CN106048015A - ET-PCR (endonuclease restriction-mediated real-time polymerase chain reaction) nucleic acid testing technology - Google Patents
ET-PCR (endonuclease restriction-mediated real-time polymerase chain reaction) nucleic acid testing technology Download PDFInfo
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Abstract
The invention discloses a PCR (polymerase chain reaction) amplification method. In the method, a restriction endonuclease sequence is added to a 5'end of an upstream primer or a downstream primer for amplification, a fluorophore is marked at the 5'end of the primer, a quenching group capable of specifically quenching the fluorophore at the 5'end is marked in the middle of the primer, the restriction endonuclease is added to a PCR amplification system and subjected to a PCR. The method has the advantages that the amplification speed is high, the reaction is sensitive, the specificity is high, and real-time, rapid and multiplex detection and analysis of nucleic acid can be realized.
Description
Technical field
The present invention relates to a kind of nucleic acid amplification method, belong to microorganism and biology field.
Background technology
At modern biology and medical domain, nucleic acid amplification is a kind of indispensable technology, is widely used in clinical inspection
The fields such as survey, basic research, archaeological research, epidemic research, transgenic research.At the nucleic acid amplification technologies developing design
In, PCR amplification technique is first isothermal DNA amplification being established, and is with historically new significance, and this technology is by extensively
General it is applied to medical science, biological and chemical association area.
Use round pcr when carrying out nucleic acid amplification, need to repeat three thermal cycle steps: nucleic and melting (95 DEG C), primer
Annealing (45-65 DEG C), extends amplification (72 DEG C).When PCR amplification terminate after, its product detection need a set of complexity flow process and
Equipment, this process is time-consuming, effort.Therefore, the application of round pcr is limited by subsequent detection condition, and these inferior positions hinder
The extensive application of round pcr, especially in area backward in economy, quick diagnosis and detection field in real time.For biological and medical science
For Related Research Domain, be highly desirable to develop one simple to operate, detect nucleic acid amplification method quick, cost-effective.
In the past over 15 years, in order to overcome the subsequent detection of traditional PCR technique, many realities based on round pcr
Time augmentation detection technology arise at the historic moment.For comparing more common round pcr, real time pcr need not follow-up detection, core
Acid amplification and detection are carried out simultaneously, and whole real-time PCR reactions process needs only to 1.5 hours, is advantageously implemented quick diagnosis.This
Outward, owing to eliminating follow-up detection process, the pollution that subsequent operation causes effectively is avoided.
Up to the present, the real time PCR amplification technology researched and developed has more than 10 to plant, and according to real-time Cleaning Principle, can use and is divided into
Following two type.The first is for relying on the detection of double-stranded DNA binding molecule, such as SYBR Green.This type of group is free
Send faint fluorescence under state, when with the minor groove binding of double chain DNA molecule after, fluorescence intensity strengthen, its intensity and double-stranded DNA
Quantity be correlated with.Owing to this type of double-strand binding molecule does not has specificity, it is impossible to identify specific double-strand, as long as double-strand will be tied
Closing luminescence, non-specific amplification or primer dimer in reacting PCR also can produce fluorescence, and therefore background is higher, so
Use clinically and there may be false positive generation.Additionally, depend on the real time pcr that double-strand combines extremely difficult to realize multiple inspection
Survey.The second is the detection relying on oligonucleotide probe, such as Taqman probe.In oligonucleotide probe, fluorophor connects
At 5 ' ends of probe, quencher is then at 3 ' ends.When probe and target sequence match, the fluorescence that fluorophor is launched because of with
3 ' the quenchers held are close and are quenched.When carrying out extension, probe is cut off by 5 ' 5 prime excision enzyme activities of polymerase so that
Fluorophor separates with quencher, launches fluorescence.The product of a part generates the generation of just fluorescence signal along with a part.
Along with the increase of amplification cycles number, the fluorophor discharged constantly accumulates.But, this type of detection is required for target sequence
Three-zone design probe, for the specific regions that some conservatives are poor, is difficult to design this type of probe and detects in real time.
Detect a nucleic acid target and need two primers and a probe, easily form dimer, impact amplification and detection.Additionally, visit
The design technology of pin and modify more complicated.Therefore, the convenience of these real time PCR amplification technology, practicality and operable
Property has much room for improvement.
In order to overcome the inferior position of existing real-time PCR correlation technique, it is achieved quick, simple, sensitive and special nucleotide sequence
Amplification, it is necessary to set up that a kind of simple to operate, economical and practical, response speed is fast, the nucleic acid amplification technologies of high specificity.
It addition, there is a bacterioid at microorganism field, they are extensive in distributed in nature, prestige the most to a great extent
Coerce human health, but its detection technique nevertheless suffers from the limitation of prior art, streptococcus pneumoniae (Streptococcus
pneumonia；S.pneumonia), staphylococcus aureus (Staphylococcus aureus；And excrement intestinal ball S.aureus)
Bacterium (Enterococcus faecalis；E.faecalis) it is exactly such bacterioid.Streptococcus pneumoniae is distributed in nature
Extensively, often living in the nasal cavity of normal person, this bacterium mainly causes lobar pneumonia and tracheitis, otitis media, meningitis, breast
The diseases such as film inflammation, endocarditis, septicemia.Staphylococcus aureus is ubiquitous in nature, empty gas and water, dust and people
All can find with in the Excreta of animal.This bacterium is modal pathogen in mankind's suppurative infection, local can be caused to suppurate and feel
Dye, it is possible to cause pneumonia, pseudomembranous enteritis, pericarditis etc., the even systemic infection such as septicemia, sepsis.Enterococcus faecalis is leather
Lan Shi is positive, hydrogen peroxide negative cocci, is one of flora in humans and animals intestinal.This bacterium is endogenous and exogenous doctor
The second largest pathogen that institute infects, recall rate is only second to escherichia coli.In the pathogenic bacterium causing urinary tract infection, enterococcus faecalis sense
Dye occupies the 2nd；Abdominal cavity, pelvic infection, enterococcus occupies the 3rd；Septicemia, enterococcus occupies the 3rd, case fatality rate be 12.6%～
57%.Additionally, these three pathogen is also the modal pathogen of Intensive Care Unit, thus by whole world hygiene department
Highest attention, become the great public health problem of various countries.At present for streptococcus pneumoniae, staphylococcus aureus and excrement intestinal
The diagnosis of coccus depends on traditional Zengjing Granule and biochemical identification, the method the most about 5 to 7 days, including increasing bacterium, choosing
Selecting and cultivate and follow-up biochemical identification, its inferior position takes time and effort, and the interpretation of chemical result depends on the subjective judgment of people, causes knot
It is poor that fruit is repeated, and easily misjudges.Along with the fast development of nucleic acid diagnostic techniques, at clinical and Basic Laboratory, round pcr by with
In diagnosis pathogen, but, this technology needs follow-up electrophoretic procedures, it is impossible to meet the needs of rapid and convenient detection.Therefore, for
Give clinical patient treat quickly and accurately, the control of hospital infection and the Epidemiological study of three kinds of pathogen, research and development
One save time, the diagnostic method that laborsaving and specificity is higher, it is possible to detect simultaneously and identify that three kinds of pathogen necessitate.
Summary of the invention
In order to realize above-mentioned target, the present invention devises a kind of novel real-time nucleic acid inspection depending on conventional PCR amplification
Survey method, the real-time nucleic acid detection method (Endonuclease depending on PCR amplification of named restricted enzyme mediation
Restriction-Mediated Real-Time Polymerase Chain Reaction, ET-PCR).In described method
In, adding a kind of restriction enzyme enzyme sequence, at the 5 of described primer for the forward primer of amplification or 5 ' ends of downstream primer
` end mark fluorescent group, the centre position labelling at primer can the quencher base of specifically fluorophor described in quencher 5` end
Group, adds described restricted enzyme in described polymerase chain amplification system, and carries out polymerase chain amplified reaction.
In a preferred embodiment, the 5 ' of the described restriction enzyme enzyme sequence that described primer 5 ' is held is being made an addition to
End adds base T.
It is further preferable that described restricted enzyme is BstUI, described restriction enzyme enzyme sequence is CGCG.
In a preferred embodiment, described fluorophor with the combination of quenching group can be: Hex and BHQ1,
Cy5 and BHQ2 or FAM and BHQ1.
In the present invention, for the specific gene ply of streptococcus pneumoniae, the specific gene nuc of staphylococcus aureus and
The specific gene Ef0027 of enterococcus faecalis verifies ET-PCR detection technique as model inspection target, separately designs three set ET-PCR
Amplimer, it is intended to verify, evaluate ET-PCR amplification technique and set up for streptococcus pneumoniae, staphylococcus aureus and excrement intestinal
Quick, the sensitive and special ET-PCR detection system of three kinds of pathogen of coccus.
In a preferred embodiment, described forward primer and downstream primer are one sequence combination:
(1) SEQ ID NO.1 and 3 (this combination is used for detecting ply gene)；
(2) SEQ ID NO.4 and 6 (this combination is used for detecting nuc gene)；Or
(3) SEQ ID NO.7 and 9 (this combination is used for detecting Ef0027 gene).
Present invention also offers said method application in multipurpose gene amplification, in described amplification system,
The fluorophor that 5 ' end labellings of every pair of forward primer of gene amplification or downstream primer for various purposes are different, and at primer
Central marker can the quencher of specifically fluorophor described in quencher 5` end.
In a preferred embodiment, described genes of interest to be amplified is 2 or 3, when described mesh to be amplified
Gene when being 2, described forward primer and downstream primer are any two kinds of combinations in following three combined sequence；When to be amplified
Described genes of interest when being 3, described forward primer and downstream primer include following all three combined sequence:
(1) SEQ ID NO.1 and 3；
(2) SEQ ID NO.4 and 6；
(3) SEQ ID NO.7 and 9.
Finally, the invention provides a kind of polymerase chain amplification kit, described test kit contains Taq enzyme, dNTP, restriction
Property restriction endonuclease BstUI, amplification buffer, for amplification forward primer and downstream primer, described forward primer or downstream primer
5 ' end be added with restricted enzyme BstUI sequence C GCG, described BstUI sequence 5 ' end be added with base T.
In a preferred embodiment, described forward primer and downstream primer combine selected from one sequence:
(1) SEQ ID NO.1 and 3；
(2) SEQ ID NO.4 and 6；Or
(3) SEQ ID NO.7 and 9.
In another preferred embodiment, described forward primer and downstream primer are to appoint in following three combined sequence
Anticipate two kinds of combinations；Or described forward primer and downstream primer include following all three combined sequence:
(1) SEQ ID NO.1 and 3；
(2) SEQ ID NO.4 and 6；
(3) SEQ ID NO.7 and 9.
In ET-PCR system, being similar to traditional round pcr, ET-PCR technology only identifies the Liang Ge district of target sequence
Territory, realizes amplification and detection in same reaction tube simultaneously.In the present invention, ET-PCR technology is proved to be a kind of amplification speed
Degree is fast, be quick on the draw, detection technique that specificity is high.As a kind of new real-time nucleic acid detection technique, it is possible to be widely used in
Biological, clinical and association area, it is achieved nucleic acid real-time, quickly and Multiple detection analysis.
Accompanying drawing explanation
Fig. 1 .ET-PCR expands principle schematic；
Fig. 2 .ET-PCR design of primers position view；
Fig. 3 .ET-PCR feasibility the result collection of illustrative plates；
Fig. 4 .ET-PCR detects single target sequence result collection of illustrative plates；
Fig. 5 .ET-PCR Evaluation on specificity collection of illustrative plates；
Fig. 6 .ET-PCR detects multisequencing result collection of illustrative plates simultaneously.
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.
Real-time nucleic acid detection technique (the Endonuclease depending on PCR amplification of restricted enzyme mediation
Restriction-Mediated Real-Time Polymerase Chain Reaction, ET-PCR) principle:
The present invention is on the basis of common PCR primers, and forward primer (F) and reverse primer (R) to PCR improve,
5` end at F and R adds one section of cleavage sequence (5`-CGCG-3`, named Ss), and this sequence can be by restricted enzyme
(BstUI) specific identification, the primer after improvement is referred to as EF and ER.Additionally, in order to protect restriction enzyme site, an extra alkali
Base T is added into the 5` end of cleavage sequence.In order to build EF and ER primer, special recognition sequence (5`-TCGCG-3`) can
It is added to the 5` end of any F or R primer.When synthesizing EF and the ER primer for different target sequences, at the 5` end of EF and ER
The fluorophor that labelling is different, the central marker quencher of primer, this quencher can specific quencher 5` end fluorescence
Group (see Figure 1A).
In the amplification reaction system of ET-PCR, it is similar to regular-PCR amplification system, uses EF or ER to substituted for original
F or R primer, ET-PCR amplification step is similar to the real-time amplification PCR reaction of two-step method.In order to illustrate to expand principle, the present invention
As a example by EF and R, only illustrate the amplification principle of ET-PCR.First, double-strandednucleic acid unwinds into single-chain nucleic acid at 95 DEG C, at 60 DEG C
Time, primer EF and R is annealed to single stranded nucleic acid template (step 1,2,3), and the primer being attached in single-stranded template is at nucleic acid amplification enzyme
Effect under start extend (step 4)；Secondly, unwinding of the double-stranded products experience new round in step 4, annealing, amplification, newly
The 5` end of product forms the special complementary series that recognition sequence (5`-TCGCG-3`) is template (step 5 and 6), this double-strand energy
Enough by the restricted enzyme BstUI specific recognition in reaction system, and cut this double-stranded sequence (5`-TCG-3` and mutually
Complementary series), fluorophor and quencher are separated by this process, cause fluorescence signal to discharge (step 7)；Product in step 7
Entering a cyclic amplification, this process includes unwinding of a new round, annealing, amplification, releasing of specific cutting and fluorescence signal
Put (step 7,8,9,10)；Additionally, from the amplified production of step 3, its course of reaction is similar to the product in step 2, this mistake
Journey also includes unwinding of double-stranded products, and primer annealing is attached to template, the amplification of fragment, specific cutting and fluorescence signal
Release (step 11,12,13,14,15) (see Figure 1B).
Can be detected by fluorescence detector by the fluorescence signal of ET-PCR amplification release, therefore, this technology realizes
Same reaction system carries out product amplification and detection, i.e. in real time PCR detection simultaneously.Additionally, for different target sequence designs
The different fluorophor of EF or ER labelling, course of reaction discharges different fluorescence signals, each fluorescence signal represents
The target sequence of each specific detection, therefore, ET-PCR achieves Multiple detection.
After ET-PCR expands, the detection method of two kinds of main flows is used for ET-PCR amplification and differentiates, first, and ET-PCR skill
The advantage of art is can carry out in real time and Multiple detection, and therefore real-time fluorescence detection is used for differentiating ET-PCR amplification.Secondly,
ET-PCR product, therefore, can produce owing to ET-PCR is similar to regular-PCR amplification by detecting amplicon after sepharose electrophoresis
Thing is also similar to the product of regular-PCR, and the amplicon of different size fragment represents different targets, can be with entering one by this technology
The accuracy of the checking ET-PCR amplification of step and specificity.
MATERIALS METHODS involved by the embodiment of the present invention
1. design of primers is according to the specific gene ply (GenBank accession SPR1793) of pneumonia streptococcus, golden yellow
Staphylococcic specific gene nuc (GenBank accession V0128.1) and the specific gene Ef0027 of enterococcus faecalis
(GenBank accessionAF454824) designs ET-PCR amplimer, and ply gene is present in all of streptococcus pneumoniae
In, its specificity is good, can strain the most close with other for streptococcus pneumoniae and Pseudomonas be distinguished.Nuc gene is present in
In all of golden yellow staphylococcus, its specificity is good, can be by staphylococcus aureus and other the most close strain and bacterium
Genus distinguishes.Ef0027 gene is present in all of enterococcus faecalis, and its specificity is good, can be close with other by enterococcus faecalis
Close strain and Pseudomonas distinguish.According to the amplification principle (Fig. 1) of ET-PCR technology, Primer Premier 5.0 is utilized to draw
Thing design software design ET-PCR primer, and the specific primer of acquisition is carried out in ncbi database sequence alignment analysis,
To get rid of, primer is that may be present with other species sequence non-specific to be mated, and the final three set ET-PCR obtained after optimizing expand
Primer.The position of design of primers and direction see that Fig. 2, sequence are shown in Table 1.
Table 1, primer sequence and modification
A.F:forward (forward primer)；R:reverse (reverse primer)；EF:the new Forward (novel forward
Primer)；S.p:S.pneumonia (streptococcus pneumoniae)；S.a:S.aureus (staphylococcus aureus)；E.f:E.faecalis
(enterococcus faecalis).
B. fluorophor Hex and the mark position of quenching group BHQ1:
5'-Hex-TCGCG-TTCT(BHQ1)GCAGAGCGTCCTTTGGTCT-3'
C. fluorophor Cy5 and the mark position of quenching group BHQ2:
5'-Cy5-TCGCG-TGT(BHQ2)ACAAAGGTCAACCAATGACA-3'
D. fluorophor FAM and the mark position of quenching group BHQ1:
5'-FAM-TCGCG-GCCACT(BHQ1)ATTTCTCGGACAGC-3'
2. in the present invention use reagent E T-PCR reaction system mixture Premix (Takara Bio, Inc., Otsu,
Japan, dNTP and buffer) it is purchased from Takara bio tech ltd, Beijing.DNA extraction kit (QIAamp DNA
minikits；Qiagen, Hilden, Germany) it is purchased from Qiagen company of Germany.PCR reaction system mixture M IX (Taq
Archaeal dna polymerase, dNTP and buffer) it is purchased from Beijing CoWin Bioscience Co., Ltd..DL50DNA Marker and
DL50DNA Marker is purchased from precious biological engineering (Dalian) company limited.Remaining reagent is commercially available typing net product.
3. the key instrument using and relating in present invention experiment: real-time fluorescence detector is Rotor-Gene Q Real-
Time System (Qiagen), Germany's Qiagen product；Electrophoresis equipment is that Jun Yi east, Beijing electrophoresis equipment company limited produces
Product；PCR instrument is Sensoquest Labcycler, Germany's Sensoquest product；Gel imaging system is Bio-Rad Gel
Dox XR, U.S.'s Bio-Rad product.
4. the extraction of bacterial genomes in the present invention, streptococcus pneumoniae, staphylococcus aureus, enterococcus faecalis and other kinds
The extraction of the genomic DNA of class bacterium.
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 concentration of group DNA and purity, streptococcus pneumoniae, staphylococcus aureus and enterococcus faecalis genomic DNA GE buffer are continuous
Dilution (from 25ng, 2.5ng, 250pg, 25pg, 2.5pg, 250fg, 25fg, to 2.5fg/ microlitre).Various genomic DNAs are the fewest
Amount subpackage ,-20 DEG C save backup.The DNA of the streptococcus pneumoniae of serial dilution, staphylococcus aureus and enterococcus faecalis is used for
The exploration of ET-PCR method and the foundation of method sensitivity.With common pathogenic bacterium and conditioned pathogen DNA (single increasing Liszt
Bacterium, vibrio cholera, vibrio parahaemolyticus, Vibrio vulnificus, enterococcus faecalis, campylobacter jejuni, Bacillus cereus, intestinal are pathogenic greatly
Enterobacteria, enterotoxigenic E.Coli, enteroinvasive E.Coli etc.) it is that template evaluates the special of ET-PCR detection reaction system
Property.Bacterial strain information is shown in Table 2.
Embodiment 1 standard ET-PCR is reacted
1. standard ET-PCR reaction system:
2. standard ET-PCR reaction Amplification
Amplification measurement result on real-time fluorescence quantitative PCR instrument:
Real-time is carried out anti-in fluorescence detector (Rotor-Gene Q Real Time System, Qiagen)
Should, after amplification terminates, take same analysis of threshold data after deduction background fluorescence signal, determine the Ct (cycle of this reaction
Threshold) value.
The feasibility of 3.ET-PCR amplified reaction
(1) fluorescence detection: under the ET-PCR reaction condition of standard, ET-PCR produces a large amount of fluorescence when DNA amplification
Signal, this signal can be detected by real-time fluorescence detector, and produce stable fluorescence signal, the intensity of its signal and amplicon
Product become positive correlation, see Fig. 3 A (for the real-time amplification signal of streptococcus pneumoniae specific gene ply, 1 represents positive amplification,
The positive DNA added is 2.5ng, and 2 represent negative control, and the distilled water of 1 microlitre replaces positive DNA to join in reaction system)；
(for the real-time amplification signal of staphylococcus aureus specific gene nuc, 1 represents positive amplification to 3C, and the positive DNA of addition is
2.5ng, 2 represent negative controls, and the distilled water of 1 microlitre replaces positive DNA to join in reaction system) and 3E (for excrement intestinal ball
The real-time amplification signal of bacterium specific gene Ef0027,1 represents positive amplification, and the positive DNA of addition is 2.5ng, and 2 represent negative right
According to, the distilled water of 1 microlitre replaces positive DNA to join in reaction system).
(2) electrophoresis assays: owing to the amplified production of ET-PCR is similar to the product of regular-PCR, therefore, its product can be used
Further confirmed by electrophoresis detection.The amplicon of different size fragment represents different targets.See Fig. 3 B (for
The electrophoresis detection of the ET-PCR amplified production of streptococcus pneumoniae specific gene ply, swimming lane 1 is DNA marker, and swimming lane 2 is positive
Amplification, it is contemplated that being observed of size, 3 is negative control)；3D (the ET-PCR for staphylococcus aureus specific gene nuc
The electrophoresis detection of amplified production, swimming lane 1 is DNA marker, and swimming lane 2 is positive amplification, it is contemplated that being observed of size, and 3 is cloudy
Property control) and 3F (for the electrophoresis detection of the ET-PCR amplified production of enterococcus faecalis specific gene Ef0027, swimming lane 1 is DNA
Marker, swimming lane 2 is positive amplification, it is contemplated that being observed of size, and 3 is negative control).
4.ET-PCR detects the sensitivity of single sample
Under the conditions of standards system, doubling dilution template DAN from 25ng, 2.5ng, 250pg, 25pg, 2.5pg, 250fg,
25fg and 2.5fg/ microlitre), 1 microlitre each concentration template adds in standard reaction system, is detected by real-time fluorescence, is stablized
Fluoroscopic examination figure, see Fig. 4.The Monitoring lower-cut of ET-PCR independent detection streptococcus pneumoniae be 250fg DNA/ reaction tube (see
Fig. 4 A and 4B), 4A (FAM passage) is to detect fluorescence signal curve in real time, and 4B is corresponding to 4A curve, for quantitative analysis
Standard curve, therefore, ET-PCR technology can be used for streptococcus pneumoniae quantitative analysis.When the base of streptococcus pneumoniae in reaction system
When being reduced to below 250fg because of group template amount, ET-PCR reaction occurs without positive amplification.According to the result of interpretation, ET-PCR skill
The detection of art independence streptococcus pneumoniae is limited to 250fg/ reaction tube.The sensitivity relatively regular-PCR of ET-PCR detection streptococcus pneumoniae
Superior (being shown in Table 3).The Monitoring lower-cut of ET-PCR independent detection staphylococcus aureus be 250fg DNA/ reaction tube (see Fig. 4 C and
4D), 4C (Cy5 passage) is to detect fluorescence signal curve in real time, and 4D is corresponding to 4C curve, bent for the standard of quantitative analysis
Line, therefore, ET-PCR technology can be used for staphylococcus aureus quantitative analysis.When staphylococcus aureus in reaction system
When genomic templates amount is reduced to below 250fg, ET-PCR reaction occurs without positive amplification.According to the result of interpretation, ET-PCR
The detection of technology independence staphylococcus aureus is limited to 250fg/ reaction tube.The sensitivity of ET-PCR detection streptococcus pneumoniae is more general
Logical PCR superior (being shown in Table 3).The Monitoring lower-cut of ET-PCR independent detection enterococcus faecalis be 250fg DNA/ reaction tube (see Fig. 4 E and
4F), 4E (Hex passage) is to detect fluorescence signal curve in real time, and 4F is corresponding to 4E curve, bent for the standard of quantitative analysis
Line, therefore, ET-PCR technology can be used for enterococcus faecalis quantitative analysis.When the genomic templates amount of enterococcus faecalis in reaction system
When being reduced to below 250fg, ET-PCR reaction occurs without positive amplification.According to the result of interpretation, ET-PCR technology independent detection
The staphylococcic Monitoring lower-cut of excrement intestinal is 250fg/ reaction tube.The sensitivity of ET-PCR detection streptococcus pneumoniae is excellent compared with regular-PCR
More (being shown in Table 3).
5.ET-PCR detects Evaluation on specificity:
With common pathogenic bacterium and conditioned pathogen DNA (streptococcus pneumoniae, staphylococcus aureus, enterococcus faecalis, cholera
Vibrio, vibrio parahaemolyticus, Vibrio vulnificus, campylobacter jejuni, Bacillus cereus, enteropathogenic E.Coli, enterotoxigenic
Escherichia coli, enteroinvasive E.Coli etc.) be template evaluate ET-PCR technology specificity (bacterial strain information refers to table 2).ET-
Round pcr can accurately differentiate streptococcus pneumoniae, staphylococcus aureus and enterococcus faecalis, and the specificity of ET-PCR method is described
Well, Fig. 5 is seen.Fig. 5 A, signal 1-6 come from streptococcus pneumoniae genomic templates amplification (FAM fluorescence channel).Fig. 5 B, signal
7-12 comes from staphylococcus aureus gene group template amplification (Cy5 passage).Fig. 5 C, signal 13-14 come from enterococcus faecalis
Genomic templates amplification (Hex passage).Reaction 15-35 is non-streptococcus pneumoniae, non-staphylococcus aureus, non-enterococcus faecalis,
Reaction 36 is negative control.Result shows, the detection target sequence that ET-PCR can be correct, and by fluorescence signal by difference target sequence
Row distinguish.
Table 2 bacterial strain information
aU, unidentified serotype (does not identifies serotype)；ATCC,American Type Culture
Collection (American Type Culture collection warehousing)；NCTC, National Collection of Type Cultures (English
State's typical strain collects preservation 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).
Embodiment 2: multiple ET-PCR reacts
The most multiple ET-PCR reaction system:
The most multiple ET-PCR Amplification
Amplification measurement result on real-time fluorescence quantitative PCR instrument:
Real-time is carried out anti-in fluorescence detector (Rotor-Gene Q Real Time System, Qiagen)
Should, after amplification terminates, take same analysis of threshold data after deduction background fluorescence signal, determine the Ct (cycle of this reaction
Threshold) value.
The Multiple detection ability of 3.ET-PCR
In order to obtain stable multi-fluorescence test pattern, standard ET-PCR system is optimized adaptation Multiple detection,
Set up Multiple detection system.Under Multiple detection system, we are simultaneously introduced three set primers and right in multiple reaction mixture
The template answered, is detected by fluorescence detector, obtains stable multi-fluorescence detection figure, sees Fig. 6.ET-PCR reaction system can
Carry out the detection of three target sequences, three targets in same reaction tube can be distinguished by correct simultaneously, it was demonstrated that ET-
The Multiple detection ability of round pcr, can be by the detection of ET-PCR Technique Popularizing to many target sequences.
The Multiple detection sensitivity of 4.ET-PCR
When application ET-PCR detects streptococcus pneumoniae, staphylococcus aureus and enterococcus faecalis simultaneously, the amplification of its detection
Circulation (CT) and detection sensitivity do not have the change of matter with simple sequence detection.ET-PCR Multiple detection system is to streptococcus pneumoniae
Monitoring lower-cut be 250fg DNA/ reaction tube (see Fig. 6 A and 6B), 6A (FAM passage) be real-time detection fluorescence signal curve, 6B is
Corresponding to 6A curve, for the standard curve of quantitative analysis, therefore, ET-PCR Multiple detection technology can be used for pneumonia streptococcus
Bacterium quantitative analysis.When in reaction system, the genomic templates amount of streptococcus pneumoniae is reduced to below 250fg, ET-PCR reacts not
Positive amplification occurs.According to the result of interpretation, the Monitoring lower-cut of multiple ET-PCR technology for detection streptococcus pneumoniae is that 250fg/ is anti-
Ying Guan.Multiple ET-PCR detection streptococcus pneumoniae sensitivity is consistent with substance detection, but superior compared with regular-PCR, is shown in Table 3.ET-
PCR Multiple detection system is 250fg DNA/ reaction tube (see Fig. 6 C and 6D), 6C to the Monitoring lower-cut of staphylococcus aureus
(Cy5 passage) is real-time detection fluorescence signal curve, and 6D is corresponding to 6C curve, for the standard curve of quantitative analysis, because of
This, ET-PCR Multiple detection technology can be used for staphylococcus aureus quantitative analysis.When Staphylococcus aureus in reaction system
When the genomic templates amount of bacterium is reduced to below 250fg, ET-PCR reaction occurs without positive amplification.According to the result of interpretation, many
The Monitoring lower-cut of weight ET-PCR technology for detection staphylococcus aureus is 250fg/ reaction tube.The golden yellow Portugal of multiple ET-PCR detection
The sensitivity of grape coccus is consistent with substance detection, but superior compared with regular-PCR, is shown in Table 3.ET-PCR Multiple detection system is to excrement intestinal ball
The Monitoring lower-cut of bacterium be 250fg DNA/ reaction tube (see Fig. 6 E and 6F), 6E (Hex passage) be real-time detection fluorescence signal curve,
6F is corresponding to 6E curve, and for the standard curve of quantitative analysis, therefore, ET-PCR Multiple detection technology can be used for excrement intestinal
Coccus quantitative analysis.When in reaction system, the genomic templates amount of enterococcus faecalis is reduced to below 250fg, ET-PCR reacts not
Positive amplification occurs.According to the result of interpretation, the detection of multiple ET-PCR technology for detection enterococcus faecalis is limited to 250fg/ reaction tube.
The sensitivity of multiple ET-PCR detection enterococcus faecalis ball is consistent with substance detection, but superior compared with regular-PCR, is shown in Table 3.
The sensitivity assessment of table 3, ET-PCR technology and regular-PCR technology
S-ET-PCR, singlex ET-PCR (substance ET-PCR detection)；M-ET-PCR, multiplex ET-PCR is (many
Weight ET-PCR detection).
The common RCR of comparative examples reacts
1. common RCR reaction system:
2.PCR reaction condition:
After reaction terminates, take 10 μ l PCR primer on the agarose gel of 2.5%, carry out electrophoresis, and in gel imaging system
Observation analysis result on system.
Claims (10)
1. a polymerase chain amplification method, it is characterised in that adding for the forward primer of amplification or 5 ' ends of downstream primer
Adding a kind of restriction enzyme enzyme sequence, at the 5` end mark fluorescent group of described primer, the centre position labelling at primer can
The specifically quencher of fluorophor described in quencher 5` end, adds described restricted in described polymerase chain amplification system
Restriction endonuclease, and carry out polymerase chain amplified reaction.
Method the most according to claim 1, it is characterised in that making an addition to the described restriction enzyme that described primer 5 ' is held
5 ' ends of enzyme sequence add base T.
Method the most according to claim 2, it is characterised in that described restricted enzyme is BstUI, described restricted interior
Cutting enzyme sequence is CGCG.
4. according to the arbitrary described method of claim 1-3, it is characterised in that the combination of described fluorophor and quenching group can
Think: Hex and BHQ1, Cy5 and BHQ2 or FAM and BHQ1.
5. according to the arbitrary described method of claim 1-3, it is characterised in that described forward primer and downstream primer are following sequence
Row combination:
(1) SEQ ID NO.1 and 3；
(2) SEQ ID NO.4 and 6；Or
(3) SEQ ID NO.7 and 9.
6. claim 1-3 arbitrary described method application in multipurpose gene amplification, it is characterised in that in described amplification
In system, the fluorescent base that 5 ' end labellings at every pair of forward primer of gene amplification or downstream primer for various purposes are different
Group, and can the quencher of specifically fluorophor described in quencher 5` end in primer central marker.
Application the most according to claim 6, it is characterised in that described genes of interest to be amplified is 2 or 3, when waiting to expand
When the described genes of interest increased is 2, described forward primer and downstream primer are any two kinds of groups in following three combined sequence
Close；When described genes of interest to be amplified is 3, described forward primer and downstream primer include following all three sequence set
Close:
(1) SEQ ID NO.1 and 3；
(2) SEQ ID NO.4 and 6；
(3) SEQ ID NO.7 and 9.
8. a polymerase chain amplification kit, described test kit contains Taq enzyme, dNTP, restricted enzyme BstUI, amplification
Buffer, the forward primer being used for amplification and downstream primer, it is characterised in that 5 ' ends of described forward primer or downstream primer add
Added with restricted enzyme BstUI sequence C GCG, the 5 ' ends in described BstUI sequence are added with base T.
Test kit the most according to claim 8, it is characterised in that described forward primer and downstream primer are selected from one sequence
Combination:
(1) SEQ ID NO.1 and 3；
(2) SEQ ID NO.4 and 6；Or
(3) SEQ ID NO.7 and 9.
Test kit the most according to claim 8, it is characterised in that described forward primer and downstream primer are following three
Any two kinds of combinations in combined sequence；Or described forward primer and downstream primer include following all three combined sequence:
(1) SEQ ID NO.1 and 3；
(2) SEQ ID NO.4 and 6；
(3) SEQ ID NO.7 and 9.
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