CN101768634A - Composition for detecting O1 group vibrio cholerae, kit and detection method - Google Patents
Composition for detecting O1 group vibrio cholerae, kit and detection method Download PDFInfo
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Abstract
The invention relates to a composition for detecting O1 group vibrio cholerae, a kit and a detection method. The composition comprises a specific primer aiming at O antigen encoding genes rfb-O1 of toxigenic O1 group vibrio cholerae and nosotoxin genes ctxA secreting cholera toxin. The composition and the kit can delicately detect the toxigenic O1 group vibrio cholerae existing in a sample, wherein the detection lower limit is 1.0*102 copies per reaction system, and the detection sensitivity to a genome DNA (deoxyribonucleic acid) is 1.0*10-1 pg per reaction system.
Description
Technical field
The present invention relates to specific detection composition, test kit and the detection method of vibrio cholerae, particularly the specific detection of O1 group cholera vibrio composition, test kit and detection method.
Background technology
Cholera is the severe intestinal communicate illness that is caused by vibrio cholerae (Vibrio chlorae), and patient's clinical symptom often shows as a large amount of thin rice gruel shapes and drains, and causes rapid dehydration.As untimely treatment, can cause hypovolemic shock, oxypathy, even the causing death.Vibrio cholerae can be divided into a plurality of groups according to serotype, but not all group can both cause the popular of disease.Wherein, can cause that the cholera popular mainly contains two kinds of serotypes: O1 group and O139 group (Kaper JB, Morris JG, Levine MM.
Cholera.Clin.Microbiol.Rev, 1995,8:48-86).Therefore in the detection of vibrio cholerae, its serotype somatotype is the important component part that effectively detects.
With regard to its pathogenic detection index, discover that in early days the strong pathogenic effects of cholera mainly is because due to the diarrhoea that vibrio cholerae excretory Toxins,exo-, cholera causes.Therefore before the cholera that the O139 group cholera vibrio causes occurred, use Toxins,exo-, cholera (cholera toxin was CT) as its pathogenic standard of detection always.Historical preceding 7 cholera are popular all to be caused by the O1 group cholera vibrio.At the year ends 1992, the cholera that causes by O139 group occurred in India, and reached circumjacent state rapidly.After the O139 group cholera vibrio causes the cholera outburst, and along with to the going deep into of O139 group and the research of O1 group cholera vibrio, find to have some non-O139 groups also non-O1 group's vibrio cholerae also express Toxins,exo-, cholera CT, but can't cause the outburst of cholera.Simultaneously, O1 and O139 group cholera vibrio under cas fortuit are not expressed Toxins,exo-, cholera owing to lack the ctx gene, can not cause the outburst of cholera simultaneously yet.So the existence that will accurately detect pathogenic vibrio cholerae need detect two indexs simultaneously: Toxins,exo-, cholera CT and serotype index (Chakraborty S, Mukhopadhyay AK, Bhadra RK, et al.
Virulence Genes in environmental strains of Vibrio cholerae.Appl.Environ.Microbiol, 2000,66:4022-4028).
In view of cholera popular hazardness is bigger, make the detection of vibrio cholerae particularly important.But traditional vibrios detection method needs to cultivate with basic protein peptone nutrient solution earlier to increase bacterium, and culture of isolated could detect after going out bacterial strain, because its needs to strain culturing always are not suitable for this traditional detection method.Because vibrio cholerae is under harsh life condition, for example microbiotic, nutrient substance shortage, weather cold etc. will enter a kind of state of surviving but can not cultivate, and are unsuitable for traditional detection method (the Lyon WJ. based on strain culturing
TaqMan PCR for detection of Vibrio cholerae O1, O139, non-O1, And non-O139 in pure cultures, raw oysters, and synthetic seawater.Appl.Environ.Microbiol, 2001,67:4685-4693).And traditional vibrios detection method is consuming time longer, is unfavorable for the rapid detection of sample is handled.Because traditional vibrio cholerae detection method wastes time and energy, and is unfavorable for rapid detection, and relates to the cultivation of bacterium to possess certain danger for operator, the PCR reaction Fast Detection Technique at cholera and Toxins,exo-, cholera begins to occur in recent years.Detect for PCR, selection of primers is most important, directly the specificity of influence detection.If primer specificity is not enough, then may cause the false positive results that detects or the appearance of false negative result.In addition, the detection for producing malicious type vibrio cholerae detects a gene separately, often is not enough to judge that it is pathogenic.Fukushima in 2003 etc. utilize the SYBR fluorescence dye to set up multi-fluorescence PCR in real time detection architecture (Hiroshi F, Yoshie T, Ryotaro S.
Duplex real-time SYBR Green PCR assays Fordetectionof17speciesof Foodorwaterbornepathogensinstools.J.Clin.Microbiol, 2003,41:5134-5146), though shortened detection time and workload, owing to adopt the single target site of non-toxin gene to detect, when being used to detect pathogenic bacterium, there is certain false positive rate in its detected result.
Detection for vibrio cholerae in the samples such as food is prevention and the important step of monitoring cholera.And the detection of vibrio cholerae is also significant for making a definite diagnosis with the somatotype of bacterial strain of patient disease.
Summary of the invention
For the existence of specific detection sample O1 group cholera vibrio more exactly, the invention provides the composition of O1 group cholera vibrio in a kind of specific detection sample.Said composition comprises following primer:
Upstream primer: 5 '-ATATTgATCCgACAAgCCCAAATg-3 ' (sequence number: 1);
Downstream primer: 5 '-CgATgTTgAggCgAAgTTTAgg-3 ' (sequence number: 2).This primer utilizes said composition at O1 group cholera vibrio genome O antigen encoding gene rfb-O1 design and through the Auele Specific Primer that preliminary experiment filters out, and can use the existence of O1 group cholera vibrio in the PCR method specific detection sample.
Wherein, can further comprise probe: 5 '-fluorophor-CgCCgCAATACgAgCCCCAAggT (sequence number: 3)-fluorescent quenching group-3 '.Thereby make said composition applicable to fluorescence real-time quantitative PCR (real time PCR) detection method, wherein said fluorophor can be Cy5, and its fluorescent quenching group can be BHQ.Those of ordinary skills all know, also can use other fluorophors known in the art and the above-mentioned probe of fluorescent quenching group mark.
Further, can to detect the pathogenic vibrio cholerae of O1 group effectively in order satisfying, to judge simultaneously that this vibrio cholerae produces the ability of cholera enterotoxin again, an embodiment of the invention provide the composition that produces malicious type O1 group cholera vibrio in a kind of specific detection sample.Comprise following two pairs of primers in the said composition:
First pair of primer:
Upstream primer: 5 '-ATATTgATCCgACAAgCCCAAATg-3 ';
Downstream primer: 5 '-CgATgTTgAggCgAAgTTTAgg-3 ';
Second pair of primer:
Upstream primer: 5 '-ATgCCAAgAggACAgAgTgAgT-3 ' (sequence number: 4),
Downstream primer: 5 '-TCAAACTAATTgAggTggAAACATATCC-3 ' (sequence number: 5).Wherein the first pair of primer is the primer at genome O antigen encoding gene rfb-O1, and the second pair of primer is the primer at Toxins,exo-, cholera gene ctxA.Use said composition, both detected its genome O antigen encoding gene rfb-O1, again specific detection Toxins,exo-, cholera gene ctxA, so utilize the said composition can be by producing the existence of malicious type O1 group cholera vibrio in the PCR method test sample.
And, can further comprise probe in the above-mentioned composition: 5 '-fluorophor-CgCCgCAATACgAgCCCCAAggT-fluorescent quenching group-3 ' and 5 '-fluorophor-TCgTgCCTAACAAATCCCgTCTgAgTTCCT (sequence number: 6)-fluorescent quenching group-3 '.Thereby make that said composition and test kit can be owing to detect the existence of producing malicious type O1 group cholera vibrio by fluorescence real-time quantitative PCR.Wherein said fluorophor and fluorescent quenching group can be Cy5 and BHQ, or FAM and ECLIPSE.And, in the different probe of a reaction system, use different fluorophors.Those of ordinary skills can select suitable fluorophor and fluorescent quenching group voluntarily according to this area knowledge.
Another embodiment of the present invention provides the PCR detection kit of O1 group cholera vibrio in a kind of specific detection sample, comprising in PCR reaction system and the above-mentioned composition any one.Preferred described PCR reaction system is the real-time quantitative PCR reaction system.Wherein preferably use TaqMan probe system to carry out mark and detection.
The present invention further provides the method for O1 group cholera vibrio in a kind of test sample, use in the above-mentioned composition any one, or in the mentioned reagent box any one, this method comprises: extract the bacterial genomes DNA in the sample; Use any one primer and/or the probe that adopts as reaction in the above-mentioned composition, or use in the mentioned reagent box any one, detect the wherein segmental existence of purpose with the PCR method, thus the existence of O1 group cholera vibrio in the judgement sample.Can adopt those skilled in the art's PCR reaction conditions as can be known, be preferably: 95 ± 2 ℃ of pre-sex change surpass 1min, and 95 ± 2 ℃ of sex change surpass 5s, and 60 ± 2 ℃ extend beyond 10s, coreaction circulation above 30 extends beyond 2min at last more alternatively under 72 ± 2 ℃.When above-mentioned PCR method is the regular-PCR method, its reaction conditions more preferably: 94 ℃ of pre-sex change 2min, 94 ℃ of sex change 30s, 60 ℃ are extended 1min, 40 circulations of coreaction, last 72 ℃ are extended 7min.When above-mentioned PCR method is the real-time quantitative PCR method, its reaction conditions more preferably: 94 ℃ of pre-sex change 2min, 94 ℃ of sex change 10s, 60 ℃ are extended 30s, 45 circulations of coreaction.
The preferred fluorescence real-time quantitative PCR method of using detects in the above-mentioned PCR method.In above-mentioned fluorescence real-time quantitative PCR, preferably use the TaqMan probe method.
Sample that this detection architecture detects need not viable bacteria, just can make vibrio cholerae dead rapidly in the cracking process of the first step DNA extraction of sample preparation, has reduced the biological hazard to operator.Present method need not to increase bacterium, and entire reaction was finished in 2 hours, has saved manpower and time greatly, can be used for rapid detection.
And, utilize preferred implementations more of the present invention, for example, when using first pair and second pair of primer to carry out PCR as mentioned above simultaneously, the existence of the product poison type O1 group cholera vibrio in can the specific detection sample, that is, not only can be to the Typing of Vibrio Cholerae in the sample, and can judge that it produces the ability of Toxins,exo-, cholera, specificity is distinguished pathogenic vibrio cholerae and non-virulent vibrio cholerae.
In the embodiment that is more preferably, adopt specific probe of the present invention, utilize TaqMan real-time fluorescence PCR technology to detect, this method is because the level of automation height, and the detection product that need not to uncap has also reduced the chance of product pollution.
When utilizing composition of the present invention, test kit and/or method that the O1 group cholera vibrio is detected, use detection sensitivity to reach 10 simultaneously at the embodiment of the common double PCR system of above-mentioned two pairs of primers of two genes (rfb-O1 and ctxA)
2Copy every reaction system; Except using above-mentioned two pairs of primers, also used the TaqMan technology, the sensing range of embodiment that adds the real-time fluorescence double PCR detection architecture of above-mentioned two specific probes is 1 * 10
2To 1 * 10
9Copy; The common double PCR embodiment that adopts above-mentioned two pairs of primers reaches 1 * 10 to the detection sensitivity of O1 group cholera vibrio genomic dna
0The every reaction system of pg, and real-time fluorescence double PCR embodiment can reach 1 * 10 to the detection sensitivity of O1 group cholera vibrio genomic dna
-1The every reaction system of pg, and this detection is fast and convenient, and entire reaction was finished in 2 hours.
Description of drawings
Fig. 1 is the agarose gel electrophoresis figure of common double PCR detection sensitivity, and wherein M is a molecular weight standard, "-" negative contrast, swimming lane 10
0～10
10By being added plasmid DNA template copy number, each PCR reaction is respectively 10
0～10
10Copy every reaction system.
Fig. 2 is the amplification curve diagram of dual real-time fluorescence PCR detection sensitivity, and wherein the curve of sign " ¤ " is a ctxA fluoroscopic examination signal, and sweep is a rfb-O1 fluoroscopic examination signal, and the template amount of being reacted by left-to-right every PCR is respectively 10
9～10
2The copy plasmid DNA; The negative contrast of NTC signal wire.
Fig. 3 is common double PCR and the real-time fluorescence double PCR detection architecture detection figure to O1 group cholera vibrio genomic dna, wherein:
(A) M is a molecular criteria, the negative contrast of swimming lane " N ", swimming lane 10
-3～10
5For each PCR reaction institute gene mentation group dna profiling number is respectively 1 * 10
-3Pg to 1 * 10
5Pg;
(B) sign " ¤ " curve is a ctxA fluoroscopic examination signal, and sweep is a rfb-O1 fluoroscopic examination signal; Template amount by left-to-right every PCR reaction is respectively 10
9Copy plasmid positive control and 1 * 10
5Pg to 1 * 10
-1The every reaction system of pg genomic dna; The negative contrast of NTC signal wire.
Fig. 4 is the specific figure of explanation real-time fluorescence double PCR detection architecture to detection O1 group cholera vibrio genomic dna, and wherein sign " ¤ " curve is a ctxA fluoroscopic examination signal, and sweep is a rfb-O1 fluoroscopic examination signal; " P " is 1 * 10
9The every reaction system of copy plasmid positive control; " O1 " is O1 group cholera vibrio genomic dna 1 * 10
3The every reaction system of pg.The negative contrast of horizontal signal lines and 19 kinds of negative control bacterium genomic dnas.
Embodiment
For specific detection O1 group cholera vibrio, the contriver has selected the genome O antigen encoding gene rfb-O1 of O1 group cholera vibrio as target sequence, at this gene design Auele Specific Primer, and selected to have most specific a pair of primer (rfbO1-F and rfbO1-R see the following form 1) through preliminary experiment and be used for the present invention.
In an embodiment of the invention, select rfbO1-F and rfbO1-R, with the existence of O1 group cholera vibrio in the PCR method test sample as Auele Specific Primer.Use this method, but the genome O antigen encoding gene rfb-O1 of O1 group cholera vibrio in the specific detection sample, and then the existence of O1 group cholera vibrio in the specific detection sample, and just make vibrio cholerae death by cracking at the detection initial stage, can not threaten to experiment operator.The preferred fluorescent real time PCR method of using detects, and has saved the gel electrophoresis equal time of regular-PCR, makes its detection and analysis easier, has shortened the time of detecting.More preferably use the fluorescent real time PCR method of TaqMan technical mark, when using the TaqMan technology, its probe is preferably the rfbO1-probe: 5 '-fluorophor-CgCCgCAATACgAgCCCCAAggT-fluorescent quenching group-3 ', this probe is at this target sequence specific probe, has strengthened the specificity that detects.Its fluorophor and fluorescent quenching group can be respectively Cy5 and BHQ.
Simultaneously, produce the O1 group cholera vibrio of malicious type for specific detection, the contriver will mainly cause the serious symptoms of cholera again---the target sequence that the gene ctxA of the Toxins,exo-, cholera of diarrhoea detects as PCR, at this gene design Auele Specific Primer.Equally, through preliminary experiment screening, select and have specific a pair of primer most (ctxA-F and ctxA-R see the following form 1) is used for the present invention.
In yet another embodiment of the present invention, adopt primer rfbO1-F and rfbO1-R simultaneously, and primer ctxA-F and ctxA-R, with the existence of producing malicious type O1 group cholera vibrio in the method test sample of double PCR.Use this method, in one-time detection, both can detect the O1 group cholera vibrio, can judge that again this bacterium produces the ability of Toxins,exo-, cholera, the malicious type O1 group cholera vibrio of the morbific product of specific detection more accurately.The preferred fluorescent dual PCR in real time method that adopts detects, more preferably adopt the TaqMan technical mark, employed probe is: the rfbO1-probe: 5 '-fluorophor-CgCCgCAATACgAgCCCCAAggT-fluorescent quenching group-3 ', with the ctxA-probe: 5 '-fluorophor-TCgTgCCTAACAAATCCCgTCTgAgTTCCT-fluorescent quenching group-3 ', wherein use different fluorophors in two probes.Use this method, not only can detect the malicious type O1 group cholera vibrio of morbific product exactly, and its operation is easier, after the required reagent of adding begins reaction, do not need uncap once more application of sample and sampling, avoided the generation of polluting.And its result can directly read, and does not need the follow-up operation such as gel electrophoresis, has saved the time of detecting greatly.And these two probes are the specific probe at the corresponding gene design, have strengthened the specificity of its detection more.Wherein said fluorophor and fluorescent quenching group are to being: Cy5 and BHQ, and FAM and ECLIPSE.But be not limited to this two pairs of fluorophors and fluorescent quenching group, those skilled in the art can select suitable fluorophor and corresponding fluorescent quenching group thereof as required.
Describe preferred implementation of the present invention in detail below in conjunction with specific embodiment.It is pointed out that the embodiment that lists only is the purpose of exemplary illustration here, and it should be interpreted as any restriction the scope of the invention.Reagent such as use therein test kit, damping fluid only are the concrete reagent of selecting in this specific embodiment, should be understood that those skilled in the art can select the corresponding reagent of other companies to realize purpose of the present invention as required.
The design of primer and TaqMan probe is with synthetic
Utilize the Blast instrument of Genbank that the ctxA gene and the rfb-O1 gene order of O1 group cholera vibrio are analyzed, select its stable conservative region, and detect target sequence design primers and probe (seeing Table 1) at these two as the detection target sequence.Primer and probe are synthetic by the precious biotech firm in Japanese TaKaRa Dalian, wherein the detection probes 5 ' of ctxA gene is held flag F AM fluorophor, 3 ' end mark ELIPSE fluorescent quenching group, detection probes 5 ' the end mark Cy5 fluorophor of rfb-O1 gene, 3 ' end mark BHQ fluorescent quenching group.
Table 1 primer and probe sequence
Detect the preparation of bacterial classification
Employed O1 group cholera vibrio inactivated vaccine is so kind as to give by the national biological product calibrating in the present embodiment.Common pathogenic bacterium in 19 kinds of other pathogen enterobacterias or the hospital infection: pathogenic colon bacillus (Escherichia coli), Aeromonas sobria (Aeromonas sobria), Aeromonas caviae (Aeromonas caviae), shigella dysenteriae (Shigella dysenteriae), Shigellae (Shigella snnei) in Song, shigella flexneri (Shigella flexneri), Salmonella typhimurtum (Salmonella typhimurium), paratyphosus A bacillus (Salmonella paratyphi A), Salmonella enteritidis (Salmonella enteritidis), enteritis Yersinia (Yersinia enteroxolitica), Plesiomonas shigelloides (Plesiomonas shigelloides), flavobacterium meningitidis (Chromobacteriummeningosepticum), Acinetobacter bauamnnii (Acinetobacter bauamnnii), intestinal bacteria (Escherichia coli), streptococcus aureus (Staphylococcus aureus), Burkholderia cepacia (Burkholderia cepacia), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Salmonella typhi (Salmonella typhi), Klebsiella Pneumoniae (Klebsiella pneumoniae) is provided by Clinical Laboratory center Microbiological Lab of the Ministry of Health.
Embodiment 1 adopts the detection of regular-PCR method
The extracting of bacterial genomes DNA
Each bacterial genomes DNA of extracting (the MiniBEST test kit of TAKARA company).Utilize ultraviolet spectrophotometer to measure purity and the concentration of each bacterial genomes DNA, O1 group cholera vibrio genomic dna is diluted to 1 * 10 with the TE damping fluid
5Pg/ μ l to 1 * 10
-3The gradient standard substance of pg/ μ l, the genomic dna of common pathogenic bacterium is diluted to 5 * 10 with the TE damping fluid in all the other 19 kinds of other pathogen enterobacterias or the hospital infection
4The all a small amount of packing of pg/ μ l, range gene group DNA ,-20 ℃ of preservations are standby.
The structure of plasmid standard and preparation
Adopt the TA clone technology to make up the purpose plasmid.After confirming the amplified fragments molecular weight through electrophoresis, the PCR product of rfb-O1 and ctxA specific sequence is connected on the pMD18-T carrier (Japanese TaKaRa company), recombinant clone is cultivated back extracting plasmid (Omega plasmid extraction kit), adopt the RVM primer of pMD18-T carrier check order (U.S. Beckman Coulter company test kit), utilize CEQ8000 software to carry out sequential analysis, and compare with former aim sequence.The right-on purpose plasmid of sequence quantitatively after (Eppendorf ultraviolet spectrophotometer), is diluted to 10 with 1 * TE (pH8.0) TE damping fluid with ultraviolet spectrophotometry
10Copy/μ l is as storage liquid, and-20 ℃ of preservations are standby, and continuous 10 times are diluted to concentration 1.0 * 10 during use
-1Copy/μ l～1.0 * 10
10Between the copy/μ l, get the template of 10 μ l standard substance, make the plasmid number form of participating in each PCR reaction become 1 to copy 10 to as real-time quantitative PCR
10The Gradient distribution of copy.
Common double PCR reaction conditions
Common double PCR reaction system is 25 μ l, get 10 * magniferous damping fluid 2.5 μ l, dNTPs (each 2.5mmol/L) 2 μ l, each 0.5 μ l (final concentration is 500nM) of 25 μ mol/L primers, template DNA 10.0 μ l, Blend Taq plus archaeal dna polymerase 1.0U, adding aqua sterilisa to end-body is 25 μ l.The PCR loop parameter: 94 ℃ of pre-sex change 2min, 94 ℃ of sex change 30s, 60 ℃ are extended 1min, 40 circulations of coreaction, last 72 ℃ are extended 7min.Get 5ul PCR product after the amplification and on 3% sepharose, carry out electrophoresis, behind the electrophoresis with ethidium bromide (EB) dyeing, and on gel imaging system observation analysis, the results are shown in Figure 1 and Fig. 3 A.
Embodiment 2 adopts the fluorescence real-time quantitative PCR reaction method to detect
Adopt bacterial genomes DNA and the plasmid standard identical with embodiment, its difference is that the PCR method that is adopted is a fluorescence real-time quantitative PCR, and has used the above-mentioned probe that designs and synthesizes.
Real-time fluorescence double PCR reaction conditions
Reaction system is 25 μ l, get each dNTP 2.0 μ l of 10 * PCR damping fluid, 2.5 μ l, 2.5mmol/L, each 0.5 μ l of 25 μ mol/L primers, each 0.5 μ l of 5 μ mol/L probes, Blend Taq plus archaeal dna polymerase 1U, template DNA 10ul, adding aqua sterilisa to end-body is 25 μ l.The PCR loop parameter: 94 ℃ of pre-sex change 2min, 94 ℃ of sex change 10s, 60 ℃ are extended 30s, 45 circulations of coreaction.Finish the back by the identical conditions analytical data in amplification, determine the Ct value of each sample.
The evaluation of the detection sensitivity of embodiment 1 and 2 detection architecture
Get the every reaction system 10 of plasmid standard
0Be copied to 10
10Copy, or with 1 * 10
-3Pg/ μ l to 1 * 10
5The O1 group cholera vibrio bacterial genomes DNA of pg/ μ l gradient concentration is a template, has estimated the detection sensitivity of common double PCR and real-time double fluorescent TaqMan PCR detection architecture, and the result is referring to Fig. 1～4.
Among the embodiment 1 to the detection sensitivity of plasmid standard
Two plasmid gradient dilution templates are carried out common dual pcr amplification rear electrophoresis and are detected demonstration, and the plasmid DNA template number of ctxA gene and rfb-O1 gene is reduced to 1.0 * 10 in every reaction system
1When reaching following the copy, the purpose band do not occur on the common PCR reaction rear electrophoresis figure, negative control is not seen amplified band (Fig. 1) yet.
Among the embodiment 2 to the detection sensitivity of plasmid standard
Two plasmid gradient dilution templates carry out showing when real-time fluorescence PCR detects, when the plasmid DNA template number of reaction system ctxA gene and rfb-O1 gene is 10
2Copy when above, its fluorescence signal intensity Δ Rn is higher than and detects thresholding (Δ Rn=30), and its sensing range is 1.0 * 10
2～1.0 * 10
9Copy every reaction system (Fig. 2).All have good dependency between the Ct value of two target gene amplified reactions and the template, the rfb-O1 dependency is 0.998, and amplification efficiency reaches 1.02, and the ctxA dependency is 0.999, and amplification efficiency is 0.97.
Among the embodiment 1 to the detection sensitivity of bacterial genomes DNA
With O1 group cholera vibrio genomic dna gradient dilution to 1 * 10
5Pg to 1 * 10
-3Common dual pcr amplification rear electrophoresis detects and shows during the every reaction system of pg, when the genomic dna gradient dilution to every reaction system 1 * 10
-1During pg on the common double PCR reaction rear electrophoresis figure ctxA and rfb-O1 purpose amplified band (Fig. 3 A) do not appear.
Among the embodiment 2 to the detection sensitivity of bacterial genomes DNA
Carry out showing when fluorescent real time PCR detects with genomic dna, 1.0 * 10 with embodiment 1 identical gradient concentration
-1Pg and when above, its fluorescence signal intensity Δ Rn are higher than and detect thresholding (Δ Rn=30), and this result shows the detection sensitivity high 10 times (Fig. 3 B) of the detection sensitivity of real-time fluorescence double PCR than common double PCR.Linear relationship between the logarithmic value of homing method analysis different genes group dna profiling amount and its CT value is found regression curve and plasmid control curves.
The evaluation of detection specificity
Genomic dna with pathogenic bacterium common in above-mentioned 19 kinds of other pathogen enterobacterias or the hospital infection is the specificity that template has been estimated dual TaqMan real-time fluorescence PCR reaction system.
Utilize the system described in the embodiment 2 to 1 * 10 of O1 group cholera vibrio inactivated vaccine genomic dna
3Visible clear and definite amplification curve when the every reaction system of pg detects is to 5 * 10 of the pathogenic bacteria gene group DNA of above-mentioned 19 kinds of Non-cholera vibrios
5The every reaction system of pg does not all produce positive amplification curve when detecting, and illustrates between probe that we use and primer and the 19 kinds of negative control bacterium that we select not have cross reaction.With concentration known (1.0 * 10
9/ every reaction system) positive plasmid standard substance and negative control carry out blind Detecting, false positive and false negative result (Fig. 4) do not occur.
Although above in a preferred manner; by the specific embodiment exemplary illustration some embodiment of the present invention; but it will be understood by a person skilled in the art that; the present invention is not limited to top disclosed embodiment; but can make amendment to it according to the knowledge of the technical field of the invention, institute makes an amendment and can not exceed the scope of protection of present invention.For example, fluorescent real time PCR used in the present invention also can adopt fluorophor and the fluorescent quenching group mark substance of pointing out among the embodiment listed in the specification sheets in addition as required, as markers such as Tet, FAM, HEX, TAMRA, ROX, Cy3, TxRd, JOE; Or other mark systems outside the use Taqman technology, for example fluorescent probe labeling techniques such as MGB probe, molecular beacon MB probe, scorpion shape probe, fluorescence double cross probe; Or use saturable dyes such as unsaturated dyestuff such as chimeric method of dyestuff such as SYBR Green I and LC Green, as long as it has used specific primer sequence of the present invention, get final product the existence of detection by quantitative goal gene, and then detect the existence of O1 group cholera vibrio specifically.So the change that these those skilled in the art understood and the replacement of customary means also fall within the scope of protection of the present invention.Protection scope of the present invention should be limited by appending claims.
Sequence table
＜110〉Cai Jianping
＜120〉specific detection of O1 group cholera vibrio composition and test kit
<130>DF10-081415
<160>6
<210>1
<211>24
<212>DNA
＜213〉vibrio cholerae (Vibrio chlorae)
<400>atattgatcc?gacaagccca?aatg?24
<210>2
<211>22
<212>DNA
＜213〉vibrio cholerae (Vibrio chlorae)
<400>cgatgttgag?gcgaagttta?gg?22
<210>3
<211>23
<212>DNA
＜213〉vibrio cholerae (Vibrio chlorae)
<400>cgccgcaata?cgagccccaa?ggt?23
<210>4
<211>22
<212>DNA
＜213〉vibrio cholerae (Vibrio chlorae)
<400>atgccaagag?gacagagtga?gt?22
<210>5
<211>28
<212>DNA
＜213〉vibrio cholerae (Vibrio chlorae)
<400>tcaaactaat?tgaggtggaa?acatatcc?28
<210>6
<211>30
<212>DNA
＜213〉vibrio cholerae (Vibrio chlorae)
<400>tcgtgcctaa?caaatcccgt?ctgagttcct?30
Claims (10)
1. composition that is used for specific detection sample O1 group cholera vibrio, comprising following primer: upstream primer: 5 '-ATATTgATCCgACAAgCCCAAATg-3 ',
Downstream primer: 5 '-CgATgTTgAggCgAAgTTTAgg-3 '.
2. composition as claimed in claim 1 wherein further comprises probe:
5 '-fluorophor-CgCCgCAATACgAgCCCCAAggT-fluorescent quenching group-3 '.
3. composition as claimed in claim 2, wherein said fluorophor are Cy5, and described fluorescent quenching group is BHQ.
4. composition as claimed in claim 1 comprises further that wherein another is to primer:
Upstream primer: 5 '-ATgCCAAgAggACAgAgTgAgT-3 ',
Downstream primer: 5 '-TCAAACTAATTgAggTggAAACATATCC-3 '.
5. composition as claimed in claim 4 wherein further comprises probe:
5 '-fluorophor-CgCCgCAATACgAgCCCCAAggT-fluorescent quenching group-3 ' and
5 '-fluorophor-TCgTgCCTAACAAATCCCgTCTgAgTTCCT-fluorescent quenching group-3 ', and in two probes, use different fluorophors respectively.
6. composition as claimed in claim 5, wherein said two pairs of fluorophors and fluorescent quenching group are: Cy5 and BHQ, and FAM and ECLIPSE.
7. test kit that is used for specific detection sample O1 group cholera vibrio is comprising each described composition in PCR reaction system and the claim 1～6.
8. test kit as claimed in claim 7, wherein said PCR reaction system is the real-time quantitative PCR reaction system.
9. the method for O1 group cholera vibrio in the specific detection sample comprises:
Extract the bacterial genomes DNA in the sample;
Use each described composition in the claim 1～6, or use claim 7 or 8 described test kits, with the PCR method described bacterial genomes DNA that increases;
Result to the PCR reaction analyzes.
10. method as claimed in claim 9, the reaction conditions of wherein said PCR method is:
95 ± 2 ℃ of pre-sex change surpass 1min, and 95 ± 2 ℃ of sex change surpass 5s, and 60 ± 2 ℃ extend beyond 10s, and coreaction circulation above 30 extends beyond 2min at last under 72 ± 2 ℃; Or
95 ± 2 ℃ of pre-sex change surpass 1min, and 95 ± 2 ℃ of sex change surpass 5s, and 60 ± 2 ℃ extend beyond 10s, coreaction circulation above 30.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103160605A (en) * | 2013-04-08 | 2013-06-19 | 北京出入境检验检疫局检验检疫技术中心 | Real-time fluorescent PCR (polymerase chain reaction) detection kit for vibrio cholerae and detecting method thereof |
| CN105648054A (en) * | 2016-01-13 | 2016-06-08 | 江苏和创生物科技有限公司 | Vibrio cholerae fluorescence PCR detection kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100395350C (en) * | 2005-12-15 | 2008-06-18 | 深圳太太基因工程有限公司 | Prime and probe sequence for detecting nucleotide fregment of comma bacillus |
| CN100386442C (en) * | 2005-12-15 | 2008-05-07 | 深圳太太基因工程有限公司 | Prime and probe sequence for detecting nucleotide fregment of 01 Group comma bacillus |
| CN101113473A (en) * | 2007-06-07 | 2008-01-30 | 天津出入境检验检疫局动植物与食品检测中心 | Method for detecting food-derived pathogenic vibrio bacteria by composite fluorescence PCR technique |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103160605A (en) * | 2013-04-08 | 2013-06-19 | 北京出入境检验检疫局检验检疫技术中心 | Real-time fluorescent PCR (polymerase chain reaction) detection kit for vibrio cholerae and detecting method thereof |
| CN105648054A (en) * | 2016-01-13 | 2016-06-08 | 江苏和创生物科技有限公司 | Vibrio cholerae fluorescence PCR detection kit |
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