CN104328113B - One group of nucleotide sequence and the application in Listeria monocytogenes is identified - Google Patents
One group of nucleotide sequence and the application in Listeria monocytogenes is identified Download PDFInfo
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Abstract
The invention discloses a kind of nucleotide sequence combination and the application in intersection isothermal amplification reactions thereof.Described intersection isothermal amplification reactions can be used for identifying Listeria monocytogenes, has detection simple, fast and easy, high specificity, highly sensitive feature.
Description
Technical field
The present invention relates to intersection constant-temperature amplification application in Bacteria Detection, particularly in Listeria monocytogenes Differential Diagnosis
In application, belong to molecular biology and microbiological art.
Background technology
Listeria monocytogenes (Listeria monocytogens, LM) be widely present in environment Gram-positive,
There are motor capacity, facultative intracellular parasitism brevibacterium, are important food-borne Zoonosis cause of disease bacterium, it is possible to cause the mankind seriously to feel
Dye.The Susceptible population of listeriosis is mainly hypoimmunity crowd and immunodeficiency crowd, and the former includes old people, new life
Youngster and anemia of pregnant woman；The latter is tumor patient, HIV sufferers and organ graft recipient etc..Mankind's listeriosis main clinic symptoms
For septicemia, meningitis, anemia of pregnant woman's miscarriage, stillbirth and heat generation gastroenteritis etc., this bacterium can pass through gut barrier, blood brain barrier and
Placental barrier.Listeria monocytogenes is sick the highest at the infection rate of the mankind, but the mortality rate of this disease is high and paid close attention to widely,
Once becoming the great public health problem of American-European countries.Domestic there is no the report being caused alimentary toxicosis to break out by Listeria monocytogenes
Road, whether physical presence fail to understand, Sporadic cases all has been reported that in multiple provinces and cities.Therefore, the quickest in order to give clinical patient
Treatment and the Epidemiological study of Listeria monocytogenes, research and develop one save time, the list that laborsaving and specificity is higher increases Liszt
Bacterium detection method necessitates.
At present traditional Zengjing Granule and biochemical identification, the method are depended on for Listeria monocytogenes isolation identification
Time-consuming about 5 to 7 days, increase bacterium including secondary, select to cultivate and follow-up biochemical identification, take time and effort, the interpretation of chemical result
Depend on the subjective judgment of people, cause result poor repeatability, easily misjudge.Along with the fast development of nucleic acid diagnostic techniques, some with
Diagnostic techniques (such as regular-PCR technology, Fluorescence PCR assay) based on PCR is used for the quick diagnosis of Listeria monocytogenes, so
And these methods depend on the instrument and equipment of costliness, need follow-up electrophoretic procedures, higher probe synthesis expense, and skillfully
Operator.Some laboratories cannot be carried out, and limits the utilization of these technology.These detection techniques are used to examine at present
The PCR method of disconnected Listeria monocytogenes and real-time PCR method, selected genes of interest (such as hly, prfA, iap) is special
The opposite sex is poor, easily occurs that false positive expands.In addition the poor sensitivity of PCR detection technique, detection process is the longest, is unfavorable for fast
Speed detection and Emergent detection.Along with the appearance of constant temperature technology, loop-mediated isothermal amplification technology (LAMP) has been used for singly increasing Lee
The detection of this special bacterium, but the design of primers of this technology is complicated, requires higher to the conservative of genes of interest, that reports at present examines
The LAMP technology of disconnected Listeria monocytogenes is mainly according to hly and prfA gene design primer, but the poor specificity of these genes,
It is easily caused false positive amplification, is unfavorable for the Accurate Diagnosis of Listeria monocytogenes.Thus, it is badly in need of development a kind of simple, quick, clever
Quick and special diagnostic techniques detects for Listeria monocytogenes.
The isothermal amplification technology (Cross priming amplification, CPA) that intersects is to be set up by Xu Gaolian etc.
A kind of new constant temperature nucleic acid amplification technology, have sensitive height, simple to operate, do not rely on expensive device, product easily detection etc.
Advantage.This technology has been widely used for molecular diagnosis field.CPA is for 5 specific regions 7 primers of design of target sequence, profit
Being catalyzed the synthesis of new chain under constant temperature with the Bst archaeal dna polymerase with strand-displacement activity, target sequence can efficiently be expanded
Increase.Article 7, among core primers 2 be cross primer, i.e. As and Sa；Article 2, for displacement primer, i.e. 4s and 5a；Article 3, draw for amplification
Thing, i.e. 3a, 2a and 1s.Cross primer As comprises 2a and 1s, i.e. 5'-2a-1s；Sa comprises 1s and 2a, i.e. 5'-1s-2a.
CPA reaction is divided into single intersection amplified reaction (S-CPA) and dual crossing amplified reaction (D-CPA).CPA reaction only needs
A constant temperature temperature between 62～66 DEG C, this temperature is the medium temperature of double-stranded DNA renaturation and extension, double-stranded DNA
Being in this temperature range in the dynamic balance state partly dissociated with quasi integration, any one primer is to the complementation of double-stranded DNA
When position carries out base pairing extension, another chain will dissociate, become strand.S-CPA is polymerized at strand-displacement activity Bst DNA
Under the effect of enzyme, with 3 ' ends of As primer 1s section as starting point, the DNA complementary series pairing with corresponding, start strand displacement DNA
Synthesis.4s primer combines, with 3 ' ends as starting point, by strand-displacement activity Bst DNA with the complementary series (4a) of 1s front end 4s
The effect of polymerase, first displaces the DNA of As primer synthesis, self DNA simultaneously synthesizing.Final 4s primer is synthesized into
DNA forms double-strand with template DNA.But the DNA first synthesized by As primer is carried out strand displacement one list of generation by 4s primer
Chain, this strand is complementary by 3s section and the primer 3a of 5 ' ends, starts DNA synthesis；2s section and primer 2 a are complementary, start chain
Displacement DNA synthesis, the release DNA containing primer 3a synthesizes chain, and this synthesis chain can be combined with primer 2 a and As complementation, starts first
Individual cyclic amplification.In addition can be replaced by the displacement primer 5a in downstream by the new chain of primer 2 a complementation synthesis, the new chain of generation occurs
Oneself's base pairing, forms single stem circulus, starts second cyclic amplification.First circulation rank of CPA reaction amplification
Section: the DNA short chain product of formation is in and unwinds and combine dynamic equilibrium, and primer can be implemented in combination with amplification with the strand dissociated and follow
Ring；Second cycle stage: in dumbbell structure, the 1s in As primer and strand 1a hybridization on ring, start new round chain and put
Changing reaction, amplify short chain DNA, the short chain product of formation is similarly in and unwinds and combine dynamic equilibrium, primer can with dissociate
Strand be implemented in combination with amplification, and produce new loop-stem structure, thus realize the cyclic amplification of CPA.S-CPA reaction can be special
Different, efficient, quickly expand target DNA, make the amount of product reach 10 within an hour9Individual copy.
D-CPA to S-CPA has similar amplification principle, simply contain in reaction system two cross primer As and
Sa, and without displacement primer 2 a.For D-CPA reacts, amplification system comprises two cross primer As and Sa, two displacements
Primer 4s and 5a, an amplimer 3a.For S-CPA reacts, amplification system comprises a cross primer As, puts for two
Change primer 4s and 5a, two amplimer 3a and 2a.
After CPA amplification, the detection of its product can be observed by sepharose electrophoresis poststaining.Relatively simple method is
Adding SYBR Green I the most in the product to dye, presenting green is positive reaction, orange red for negative reaction.Can also lead to
The turbidity crossing amplification by-product magnesium pyrophosphate precipitation judges, liquid is muddy, centrifugal or have white precipitate for positive reaction,
Without this phenomenon is then negative reaction.The simplest method is to add visible dyes in the reactive mixture now, positive anti-
The color of pipe should become green from light gray, negative reaction pipe then keeps original light gray.
In view of routine techniquess such as such as PCR, when detecting pathogenic microorganism, to there is susceptiveness poor, the highest scarce of specificity
Point, how using CPA technology to solve some pathogenic microorganisms these problems present in the Differential Diagnosis has become the micro-life of cause of disease
The demand urgently to be resolved hurrily of analyte detection technical field.
Summary of the invention
It is an object of the invention to provide one and can be applied to the detection sequence of Bacteria Detection, particularly isothermal amplification technology
Row, and further provide for constant temperature and intersect the method for amplification technique detection Listeria monocytogenes, and a kind of for this pathogen
Quickly, sensitive and special detection of nucleic acids system.
Based on foregoing invention purpose, the kind specific gene lmo0733 that the present invention is directed to Listeria monocytogenes designs a set of friendship
Fork reaction amplimer.Therefore, present invention firstly provides a kind of nucleotide sequence combination, be made up of SEQ ID NO:1,3-7,
Wherein SEQ ID NO:4 and 7 exists time different.The various primers that these nucleotide sequences intersect in amplified reaction as constant temperature make
With.
In a preferred technical scheme, described combination is made up of SEQ ID NO:1,3-6.This group primer is used for S-
CPA expands.
In a preferred technical scheme, described combination by SEQ ID NO:1,3,5-7 forms.This group primer is used for D-
CPA expands.
Present invention also offers the application in intersection isothermal amplification reactions of the above-mentioned nucleotide sequence combination.
In a preferred technical scheme, described intersection isothermal amplification reactions can be divided into following steps:
(1) DNA profiling of sample to be detected is extracted；
(2) DNA profiling that step (1) obtains is put in intersection isothermal amplification reactions system and expand；
(3) amplified production that detecting step (2) obtains.
Preferably, described intersection isothermal amplification reactions is S-CPA amplification, and reaction system cumulative volume is 20 μ l, wherein contains:
0.3mM SEQ ID NO:1,1.44mM SEQ ID NO:3,1.44mM SEQ ID NO:4,0.3mM SEQ ID NO:5,
2.4mM SEQ ID NO:6, and 20mM Tris-HCl (pH 8.8), 10mM KCl, 4mM MgSO4,10mM(NH4)2SO4,
0.1%Tween 20,0.8M glycine betaine, 1.4mM dNTPs, 1 μ l of Bst archaeal dna polymerase (8U/mL), 1 μ l Loopamp
Fluorescent dye (FD) and 1 μ l DNA profiling, reaction temperature 64 DEG C, the response time is 90 minutes.
Preferably, described intersection isothermal amplification reactions is D-CPA amplification, and reaction system cumulative volume is 20 μ l, wherein contains:
0.3mM SEQ ID NO:1,1.44mM SEQ ID NO:3,0.3mM SEQ ID NO:5,2.4mM SEQ ID NO:6,
1.44mM SEQ ID NO:7, and 20mM Tris-HCl (pH 8.8), 10mM KCl, 4mM MgSO4,10mM(NH4)2SO4,
0.1%Tween 20,0.8M glycine betaine, 1.4mM dNTPs, 1 μ l of Bst archaeal dna polymerase (8U/mL), 1 μ l Loopamp
Fluorescent dye (FD) and 1 μ l DNA masterplate, reaction temperature 64 DEG C, the response time is 90 minutes.
In a preferred technical scheme, described step (3) can be range estimation, transmissometer detection or agarose gel
Electrophoresis detection.
In a preferred technical scheme, described sample to be detected can be bacterial cultures, body fluid or human secretory
Thing sample.
What the present invention provided applies the intersection isothermal amplification reactions of above-mentioned nucleotide sequence to detect for Listeria monocytogenes
With regular-PCR ratio, there is significant advantage.CPA amplification reaction condition is simple, and temperature is single, react and result is studied and judged quickly, easily
In carrying out at laboratories.It addition, the remolding sensitivity regular-PCR method of CPA amplified reaction is high 10 times.Causing a disease with common
Bacterium and conditioned pathogen DNA (listeria ivanovii, Yi Luoke Listerella, Weir Listerella, Xi Er Listerella, grignard
Listerella, Bacillus cereus, enteropathogenic E.Coli, enterotoxigenic E.Coli, enteroinvasive E.Coli etc.) be
Template is evaluated in the specific experiment of CPA reaction system, and this system is in 25 kinds of other common pathogens of detection and opportunistic diseases
Specific amplification does not occurs during bacterium, illustrates that the specificity of this detection system is good.
The display of above advantage is the present invention have broad application prospects.
Accompanying drawing explanation
Fig. 1, the position of design of primers and direction schematic diagram；
Fig. 2, S-CPA reaction result figure；
Fig. 3, D-CPA reaction result figure；
Fig. 4, CPA Evaluation on specificity result figure；
Fig. 5, regular-PCR detection sensitivity evaluation result figure；
Fig. 6, S-CPA and D-CPA real-time transmissometer testing result figure；
Fig. 7, S-CPA detection sensitivity evaluation result figure；
Fig. 8, D-CPA detection sensitivity evaluation result figure.
Detailed description of the invention
Below in conjunction with specific embodiment further describe the present invention, advantages of the present invention and feature will be with describe and
Apparent.But these embodiments are only exemplary, protection scope of the present invention is not constituted any restriction.
Sequential design
According to Listeria monocytogenes species-specific genes lmo0733 (NC_003210.1, Listeria
Monocytogenes, LM) design cross reaction primer, this gene is present in all serotype of Listeria monocytogenes, and it is special
Property good, can be by Listeria monocytogenes and other the most close strains (such as listeria ivanovii, Weir Listerella, Yi Luo
Gram Listerella) and Pseudomonas (such as bacillus) distinguish.Multiple Sequence Alignment software ClustalX 1.83 and primer is utilized to set
Meter software Primer Premier 5.0 designs cross reaction primer, and is entered in ncbi database by the specific primer of acquisition
Row sequence alignment analysis, to get rid of, primer is that may be present with other species sequence non-specific to be mated, final obtain optimize after
A set of constant temperature cross reaction primer.The position of design of primers and direction see that Fig. 1, sequence are shown in Table 1.
Table 1: constant temperature cross reaction primer sequence
Note: R=A/G；N=A/T/C/G.
With the reagent related to used in the present invention:
Loopamp Kit (Eiken Chemical Co.Ltd., Tokyo, Japan) is purchased from Rong Yan company of Japan.DNA carries
Take test kit (QIAamp DNA minikits；Qiagen, Hilden, Germany) it is purchased from Qiagen company of Germany.PCR reacts
System mixture M IX (Taq archaeal dna polymerase, dNTP and buffer) is purchased from Beijing CoWin Bioscience Co., Ltd..
DL50 DNA Marker and DL50DNA Marker is purchased from precious biological engineering (Dalian) company limited.Remaining reagent is commercially available
Analytical pure level product.
The key instrument using and relating in present invention experiment: Loopamp real-time transmissometer LA-320C (Eiken
Chemical Co., Ltd, Japan) it is purchased from Rong Yan company of Japan.DK-450B type electric heating constant temperature instrument is purchased from Shanghai gloomy letter experiment instrument
Device company limited；PCR instrument is Sensoquest Labcycler, Germany's Sensoquest product；Electrophoresis equipment is Beijing monarch meaning
East electrophoresis equipment company limited product；Gel imaging system is Bio-Rad Gel Dox XR, U.S.'s Bio-Rad product.
Embodiment 1:S-CPA detects
Extract the genomic DNA of Listeria monocytogenes, use the DNA extracted to carry out S-CPA and D-CPA as template anti-
Should, finally use different detection meanss that result is carried out interpretation.
1. genome extracts: DNA extraction kit (the QIAamp DNA extracting use Qiagen company of bacterial genomes
minikits；Qiagen, Hilden, Germany), operate to specifications.Ultraviolet spectrophotometer is utilized to measure gene
The group concentration of DNA and purity, Listeria monocytogenes genomic DNA with GE buffer serial-dilution (from 25ng, 2.5ng, 250pg,
25pg, 2.5pg, 250fg, 125fg to 62.5fg).The all a small amount of subpackage of various genomic DNAs ,-20 DEG C save backup.Intersect anti-
Answer system:
The reaction system of 2.S-CPA: cumulative volume is 20 μ l, comprises 2.4mM cross primer As (2a+1s), 1.44mM amplification
Primer 3a and 2a, 0.3mM replace primer 4s and 5a, 20mMTris-HCl (pH 8.8), 10mM KCl, 4mM MgSO4,10mM
(NH4)2SO4, 0.1%Tween 20,0.8M glycine betaine, 1.4mM dNTPs, 1 μ l of Bst archaeal dna polymerase (8U/mL), 1 μ l
Loopamp fluorescent dye (FD) and 1 μ l DNA profiling.
3. amplified reaction
Isothermal reaction (Shanghai gloomy reliable test Instrument Ltd.), reaction temperature is carried out in DK-450 Type B electric heating constant temperature instrument
Spending 64 DEG C, the response time is 90min.
4. result interpretation
After reaction terminates, by naked eyes interpretation, reaction system is that green is positive for amplification.
Take 5 μ l amplified productions on the agarose gel of 2.5%, carry out electrophoresis, and observation analysis on gel imaging system
Result.
Transmissometer interpretation: in Loopamp real-time transmissometer LA-320C (Eiken Chemical Co., Ltd, Japan, day
This Rong Yan company) on carry out.
Embodiment 2:D-CPA detects
1. genome extracts
With embodiment 1
The reaction condition of 2.D-CPA: cumulative volume is 20 μ l, comprises 2.4mM cross primer As (2a+1s), 1.44mM and intersects
Primer Sa (1s+2a), 1.44mM amplimer 3a, 0.3mM displacement primer 4s and 5a, 20mM Tris-HCl (pH 8.8), 10mM
KCl,4mM MgSO4,10mM(NH4)2SO4, 0.1%Tween 20,0.8M glycine betaine, 1.4mM dNTPs, 1 μ l of Bst
Archaeal dna polymerase (8U/mL), 1 μ l Loopamp fluorescent dye (FD) and 1 μ l DNA masterplate.
3. amplified reaction
Isothermal reaction (Shanghai gloomy reliable test Instrument Ltd.), reaction temperature is carried out in DK-450 Type B electric heating constant temperature instrument
Spending 64 DEG C, the response time is 90min.
4. result interpretation
After reaction terminates, by naked eyes interpretation, reaction system is that green is positive for amplification.
Take 5 μ l amplified productions on the agarose gel of 2.5%, carry out electrophoresis, and observation analysis on gel imaging system
Result.
Transmissometer interpretation: in Loopamp real-time transmissometer LA-320C (Eiken Chemical Co., Ltd, Japan, day
This Rong Yan company) on carry out.
Embodiment 3. comparative examples: common RCR detection
1. genome extracts with embodiment 1
2. common RCR reaction system:
3. amplified reaction
PCR reaction condition:
PCR primer sequence: SEQ ID NO:1 and SEQ ID NO:5
4. result interpretation
Take 6 μ l amplified productions on the agarose gel of 2.5%, carry out electrophoresis, and observation analysis on gel imaging system
Result.
The interpretation of testing result with compare
1. prove the availability of the cross reaction primer of design
As shown in Figures 2 and 3, there is the change of obvious color in S-CPA (Fig. 2) and CPA (Fig. 3) positive reaction pipe, from shallow
Lycoperdon polymorphum Vitt becomes green (black-and-white photograph display turbidity is higher), and negative reaction pipe color keeps constant, and (black and white is shone to remain as light gray
Sheet display turbidity is relatively low).By agarose gel electrophoresis, positive reaction product is special stepped (swimming lane 2), and negative anti-
Any band (swimming lane 3) should be occurred without.
The detection specificity of 2.CPA system
With common pathogenic bacterium and conditioned pathogen (listeria ivanovii, Yi Luoke Listerella, Weir Listerella,
Xi Er Listerella, Listera grayi, Bacillus cereus, enteropathogenic E.Coli, enterotoxigenic E.Coli, intestinal is invaded
Attacking property escherichia coli etc.) DNA be template evaluate CPA reaction system specificity.
As shown in Figure 4, when S-CPA 25 kinds of other common pathogens of detection and opportunist, specific amplification does not occurs,
Illustrate that the specificity of this detection system is good.Strain background see table.
Table 2: for the strain background situation catalog of detection
Note U: do not identify bacterial strain；
ATCC: American Type Culture Collecti；
NCTC: Britain's Type Culture Collection；
ICDC: Inst of Infection Disease Prevention and Control, Chinese Diseases Prevention an.
3. the sensitivity of regular-PCR detection system
The list using serial dilution increases Liszt's genomic DNA and carries out display after regular-PCR amplification: when base in reaction system
When being reduced to below 25pg because of group template amount, there is not purpose band (see Fig. 5) in regular-PCR amplification rear electrophoresis figure.Figure is swum
Road M is DNA molecular amount marker DL50, swimming lane 2 to 9 be respectively template amount be the genomic DNA of 25ng～62.5fg.
The detection sensitivity of 4.CPA system
The list using serial dilution increases result after Liszt's genomic DNA carries out constant temperature cross reaction amplification and shows: S-CPA
It is 25ng～2.5pg with the detection range of D-CPA, when in reaction system, genomic templates amount is reduced to below 2.5pg, constant temperature
The amplification that intersects occurs without positive amplification.
In order to use real-time transmissometer to read S-CPA and D-CPA amplification, (A is S-CPA amplification to Fig. 6, and B is D-CPA
Amplification).
Fig. 7 is shown as S-CPA amplification, and A is the result shown by macroscopic method, and swimming lane 1-5 amplification is the positive,
B is the result shown by agarose gel electrophoresis, and in figure, swimming lane M is DNA molecular amount marker DL50；Swimming lane 2 to 9 is respectively
Be template amount be the genomic DNA of 25ng～62.5fg.
Fig. 8 is shown as D-CPA amplification, and A is the result shown by macroscopic method, and swimming lane 1-5 amplification is the positive,
B is the result shown by agarose gel electrophoresis, and in figure, swimming lane M is DNA molecular amount marker DL50；Swimming lane 2 to 9 is respectively
Be template amount be the genomic DNA of 25ng～62.5fg.
Testing result shows: the detection range of S-CPA and D-CPA is 25ng～2.5pg, when genome in reaction system
When DNA profiling amount is reduced to below 2.5pg, constant temperature intersection amplification occurs without positive amplification, and according to the result of interpretation, CPA expands
The remolding sensitivity regular-PCR method of reaction is high 10 times.
Claims (7)
1. a nucleotide sequence combination, described combination be made up of SEQ ID NO:1,3-6 or by SEQ ID NO:1,3,5-7
Composition.
Nucleotide sequence combination the most according to claim 1 answering in the intersection isothermal amplification reactions of non-diagnostic purpose
With.
Application the most according to claim 2, described intersection isothermal amplification reactions can be divided into following steps:
(1) DNA profiling of sample to be detected is extracted；
(2) DNA profiling that step (1) obtains is put in intersection isothermal amplification reactions system and expand；
(3) amplified production that detecting step (2) obtains.
Application the most according to claim 3, the reaction system cumulative volume of described intersection isothermal amplification reactions is 20 μ l, wherein
Contain: 0.3mM SEQ ID NO:1,1.44mM SEQ ID NO:3,1.44mM SEQ ID NO:4,0.3mM SEQ ID NO:
5,2.4mM SEQ ID NO:6, and 20mM Tris-HCl, 10mM KCl, the 4mM MgSO of pH 8.84,10mM(NH4)2SO4, 0.1%Tween 20,0.8M glycine betaine, the 8U/mL Bst archaeal dna polymerase of 1.4mM dNTPs, 1 μ l, 1 μ l Loopamp
FD fluorescent dye and 1 μ l DNA profiling, reaction temperature 64 DEG C, the response time is 90 minutes.
Application the most according to claim 3, the reaction system cumulative volume of described intersection isothermal amplification reactions is 20 μ l, wherein
Contain, 0.3mM SEQ ID NO:1,1.44mM SEQ ID NO:3,0.3mM SEQ ID NO:5,2.4mM SEQ ID NO:
6,1.44mM SEQ ID NO:7, and the 20mM Tris-HCl of pH 8.8), 10mM KCl, 4mM MgSO4,10mM(NH4)2SO4, 0.1%Tween 20,0.8M glycine betaine, the 8U/mL Bst archaeal dna polymerase of 1.4mM dNTPs, 1 μ l, 1 μ l Loopamp
FD fluorescent dye and 1 μ l DNA masterplate, reaction temperature 64 DEG C, the response time is 90 minutes.
Application the most according to claim 3, described step (3) can be range estimation, transmissometer detection or agarose gel electricity
Swimming detection.
Application the most according to claim 3, described sample to be detected can be bacterial cultures, body fluid or human secretion
Sample.
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