CP1* first synthesizes and produces single-stranded, single-stranded D1s, C1s, the R1s,
P2s, F2s region can be combined with amplimer D1, C1*, R1, cross primer CP2 and displacement primers F 2 successively, and is played chain and put
Change amplification effect (step 1,2).C1* primers expand and replace the amplification chain of D1, generate short-movie section C1s-D1 product, the product energy
It is enough to be combined with C1* and CP1* primers, start strand displacement amplification, into cyclic amplification 1 (step 3 and circulation 1).R1 primers are expanded simultaneously
The amplification chain of displacement C1*, generates short-movie section C1s-C1* product, and the product can be combined with C1* and CP1* primers, starts chain and puts
Amplification is changed, into cyclic amplification 1 (step 4 and circulation 2).It is in cyclic amplification 2, with the carrying out that MCDA is expanded, substantial amounts of double
Mark product is formed, CP1* ends labelling biotin, C1* ends labelling Digoxin (Figure 1A).This pair of target product can be given birth to by gold nano
Thing sensor is detected, so as to carry out visual detection.Follow-up MCDA amplifications, including step 5 and 6, the early stage for referring to inventor is special
Sharp CN104946744A.Further, since the amplification procedure of CP2* and C2* is similar to the amplification procedure of CP1* and C1*, expand in MCDA
In increasing system, substantial amounts of pair of target detectable product can be equally formed.CP2* ends labelling biotin, C2* ends labelling Digoxin.
This pair of target product also can be detected by gold nano biosensor (LFB), so as to carry out visual detection.Therefore, utilizing MCDA-
When LFB technologies carry out target sequence detection, detectable product is built using CP1* and C1* primers, be possible with CP2* and C2* draws
Thing builds detectable product.
(2) the biological design and the detection to amplified production for passing detector (LFB)
Figure 1B is shown in the design of biosensors (LFB).LFB include five parts, sample pad 1, gold standard pad 2, fibrous membrane 3,
Adsorptive pads 4 and backboard 5, are assembled in sample pad, gold standard pad, fibrous membrane and adsorptive pads on backboard first successively, then by SA-G
(biotin is coupled for (the streptomycin Avidin that golden nanometer particle is coupled), anti-Dig (anti digoxin antibody) and Biotin-BSA
Bovine serum albumin) be coated on gold standard pad, detection line (TL) 6, and control line (CL) 7 respectively, it is to be dried after it is standby.
The Cleaning Principle of LFB：MCDA target genes amplified matter is directly added drop-wise to into the sample pad area (target gene of LFB
Amplified matter is by biotin and Digoxin while labelling), the detection buffer of 120 μ l is added to into sample pad area, MCDA then
Product moves (moving to adsorptive pads direction from sample pad) under siphonage from the bottom up.When MCDA products reach gold standard pad
Afterwards, one end (i.e. biotin labeling end) of double mark products and SA-G (the streptomycin Avidin that golden nanometer particle is coupled) reaction.When
Product continues motion, the other end (i.e. digoxigenin labeled end) of double mark products and the antibodies in detection line region, and double marks are produced
Thing is fixed on detection line region.As product is in the accumulation in detection line region, by the SA-G of the other end, (golden nanometer particle is coupled
Streptomycin Avidin) carry out chromogenic reaction, so as to carry out Visual retrieval to MCDA products.Additionally, SA-G (the Jenners of surplus
The streptomycin Avidin that rice corpuscles are coupled) can with the Biotin-BSA in control line region (bovine serum albumin that biotin is coupled),
Direct chromogenic reaction is carried out, judges whether the function of LFB is normal.
The interpretation (Fig. 1 C) of LFB results：Only there are red stripes in control line region, represents negative control, does not have the positive
Product；There are red stripes in control line and detection line region, represent the test positive result for target；When LFB does not go out
During existing red line band, LFB failures are represented；Only when red stripes occurs in p-wire, CL redfree bands, representing result can not
OK, need to detect again.
2. the reagent and instrument in the present invention involved by embodiment：
Anti digoxin antibody (anti-Dig), the streptomycin Avidin (SA-G) and biotin that golden nanometer particle is coupled are coupled
Bovine serum albumin (B-BSA) be purchased from Resenbio companies.Backboard, sample pad, gold standard pad, fibrous membrane and adsorptive pads are purchased from Jie-
Yi companies.Loopamp Kit (Eiken Chemical Co.Ltd., Tokyo, Japan) are purchased from Japanese Rong Yan companies.DNA is carried
Take test kit (QIAamp DNA minikits；Qiagen, Hilden, Germany) it is purchased from German Qiagen companies.PCR reacts
System mixture M IX (Taq archaeal dna polymerases, dNTP and buffer) is purchased from Beijing CoWin Bioscience Co., Ltd..
DL50DNA Marker are purchased from precious biological engineering (Dalian) company limited.Remaining reagent is commercially available analysis net product.
Key instrument used in present invention experiment：The real-time transmissometer LA-320C of constant temperature (Eiken Chemical Co.,
Ltd, Japan) it is purchased from Japanese Rong Yan companies.PCR instrument be Sensoquest Labcycler, German Sensoquest products；Electricity
Swimming equipment is Beijing Jun Yi east electrophoresis equipment company limited product；Gel imaging system is Bio-Rad Gel Dox XR, beautiful
State's Bio-Rad products.
3. the method in the present invention involved by embodiment
Genome is extracted：DNA of the extraction of the genomic DNA of Listeria monocytogenes and other antibacterials using Qiagen companies
Extracts kit (QIAamp DNA minikits；Qiagen, Hilden, Germany), operated to specifications.Utilize
Ultraviolet spectrophotometer determines the concentration and purity of genomic DNA, the DNA GE buffer serial-dilutions of Listeria monocytogenes
(from 10ng, 10pg, 10fg, 1fg to 0.1fg/ microlitre).The a small amount of subpackage of various genomic DNAs, -20 DEG C save backup.
The Listeria monocytogenes DNA of serial dilution is used for the exploration of optimum temperature and building for amplification system of MCDA amplifications
It is vertical.L.monocytogenes-MCDA-LFB technology special is evaluated as template with common pathogenic bacterium and conditioned pathogen DNA
Property.Bacterial strain information is shown in Table 2.
4. the design of primers involved by the embodiment of the present invention
In order to verify, evaluate MCDA-LFB technologies and set up for the quick, sensitive and special of Listeria monocytogenes pathogen
Different MCDA-LFB detection system.The present invention designs a set of MCDA amplifications for the specific gene lmo0733 of Listeria monocytogenes
Primer, it is intended to verify feasibility, sensitivity, specificity and the reliability of MCDA-LFB technologies.
Lmo0733 is present in all of Listeria monocytogenes, and its specificity is good, can by Listeria monocytogenes and other
Closely close strain is distinguished.Using primer-design software PrimerExplorer V4 (Eiken Chemical)
(http://primerexplorer.jp/e/) and the design MCDA primers of primer-design software Primer Premier 5.0, and
By the specific primer for obtaining in ncbi database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) in carry out
Sequence alignment analysis, are matched with excluding primer non-specificity that may be present with other species sequences, after finally being optimized
MCDA amplimers.The position and direction of design of primers sees that Fig. 2, sequence and modification are shown in Table 1.
1 primer sequence of table and modification
aCP1*, the primer are used for MCDA-LFB detection system, hold labelling biotin 5 '；C1*, the primer are used for
MCDA-LFB detection system, holds labelling Digoxin 5 '；bDig, Digoxin；Biotin, biotin；cMer, monomeric
Unit (monomeric unit)；Nt, nucleotide (nucleotide).
The feasibility of embodiment 1.MCDA-LFB amplification
The MCDA reaction systems of standard：The concentration of cross primer CP1* and CP1 be 30pmol, the concentration of cross primer CP2
For 60pmol, the concentration for replacing primers F 1 and F2 is 10pmol, amplimer R1, and the concentration of R2, D1 and D2 is 30pmol, is expanded
The concentration of primer C1* and C2 is 20pmol, the Betain of 10mM, the MgSO of 6mM4, the dNTP of 1mM, the 10 × Bst of 12.5 μ L
DNA polymerase buffer liquid, the strand displacement archaeal dna polymerase of 10U, the template of 1 μ L add deionized water to 25 μ L.Whole reaction is permanent
Temperature 65 DEG C 1 hour, 85 DEG C of 5min terminating reactions.
After MCDA amplifications, three kinds of detection methods are used for MCDA amplifications and differentiate, first, add in the reactive mixture visual
Dyestuff (such as FD reagents, bright general fluorescent visual reagent), the color of positive reaction pipe are changed into green, negative reaction Guan Ze from light gray
Keep original light gray.Secondly, MCDA products can detect amplicon after sepharose electrophoresis, due to containing in product
Different size of amplified fragments, therefore the electrophoretogram of positive amplification product is stepped in specificity, negative reaction occurs without any
Band.More direct simple method is product to be detected by LFB.
Visible color method of changing：MCDA produces substantial amounts of pyrophosphate ion while synthetic DNA, and the ion can be taken by force
The manganese ion combined with calcein is taken, calcein is recovered free state and is fluoresced.The light-emitting admixture can with it is anti-
The magnesium ion that middle should be produced is combined, and is strengthened fluorescence.Can be positive by fluorescence visual detection color change sentence read result
Reaction tube is changed into green from light gray, and negative reaction keeps light grey constant, sees Fig. 3 A.A1 represents positive amplification (in reaction tube
The Listeria monocytogenes template of 10pg is added, as positive control), A2 represents that negative amplification (adds 10pg's in reaction tube
Gram+Listeria ivanovii template, acts on negative control), A3 represents that negative amplification (adds the Gram- of 10pg husky in reaction tube
Door bacterium template, acts on negative control), A4 represents that (1 microlitre of distilled water replaces the template of 10pg, used as sky for blank reaction
In vain to correlating).Only there is positive amplification in positive control, illustrates the detection Listeria monocytogenes for lmo0733 designs
MCDA primers can use.
Electrophoresis assays：The product of Fig. 3 A is carried out into electrophoresis detection, as the amplified production of MCDA contains many sizes
Loop-stem structure and the DNA fragmentation of multi-ring Brassica oleracea L. var. botrytis L. spline structure that the short-movie section for differing and a series of target sequence of inverted repeats are constituted
Mixture, manifests the staged collection of illustrative plates of different size zone composition on gel, sees Fig. 3 B after electrophoresis.Sentenced by electrophoresis assays
Read MCDA amplifications, positive reaction occurs in that expected result, and any amplification bar do not occur in negative reaction and blank
Band, the MCDA primers further demonstrated designed by this research are feasible, can be used for target sequence amplification detection.
LFB is detected：The product of Fig. 3 A is carried out into LFB detections.Due to the MCDA primer marks for Listeria monocytogenes detection
The hapten of note is Dig (Digoxin), therefore, Listeria monocytogenes detection sun is expressed as when red stripes occur in TL and CL
Property.By LFB detection method interpretation MCDA amplifications, positive reaction occurs in that expected result, and negative reaction and blank are right
According to only there are CL red stripes, the designed LFB of this research is demonstrated, MCDA-LFB technologies and MCDA primers are feasible, Neng Gouyong
In the detection (Fig. 3 C) of purpose target sequence.
Embodiment 2. determines the optimal reaction temperature of MCDA technologies
Under standard reaction system condition, Listeria monocytogenes DNA profiling is added and for the correspondence designed by lmo0733
MCDA primers, its template concentrations be 10pg/ μ l.Reaction is carried out (60-67 DEG C) under constant temperature, the real-time turbidity of application of results
Instrument is detected, obtains different dynamic curve diagrams at different temperature, see Fig. 4.60-63 DEG C is proposed as MCDA primers
Optimal reaction temperature.In the temperature range, amplification rate is most fast.Subsequent authentication in the present invention selects 61 DEG C as constant temperature
Condition carries out MCDA amplifications.Fig. 4 represents that the MCDA primers temperature dynamic of the detection Listeria monocytogenes for lmo0733 designs is bent
Line chart.
Embodiment 3.MCDA-LFB detects the sensitivity of single target
Carry out after the MCDA amplified reactions of standard, with LFB with the good Listeria monocytogenes genomic DNA of serial dilution
Detection shows：The detection range of MCDA-LFB is 10ng～10fg, and LFB red line (Fig. 5 A) occurs in TL and CL regions.When anti-
When answering genomic templates amount in system to be reduced to below 10fg, only there is red line in CL regions in LFB, represents negative findings (figure
5A4-A5).Fig. 5 A read MCDA amplifications with LFB visualizations；Fig. 5 A1 to A5 represent that the template amount of Listeria monocytogenes is
10ng, 10pg, 10fg, 1fg and 0.1fg, Fig. 5 A6, A7, A8 represent listeria ivanovii (10pg), Salmonella template respectively
(10pg), blank (1 microlitre of distilled water).
In order to further verify that MCDA-LFB detects the sensitivity of Listeria monocytogenes, 3 kinds of detection methods are used in addition
MCDA amplifications differentiate.First, visible dyes (such as FD reagents, bright general fluorescent visual reagent) are added in the reactive mixture,
The color of positive reaction pipe is changed into green from light gray, and negative reaction pipe then keeps original light gray.Detection shows：
The detection range of L.monocytogenes-MCDA is 10ng～10fg, and positive amplification pipe is changed into green (Fig. 5 B1-B3).When anti-
When answering genomic templates amount in system to be reduced to below 10fg, reaction occurs without green, remains as light gray, represents negative findings
(Fig. 5 B4-B5).Fig. 5 B read MCDA amplifications with visual method：Fig. 5 B1 to B5 represent that the template amount of Listeria monocytogenes is
10ng, 10pg, 10fg, 1fg and 0.1fg, Fig. 5 B6, B7, B8 represent listeria ivanovii template (10pg), Salmonella respectively
Template (10pg), blank (1 microlitre of distilled water).Second, MCDA products can detect amplicon after sepharose electrophoresis,
As different size of amplified fragments are contained in product, therefore the electrophoretogram of positive amplification product is stepped in specificity, cloudy
Property reaction occur without any band.Detection shows：The detection range of L.monocytogenes-MCDA is 10ng～10fg, positive
There is ladder strip band (Fig. 5 C1-C3) in reaction.When genomic templates amount is reduced to below 10fg in reaction system, spy is occurred without
Different ladder strip band, represents negative findings (Fig. 5 C4-C5).Fig. 5 C read MCDA amplifications with electrophoresis method；Fig. 5 C1 to C5
The template amount of expression Listeria monocytogenes is 10ng, 10pg, 10fg, 1fg and 0.1fg, and Fig. 5 C6, C7, C8 represent Lee Yi Shi respectively
This special bacterium (10pg), Salmonella template (10pg), blank (1 microlitre of distilled water).Third, transmissometer is used to analyze in real time
MCDA expands (Fig. 5 D).Carry out after the MCDA amplified reactions of standard with the good Listeria monocytogenes genomic DNA of serial dilution,
Result judgement is carried out with real-time transmissometer, detection shows：The detection range of L.monocytogenes-MCDA be 10ng～
10fg, positive amplification Haze curve are observed (Fig. 5 D1-D3).When in reaction system genomic templates amount be reduced to 10fg with
When lower, positive amplification Haze curve is occurred without, represent negative findings (Fig. 5 D4-D5).Fig. 5 D read with the visualization of real-time transmissometer
Take MCDA amplifications；Fig. 5 D1 to D5 represent Listeria monocytogenes template amount be 10ng, 10pg, 10fg, 1fg and 0.1fg,
D6, D7, D8 represent listeria ivanovii (10pg), salmonella template (10pg), blank (1 microlitre of distilled water) respectively.
Embodiment 4. determines the optimum reacting time of MCDA-LFB technologies
In the case where standard answers system condition, add and draw for Listeria monocytogenes DNA profiling and designed corresponding MCDA
Thing, with the good Listeria monocytogenes genomic DNA of serial dilution as template.Reaction carries out (61 DEG C) under constant temperature,
Constant temperature time is distinguished 10 minutes, 15 minutes, 20 minutes and 25 minutes.Show with LFB detections：MCDA-LFB technologies are single in detection
When increasing Listerella, optimum reacting time is 20 minutes (Fig. 6).It is when MCDA systems were in amplification step constant temperature 20 minutes, minimum
The template of test limit level can be detected (Fig. 6 C).In Fig. 6 C, LFB detection ranges are 10ng～10fg, and LFB is in TL and CL areas
There is red line (LFB1-LFB3) in domain.When in reaction system, genomic templates amount is reduced to below 10fg, LFB Jin CL areas
There is red line in domain, represents negative findings (LFB4-LFB5).Fig. 6 reads MCDA systems from 10 minutes to 25 with LFB visualizations
The amplification of minute；LFB1 to LFB5 represent Listeria monocytogenes template amount be 10ng, 10pg, 10fg, 1fg and 0.1fg,
LFB6, LFB7, LFB8 represent listeria ivanovii template (10pg), salmonella template (10pg), (1 microlitre of blank respectively
Distilled water).
Embodiment 5. determines the specificity of MCDA-LFB technologies
With common pathogenic bacterium and conditioned pathogen DNA (Listeria monocytogenes, salmonella, shigella dysenteriae, vibrio cholera, pair
Hemolytic vibrio, Vibrio vulnificus, enterococcus faecalis, staphylococcus aureuses, campylobacter jejuni, Bacillus cereuss, intestinal are pathogenic
Escherichia coli, enterotoxigenic E.Coli, enteroinvasive E.Coli etc.) for template evaluate MCDA-LFB technologies specificity.
(bacterial strain information refers to table 2).MCDA-LFB technologies can accurately differentiate Listeria monocytogenes, illustrate the special of MCDA-LFB methods
Property good (see Fig. 7).In Fig. 7, LFB1-12：Listeria monocytogenes template；LFB13-39, non-Listeria monocytogenes template；
LFB40, blank.As a result show, MCDA-LFB can correctly detect target sequence.
2. bacterial strain information of table
aU, unidentified serotype (unidentified serotype)；ATCC, American Type Culture
Collection (American Type Culture Collection)；Slcc,Seeliger’s Special Listeria Culture
Collection (Seeley lattice Liszt cultivates collection)；NCTC,National Collection of Type Culture
(British Standard type culture collection center).ICDC, National Institute for Communicable Disease
Control Disease Control and Prevention, Chinese Center for Disease Control and
Prevention (Inst of Infection Disease Prevention and Control, Chinese Diseases Prevention an).
Sequence table
<110>Inst of Infection Disease Prevention and Control, Chinese Diseases Prevention an
<120>It is intersect the method that amplification detects Listeria monocytogenes with reference to gold nano bio-sensing more
<160> 12
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213> Listeria monocytogenes
<400> 1
aaggactttc gcaagaaga 19
<210> 2
<211> 18
<212> DNA
<213> Listeria monocytogenes
<400> 2
ggcggtcttt ctttgagc 18
<210> 3
<211> 39
<212> DNA
<213> Listeria monocytogenes
<400> 3
aggaactgac agtccttgct atcaaactga atgtggtgc 39
<210> 4
<211> 39
<212> DNA
<213> Listeria monocytogenes
<400> 4
aggaactgac agtccttgct atcaaactga atgtggtgc 39
<210> 5
<211> 50
<212> DNA
<213> Listeria monocytogenes
<400> 5
cactttgctt ggagaaactg ttatgaagtt tataatctcc agtttttcgg 50
<210> 6
<211> 20
<212> DNA
<213> Listeria monocytogenes
<400> 6
aggaactgac agtccttgct 20
<210> 7
<211> 20
<212> DNA
<213> Listeria monocytogenes
<400> 7
aggaactgac agtccttgct 20
<210> 8
<211> 25
<212> DNA
<213> Listeria monocytogenes
<400> 8
cactttgctt ggagaaactg ttatg 25
<210> 9
<211> 21
<212> DNA
<213> Listeria monocytogenes
<400> 9
cccatttaga gattgtttgt c 21
<210> 10
<211> 22
<212> DNA
<213> Listeria monocytogenes
<400> 10
caaaggttga tgatgtaaaa gc 22
<210> 11
<211> 21
<212> DNA
<213> Listeria monocytogenes
<400> 11
ggagattaac atatcggaat c 21
<210> 12
<211> 20
<212> DNA
<213> Listeria monocytogenes
<400> 12
gaagtgcttg aaacaccagt 20
Claims (10)
1. a kind of application intersects the method that amplification combines gold nano bio-sensing testing goal gene, and methods described includes following
Step：
(1) rise from the 3 ' of genes of interest fragment ends, the first arbitrary sequence F1s of setting, the second arbitrary sequence P1s, from described
The 5 ' of genes of interest fragment are held, and set the 3rd arbitrary sequence F2, the 4th arbitrary sequence P2, in second arbitrary sequence P1s
5 ' end setting the 5th arbitrary sequences C1, and/or the 4th arbitrary sequence P2 3 ' end setting the 6th arbitrary sequences C2s,
The 7th arbitrary sequence D1 and the 8th arbitrary sequence R1 are set respectively at the 3 ' ends and 5 ' ends of sequence C 1, at 5 ' ends of sequence C 2s and
3 ' ends set the 9th arbitrary sequence D2s and the tenth arbitrary sequence R2s respectively；
(2) displacement primers F 1 is provided, the primers F 1 is containing the sequence complementary with sequence F1s, there is provided cross primer CP1, described
Primer CP1 is from 5 ' ends successively containing sequence C 1 and sequence P1 complementary with sequence P1s, there is provided displacement primers F 2, the primer contain
There is sequence F2, there is provided cross primer CP2, the primer CP2 is from 5 ' ends successively containing the sequence C 2 complementary with sequence C 2s and sequence
P2, the 5 ' ends for being simultaneously provided in cross primer CP1 or CP2 are marked with the cross primer CP1* or CP2* of biotin；
(3) provide amplimer, the amplimer includes the amplimer C1 containing sequence C 1, and/or containing with sequence C 2s
Complementary amplimer C2, the amplimer D1 containing sequence D 1, the amplimer R1 containing sequence R1, containing with sequence D 2s
Complementary amplimer D2 and containing the amplimer R2 complementary with sequence R2s, is simultaneously provided in the amplimer C1 or C2
5 ' the end haptenic amplimer C1* or C2* of labellings；
(4) in the presence of chain shift-type polymerase (Bst), melting temperature regulator, primer, application target genetic fragment is used as mould
Plate constant-temperature amplification DNA；
(5) using the amplified production of gold nano biosensor detecting step (4).
2. method according to claim 1, it is characterised in that in 5 ' end institute labellings of the amplimer C1* or C2*
Hapten is Digoxin.
3. method according to claim 2, it is characterised in that in 5 ' the end labelling biotin of cross primer CP1*, in institute
State amplimer C1*5 ' ends labelling Digoxin.
4. method according to claim 3, it is characterised in that the gold nano biosensor includes a backboard, in institute
State, on the nitrocellulose filter according to
Secondary setting detection line and control line, are coated with the chain of golden nanometer particle coupling successively in gold standard pad, detection line and control line region
The bovine serum albumin that mycin Avidin, anti digoxin antibody and biotin are coupled.
5. method according to claim 1, it is characterised in that the constant-temperature amplification is carried out in 60-63 DEG C of environment
's.
6. method according to claim 4, it is characterised in that the constant-temperature amplification is carried out in 61 DEG C of environment.
7. according to the arbitrary described method of claim 1-6, it is characterised in that the genes of interest is Listeria monocytogenes
lmo0733。
8. described method according to claim 7, it is characterised in that the sequence of the displacement primers F 1 such as SEQ ID NO:1
It is shown, the sequence such as SEQ ID NO of the displacement primers F 2:Shown in 2；The sequence of the cross primer CP1 such as SEQ ID NO:3
It is shown, the sequence such as SEQ ID NO of the cross primer CP1* of biotin are marked with the 5 ' ends of the cross primer CP1:4
It is shown, the sequence such as SEQ ID NO of the cross primer CP2:Shown in 5, the sequence such as SEQ ID NO of primer C1:Shown in 6,
5 ' the ends of the primer C1 are marked with the sequence such as SEQ ID NO of the primer C1* of Digoxin:Shown in 7, the sequence of primer C2 is such as
SEQ ID NO:Shown in 8, the sequence such as SEQ ID NO of primer D1:Shown in 9, the sequence such as SEQ ID NO of primer D2:Shown in 10,
The sequence of primer R1 such as SEQ ID NO:Shown in 11, the sequence such as SEQ ID NO of primer R2:Shown in 12.
9. one group be used for constant-temperature amplification Listeria monocytogenes lmo0733 genes primer sequence, it is characterised in that the sequence bag
Include：Such as SEQ ID NO:Displacement primers F 1 shown in 1, such as SEQ ID NO:Displacement primers F 2 shown in 2, such as SEQ ID NO:3
Shown cross primer CP1, such as SEQ ID NO:Cross primer CP2 shown in 5, such as SEQ ID NO:Amplimer shown in 6
C1, such as SEQ ID NO:Amplimer C2 shown in 8, such as SEQ ID NO:Amplimer D1 shown in 9, such as SEQ ID NO:10
Shown amplimer D2, such as SEQ ID NO:Amplimer R1 shown in 11, such as SEQ ID NO:Amplimer shown in 12
R2, the 5 ' ends for being simultaneously provided in cross primer CP1 or CP2 are marked with the cross primer CP1* or CP2* of biotin, and in institute
State the haptenic amplimer C1* or C2* of 5 ' end labellings of amplimer C1 or C2.
10. primer sequence according to claim 9, it is characterised in that in 5 ' the end labelling biotin of cross primer CP1*,
Digoxin is marked with the 5 ' ends of amplimer C1*.
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