CN109022550A - Detect the real-time fluorescence quantitative PCR nucleic acid sequence and kit of A type clostridium botulinum - Google Patents
Detect the real-time fluorescence quantitative PCR nucleic acid sequence and kit of A type clostridium botulinum Download PDFInfo
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- CN109022550A CN109022550A CN201810993779.XA CN201810993779A CN109022550A CN 109022550 A CN109022550 A CN 109022550A CN 201810993779 A CN201810993779 A CN 201810993779A CN 109022550 A CN109022550 A CN 109022550A
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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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
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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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The present invention provides a kind of real-time fluorescence quantitative PCR nucleic acid sequence and kit for detecting A type clostridium botulinum, nucleic acid sequence includes upstream primer A-F, downstream primer A-R and probe A-P, A type clostridium botulinum is detected with primer and probe provided by the invention, accuracy rate reaches 100%, when being expanded to other 25 kinds of pathogenic entero becterias and common bacteria DNA, it is showed no specific amplification curve appearance, specificity is good.It can determine that it is 5.04 × 10 to A type clostridium botulinum detection sensitivity according to the quantitative fluorescent PCR standard curve that recombinant plasmid standard items construct2Copy/μ l.Separately in fluorescence quantitative PCR detection system, the recombinant plasmid concentration of the toxin gene containing A is 102~109Within the scope of copy/μ l, there are good linear relationships between the Ct value and template concentration of amplified reaction.The present invention has the characteristics that be quick on the draw, specificity is high, quick, convenient, high-throughput, for the screening of A type clostridium botulinum, quarantines on a large scale and epidemiological survey provides a kind of effective detection means.
Description
Technical field
The present invention relates to the detections of the molecules of clostridium botulinum, and in particular to a kind of real-time fluorescence for detecting A type clostridium botulinum
Quantitative PCR nucleic acid sequence and kit.
Background technique
Clostridium botulinum (C. botulinum) be a kind of Gram-positive, anaerobism, amphitrichous, without pod membrane, gemma ellipse
Tubbiness bacillus.It was initially found from sausage in 1896 by Belgian scholar Van Ermengemcong, and confirms that it can be produced
Raw exotoxin-botulinum toxin (Botulinum toxin), causes humans and animals to be poisoned.This bacterium be widely present in soil, ocean,
In the sediment in lake and mammal, birds, the enteron aisle of fish, feed and food.Clostridium botulinum cannot be in organism living
Growth, when environment anaerobism, when having proper nutrition, can growth and breeding generate botulinum toxin, people and animals eat food containing this toxin,
When feed, poisoning symptom can occur.Clostridium botulinum is divided into 7 types of A-G according to creotoxin antigenicity, wherein A, B, E, F are
People's poisoning type, C, D type are animal and poultry poisoning type, and G type is seldom shown in.Find H-type again in baby's meat poisoning case recently.
Botulinum toxin is to be currently known most violent neurotoxin, and caused food poisoning is in province of more than ten of China, area
It is found, case fatality rate is high, endangers food safety.It is mainly separately cultured for the detection method of clostridium botulinum at present, is small
Mouse zoopery, for molecular biology methods such as the immunological methods such as the indirect ELISA of toxin and regular-PCRs, exist more
The disadvantages of time-consuming and laborious and sensitivity is not high.
Real-time fluorescence quantitative PCR (real-time fluorescent quantitative polymerase chain
Reaction, qPCR) technology, refer to and fluorophor is added in PCR reaction system, it is whole using fluorescence signal accumulation real-time monitoring
A PCR process, the method that quantitative analysis is carried out to unknown template finally by standard curve.Since qPCR is Real_time quantitative detection
Disease-producing pathogens gene nucleic acid, therefore it is more original more excellent than immunological methods such as chemiluminescence, time resolution, protein chips
Gesture.The present invention devises the primer and probe for A type clostridium botulinum toxin gene, and it is quick to establish real-time fluorescence quantitative PCR
Detection architecture provides a kind of new technological means for the quick detection of A type clostridium botulinum.
Summary of the invention
The purpose of the present invention is to provide it is a kind of detect A type clostridium botulinum real-time fluorescence quantitative PCR nucleic acid sequence and
Kit provides a kind of new technological means for the quick detection of A type clostridium botulinum.
In order to achieve the above object, the present invention is achieved through the following technical solutions:
The nucleic acid sequence of A type clostridium botulinum is detected, the nucleic acid sequence includes primer and probe, and the primer is detection meat poisoning shuttle
The upstream primer A-F and downstream primer A-R of bacterium A toxin gene, the probe are the probe A- for detecting clostridium botulinum A toxin gene
P, upstream primer A-F, downstream primer A-R and probe A-P nucleotide sequence respectively such as SEQ ID No:1, SEQ ID No:2 and
Shown in SEQ ID No:3, specifically:
Upstream primer A-F:5'- taataaaatatgggttattccagaaagag-3', (SEQ ID No:1)；
Downstream primer A-R:5'- tgttgaatcataatatgaaactggaact-3', (SEQ ID No:2)；
Probe A-P:5'- tcctgaagaaggagatttaaatccaccaccag -3', (SEQ ID No:3)；The probe A-
The end the 5' label reporter fluorescence group end FAM, 3' of P marks quenching fluorescence group BHQ1.The target patch segment length of above-mentioned primer amplification
Degree is 111bp.
The present invention also provides a kind of real-time fluorescence quantitative PCR kits for detecting A type clostridium botulinum, including following components:
(1) qPCR reaction system composition, including upstream primer A-F, such as sequence SEQ as shown in sequence SEQ ID No:1
The end 5' of downstream primer A-R shown in ID No:2 and the probe A-P as shown in sequence SEQ ID No:3, middle probe A-P mark
Remember that the reporter fluorescence group end FAM, 3' marks quenching fluorescence group BHQ1.
(2) negative control, using TE buffer, the TE buffer is 10mMTris-HCl, 1mM EDTA.
(3) positive control, A type clostridium botulinum recombinant plasmid standard items.
(4) dilution buffer.Select the TransNGS of Quan Shijin biotech firmTM Library Dilution Buffer。
Further, above-mentioned qPCR reaction system composition further includes reagent and enzyme required for qPCR reacts, it is preferable that institute
Stating qPCR reaction system further includes qPCR SuperMix, the qPCR SuperMix containing dNTPs, Taq archaeal dna polymerase and
PCR reaction buffer.QPCR SuperMix is purchased from Quan Shijin biotech firm.
The present invention also provides detection A type clostridium botulinum type real-time fluorescence quantitative PCR detection methods, comprising the following steps:
(1) sample to be tested bacterial genomes DNA is extracted；
The extracting of bacterial genomes DNA uses the kit of Promega Technology Co., Ltd., is operated to specifications, -20
It DEG C saves backup.The method well known in the prior art for extracting DNA can also be used to extract.
(2) qPCR amplification is carried out to the sample DNA of extraction；Wherein drawn using the upstream as shown in sequence SEQ ID No:1
Object A-F, the downstream primer A-R as shown in sequence SEQ ID No:2 and the probe A-P as shown in sequence SEQ ID No:3, it is described
The end the 5' label reporter fluorescence group end FAM, 3' of probe A-P marks quenching fluorescence group BHQ1.
1 qPCR reaction system of table:
| Title | Final concentration | Sample-adding amount (μ l) |
| qPCR SuperMix | 2× | 12.5 |
| Primer A-F | 10µM | 0.25 |
| Primer A-R | 10µM | 0.25 |
| Probe A-P | 10µM | 0.5 |
| Template DNA | ≥103Copy/μ l | 0.5 |
| ddH2O | 10.5 | |
| It amounts to | 25 |
Reaction condition are as follows: 95 DEG C of initial denaturations 2min, 95 DEG C of denaturation 10s, 60 DEG C of extension 30s, totally 45 recycle.
(3) result judgement:
The setting of interpretation of result condition: threshold value setting principle is just above the highest of normal negative control amplification curve with threshold line
Point.Value≤35 Ct, and apparent amplification curve occur is the positive, shows that there are A type clostridium botulinums in sample；Without Ct value, and
Without amplification curve, for feminine gender.If value > 35 sample Ct, it is proposed that reform, otherwise it is the positive that reforming result without Ct value, which is feminine gender,.
Beneficial effects of the present invention:
1 primer and probe provided by the present invention can be used for the Qualitative and quantitative analysis of A type clostridium botulinum, be quick on the draw, specifically
Property is good.
2 can simultaneously detect a large amount of samples, can be observed in 2h as a result, having quick, sensitive, convenient, high pass
The features such as amount, provides a kind of effective detection hand for the screening of A type clostridium botulinum, extensive quarantine and epidemiological survey
Section.
Detailed description of the invention
Fig. 1 is A toxin gene recombinant plasmid standard items fluorescence signal figure.
Fig. 2 is the corresponding standard curve of A toxin gene recombinant plasmid standard items logarithm-Ct.
Specific embodiment
The present invention is further elaborated combined with specific embodiments below, but these embodiments are only limitted to illustrate the present invention
Rather than it limits the scope of the invention.
Experimental method in following embodiment is conventional method unless otherwise instructed.
Material therefor, reagent etc. are commercially available without special instruction in following embodiment.
Embodiment 1 detects the design of the primer special and probe of A type clostridium botulinum
According to the Clostridium botulinum type A toxin gene sequence announced on GenBank, using CLUSTLAW software to the A of clostridium botulinum
Toxin gene carries out multiple alignment, and the conservative region for selecting its stable is directed to this sequence design as detection target sequence
Pair of primers and a probe, wherein the sequence of primer A-F is as shown in SEQ ID No:1；The sequence of primer A-R such as SEQ ID
Shown in No:2；The sequence of probe A-P is as shown in SEQ ID No:3.The end 5' of probe A-P marks the end reporter fluorescence group FAM, 3'
Mark quenching fluorescence group BHQ1.After finally carrying out the specificity that blast compares determining primer and probe with GenBank, transfer to
Shanghai Sheng Gong Bioisystech Co., Ltd is synthesized.
Embodiment 2 detects the composition of the kit of the quantitative fluorescent PCR of A type clostridium botulinum
(1) qPCR reaction system composition, including upstream primer A-F, such as sequence SEQ as shown in sequence SEQ ID No:1
The end 5' of downstream primer A-R shown in ID No:2 and the probe A-P as shown in sequence SEQ ID No:3, middle probe A-P mark
Remember that the reporter fluorescence group end FAM, 3' marks quenching fluorescence group BHQ1；The qPCR reaction system further includes qPCR
SuperMix, the qPCR SuperMix contain dNTPs, Taq archaeal dna polymerase and PCR reaction buffer.qPCR SuperMix
Purchased from Quan Shijin biotech firm.
(2) negative control is formed as follows: 10mMTris-HCl, 1mM EDTA using TE buffer
(3) positive control, A type clostridium botulinum recombinant plasmid standard items.
(4) dilution buffer.Select the TransNGS of Quan Shijin biotech firmTM Library Dilution Buffer。
The building and preparation of 3 plasmid standard of embodiment
Using A-F and A-R as primer, PCR amplification is carried out, purpose product segment is connected to pMD-18T after cutting glue purification recycling
Carrier, converts JM109 competent cell, and screening positive clone is verified with PCR and final confirmation is sequenced.
The foundation of 4 real-time fluorescence quantitative PCR reaction system of embodiment
Use TransStart Probe qPCR SuperMix(Quan Shijin biotech firm) carry out qPCR nucleic acid amplification.Reactant
System establishes according to table 1, and using Rotor-Gene Q real-time fluorescence quantitative PCR instrument, amplification condition is 95 DEG C of initial denaturation 2min, and 95 DEG C
10s, 60 DEG C of extension 30s are denaturalized, totally 45 circulations, are determined as the positive for the amplification of value≤35 Ct.
The foundation of 5 standard curve of embodiment and Repeatability checking
Plasmid (Omega company) is extracted according to plasmid extraction kit specification, measures extracting plasmid with ultraviolet specrophotometer
Concentration, according to the molecular weight of plasmid by Plasmid samples concentration conversion be copy Particle density: in every μ l sample detect gene copy
× avogadro coustant × 10 number=concentration (ng/ μ l)-9/ (660 × recombinant plasmid base number).With dilution buffer (Quan Shijin
Biotech firm TransNGSTMLibrary Dilution Buffer.) above-mentioned plasmid is successively diluted to 1.0 × 102Copy/μ
L~1.0 × 109Totally 8 concentration gradients, each gradient standard items set 3 repetitions to copy/μ l, and negative control template is buffered with TE
Liquid carries out real-time fluorescence quantitative PCR detection.
When target gene recombinant plasmid concentration is 5.04 × 102When copy/μ l or more, fluorescence signal intensity is higher than inspection
Survey threshold value.When target gene recombinant plasmid concentration is less than 5.04 × 102When copy/μ l, do not occur amplification fluorescent signal, so
Method is 5.04 × 10 to the Monitoring lower-cut containing target gene recombinant plasmid2Copy/μ l.Separately in the PCR detection architecture, contain A
The recombinant plasmid concentration of toxin gene is 102~109Within the scope of copy/μ l, between the Ct value and template concentration of amplified reaction
There are good linear relationships, as depicted in figs. 1 and 2.
Embodiment 6 detects Evaluation on specificity
Method Evaluation on specificity, comprising the following steps:
(1) sample source
2 experimental strain of table
Separation strains (Isolated strain) are all from Inst of Infection Disease Prevention and Control, Chinese Diseases Prevention an's reality in table 2
It tests room and saves bacterium.
(2) bacterial genomes DNA is extracted；
The extracting of bacterial genomes DNA uses the kit of Promega company, is operated to specifications, -20 DEG C of preservations are standby
With.
(3) specific detection is tested
Using the DNA of bacteria obtained in step (1) and step (2) as template, water is blank control, carries out qPCR reaction, reaction
System is established according to table 1, and using Rotor-Gene Q real-time fluorescence quantitative PCR instrument, amplification condition is 95 DEG C of initial denaturation 2min, and 95
DEG C denaturation 10s, 60 DEG C of extensions 30s, totally 45 recycle.
As the result is shown with the system to Bacillus cercus, clostridium difficile, clostridium tertium, C.perfringens, Soxhlet shuttle
Bacterium, enterobacter cloacae, Streptococcus suis, small intestine colon Yersinia, enterococcus faecalis, enterococcus faecium, Listeria monocytogenes, enterorrhagia
Property Escherichia coli (EHEC), enteropathogenic E.Coli (EPEC), enteroaggrerative E.coli (EaggEC), enterotoxigenic large intestine
Bacillus (ETEC), enteroinvasive E.Coli (EIEC), campylobacter jejuni, comma bacillus, Salmonella typhi, hog cholera sramana
25 kinds of cause of diseases such as bacterium, Citrobacter freundii, Enterobacter sakazakii, Shigella flexneri, shigella sonnei, staphylococcus aureus
Bacterium genomic DNA does not generate specific amplification curve when being detected, and illustrates that the specificity of the detection architecture is good.
The preparation of 7 Artificial stool defecography sample of embodiment and detection
Healthy babies excrement is taken, 0.1 g/pipe is dispensed, totally 5 pipe.Wherein 100 μ lddH are added in a pipe2O as blank control,
Remaining 4 pipe 60 μ lddH of each addition2O boils 5 minutes, and 13000rpm is centrifuged 5 minutes, takes supernatant, every pipe sequentially adds 103~
106The 40 μ l of A type clostridium botulinum recombinant plasmid standard solution of doubling dilution.Each concentration sets 3 repetitions, carries out glimmering in real time
Fluorescent Quantitative PCR detection.
The results show that when target gene recombinant plasmid concentration is A type 1.7142 × 103It is glimmering when copy/μ l or more
Light signal strength is higher than detection threshold value.When target gene recombinant plasmid concentration is less than 1.7142 × 103Copy/μ l does not expand
Increase fluorescence signal, so method is 1.7142 × 10 to the Monitoring lower-cut containing target gene recombinant plasmid in Artificial stool defecography sample3
Copy/μ l.
Obviously, the above embodiment of the present invention be only to clearly illustrate example of the present invention, and not be pair
The restriction of embodiments of the present invention.For those of ordinary skill in the art, it can also do on the basis of the above description
Various forms of variations or variation out.Here all embodiments can not be exhaustive.It is all to belong to technology of the invention
The obvious changes or variations that scheme is extended out are still in the scope of protection of the present invention.
Sequence table
<110>Inst of Infection Disease Prevention and Control, Chinese Diseases Prevention an
<120>the real-time fluorescence quantitative PCR nucleic acid sequence and kit of A type clostridium botulinum are detected
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 29
<212> DNA
<213>primer A-F (artificial sequence)
<400> 1
taataaaata tgggttattc cagaaagag 29
<210> 2
<211> 28
<212> DNA
<213>primer A-R (artificial sequence)
<400> 2
tgttgaatca taatatgaaa ctggaact 28
<210> 3
<211> 32
<212> DNA
<213>probe A-P (artificial sequence)
<400> 3
tcctgaagaa ggagatttaa atccaccacc ag 32
Claims (10)
1. detecting the nucleic acid sequence of A type clostridium botulinum, the nucleic acid sequence includes primer and probe, it is characterized in that the primer is
The upstream primer A-F and downstream primer A-R of clostridium botulinum A toxin gene are detected, the probe is detection clostridium botulinum A toxin base
The probe A-P of cause, upstream primer A-F, downstream primer A-R and probe A-P nucleotide sequence respectively as sequence SEQ ID No:1,
Shown in SEQ ID No:2 and SEQ ID No:3, the end the 5' label reporter fluorescence group end FAM, the 3' label of the probe A-P is quenched
Go out fluorophor BHQ1.
2. the nucleic acid sequence of detection A type clostridium botulinum according to claim 1, it is characterized in that the target fragment of primer amplification
Length is 111bp.
3. a kind of kit for detecting A type clostridium botulinum, it is characterized in that the kit includes:
(1) qPCR reaction system composition, including upstream primer A-F, such as sequence SEQ as shown in sequence SEQ ID No:1
The end 5' of downstream primer A-R shown in ID No:2 and the probe A-P as shown in sequence SEQ ID No:3, the probe A-P mark
Remember that the reporter fluorescence group end FAM, 3' marks quenching fluorescence group BHQ1；
(2) negative control；
(3) positive control.
4. kit according to claim 3, it is characterized in that the qPCR reaction system composition further includes qPCR
SuperMix, the qPCR SuperMix contain dNTPs, Taq archaeal dna polymerase and PCR reaction buffer.
5. kit according to claim 3 or 4, it is characterized in that the negative control is TE buffer, TE buffer
For 10mMTris-HCl, 1mM EDTA.
6. kit according to claim 3 or 4, it is characterized in that the positive control is A type clostridium botulinum recombinant plasmid
Standard items.
7. kit according to claim 3 or 4, it is characterized in that the kit further includes dilution buffer.
8. a kind of detection A type clostridium botulinum type real-time fluorescence quantitative PCR detection method, it is characterized in that the following steps are included:
(1) sample to be tested bacterial genomes DNA is extracted；
(2) qPCR amplification is carried out to the sample DNA of extraction；Wherein use the upstream primer A- as shown in sequence SEQ ID No:1
F, the downstream primer A-R as shown in the sequence SEQ ID No:2 and probe A-P as shown in sequence SEQ ID No:3, the probe
The end the 5' label reporter fluorescence group end FAM, 3' of A-P marks quenching fluorescence group BHQ1；
(3) PCR product is analyzed.
9. detection method according to claim 8, it is characterized in that 25 μ l of PCR reaction system is calculated as:
0.5 μ l of template DNA
10 μM of 0.5 μ l of primer A-F
10 μM of 0.5 μ l of primer A-R
10 μM of 0.5 μ l of probe A-P
2×qPCR SuperMix 12.5µl
ddH2O 10.5µl。
10. detection method according to claim 8 or claim 9, it is characterized in that PCR reaction condition are as follows: after 95 DEG C of 2min, 95 DEG C of 10s,
60 DEG C of 30s mono- circulations, totally 45 recycle.
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| CN112342279A (en) * | 2020-11-06 | 2021-02-09 | 中国科学院城市环境研究所 | Kit and method for simultaneously detecting specific genes of cyanobacteria bloom Ralstonia and prototheca toxin |
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| CN112342279A (en) * | 2020-11-06 | 2021-02-09 | 中国科学院城市环境研究所 | Kit and method for simultaneously detecting specific genes of cyanobacteria bloom Ralstonia and prototheca toxin |
| CN112342279B (en) * | 2020-11-06 | 2022-10-14 | 中国科学院城市环境研究所 | Kit and method for simultaneously detecting specific genes of cyanobacteria bloom Ralstonia and prototheca toxin |
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Application publication date: 20181218 |