CN102719542B - Simultaneous amplification and testing reagent kit for VC (vibrio cholerae) - Google Patents

Simultaneous amplification and testing reagent kit for VC (vibrio cholerae) Download PDF

Info

Publication number
CN102719542B
CN102719542B CN201210203302.XA CN201210203302A CN102719542B CN 102719542 B CN102719542 B CN 102719542B CN 201210203302 A CN201210203302 A CN 201210203302A CN 102719542 B CN102719542 B CN 102719542B
Authority
CN
China
Prior art keywords
sequence
rna
mark
primer
liquid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201210203302.XA
Other languages
Chinese (zh)
Other versions
CN102719542A (en
Inventor
李豪
张长明
于明辉
居金良
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Rendu Biotechnology Co., Ltd
Original Assignee
SHANGHAI RENDU BIOTECHNOLOGY CO Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by SHANGHAI RENDU BIOTECHNOLOGY CO Ltd filed Critical SHANGHAI RENDU BIOTECHNOLOGY CO Ltd
Priority to CN201210203302.XA priority Critical patent/CN102719542B/en
Publication of CN102719542A publication Critical patent/CN102719542A/en
Application granted granted Critical
Publication of CN102719542B publication Critical patent/CN102719542B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • YGENERAL 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
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a simultaneous amplification and testing reagent kit for VC (vibrio cholerae). The simultaneous amplification and testing reagent kit for the VC comprises a capture probe, a VC testing primer T7, a VC testing primer nT7, a VC testing probe, an M-MLV reverse transcriptase, a T7 RNA (ribonucleic acid) polymerase and the like. The simultaneous amplification and testing reagent kit for the VC can test VC RNA in food, has the advantages of high specificity, high sensitivity (capable of reaching 100 CFU/ml), low contamination (an amplified product RNA is easy to degrade in the natural environment) and rapidness in testing (testing can be completed within 50 minutes conventionally), plays an important role in rapid testing of the VC and is wide in application prospect.

Description

The real-time fluorescence nucleic acid constant-temperature amplification detection kit of vibrio cholerae (VC)
Technical field
The present invention relates to the Measurement for Biotechnique of pathogenic bacterium, be specifically related to the primer, probe and the related kit that during the real-time fluorescence nucleic acid constant-temperature amplification of the vibrio cholerae (VC) of specificity target capture technique and the combination of real-time fluorescence nucleic acid constant-temperature amplification detection technique is detected, use.
Background technology
Vibrio cholerae (V.cholera) is the pathogenic agent of mankind's cholera, and cholera is a kind of ancient and popular one of deadly infectious disease widely.Once caused repeatedly and be very popular in the world, main manifestations is violent vomiting, diarrhoea, and dehydration, mortality ratio is very high, belongs to international quarantine transmissible disease.The mankind are unique susceptible persons of vibrio cholerae in natural situation, main by the water source or the especially fishery products peroral infection of food that pollute.The continuous aggravation globalizing along with international trade in recent years, the foreign trade increase year after year of China.And in the food of import, going wrong maximum is fresh and alive or freezing fishery products.For example, in 2005 and import fishery products in 2006, the recall rate of Vibrio parahemolyticus is 9% left and right, and the recall rate of vibrio cholerae is 2%~3%, and other vibrios detects also time as Vibrio vulnificus etc.And the food habits that formed for a long time of China, to some fishery products as lobster, eat something rare as pulling out freshwater mussel and salmon etc., thereby human consumer's health is caused to potential threat, so be necessary to strengthen the detection to Common Pathogenic vibrios in fishery products.
Vibrio cholerae comprises multiple virulence correlation factor, and wherein Toxins,exo-, cholera (CT) is its main virulence factor.In environment, isolated vibrio cholerae majority does not produce CT.01 group and 0139 group cholera vibrio are to cause the pathogenetic two kinds of main serotypes of disease, and in addition, non-zero 1, non-zero 139 the vibrio cholerae of part that studies show that in recent years also can produce CT.Therefore, the vibrio cholerae in isolation identification food, to protection people ' s health, ensures that the safety of fishery products has great importance.
Conventional vibrio cholerae detection method comprises at present: 1) traditional cultural method.Totally can be divided into 4 stages or step.Pre-bacterium, selective enrichment, separation, the biochemical identification of increasing.Need 4~7 days, just can draw clear and definite diagnostic result.2) immunolabelling technique.Utilize the specific reaction of antigen-antibody, and detect in conjunction with some biological chemistries or physics method, but the sensitivity of the method and specificity are all poor.3) molecular biology for detection.There is at present round pcr, real-time fluorescence quantitative PCR and LAMP method.Above several method easily causes the pollution of amplified material, often causes false positive or the false negative phenomenon of experimental result, and can not distinguish viable bacteria and dead bacterium, has hindered its large-scale promotion and has used.
Real-time fluorescence nucleic acid constant-temperature amplification detection technique (Simultaneous Amplification and Testing, abbreviate SAT) be the method for direct rapid detection RNA a kind of, compared with detecting the real-time fluorescence PCR of DNA, difference, the step of a reverse transcription reaction that the former detection system is many, nucleic acid amplification carries out (42 DEG C) at a temperature, without thermal cycling.Adopt M-MLV reversed transcriptive enzyme and T7RNA polymerase to carry out nucleic acid amplification, with respect to other nucleic acid amplification technologies, reaction inhibition still less, can effectively reduce false negative result.But it is different that SAT technology is applied faced problem in the detection of different sorts pathogenic bacterium, need to make a concrete analysis of the characteristic of pathogenic bacterium and carry out specialized designs.There is no at present the research report for the real-time fluorescence nucleic acid constant-temperature amplification detection technique of vibrio cholerae (VC).
Summary of the invention
Lower for solving the sensitivity of existing vibrio cholerae (VC) detection method, sense cycle is grown, is easily caused that the pollution of amplified material causes false positive or false negative and the higher problem of testing cost of experimental result, the invention provides that a kind of sense cycle is short, highly sensitive, high specific, low pollution, stable reaction and testing cost is low, the real-time fluorescence nucleic acid constant-temperature amplification detection technique of the vibrio cholerae (VC) that is easy to apply, comprise primer special, probe, test kit and use thereof.
The real-time fluorescence nucleic acid constant-temperature amplification detection kit of vibrio cholerae provided by the present invention (VC), include a capture probe, the a pair of VC for the DNA copy that produces VC target nucleic acids (VC RNA) under the effect of M-MLV ThermoScript II detects primer T7 and nT7, and a VC detection probes for the RNA copy specific combination that produces with the DNA copy according to described VC target nucleic acids (VC RNA) under t7 rna polymerase effect.
Described capture probe can be combined with target nucleic acids (VC RNA) sequence specific of the vibrio cholerae (VC) as shown in sequence in sequence table 1, in the time having in VC mark (VC IC RNA), its preferably also can with this VC interior label sequence specific combination, the nucleotide sequence of described capture probe is as shown in sequence in sequence table 2; Described VC detects primer and is made up of T7 primer and nT7 primer, and T7 primer sequence is as shown in sequence in sequence table 3, and nT7 primer sequence is as shown in sequence in sequence table 4; The nucleotide sequence of described VC detection probes is as shown in sequence in sequence table 5, and 5 ' holds flag F AM fluorophor, 3 ' end mark DABCYL quenching group.
Further, described test kit also includes M-MLV ThermoScript II and t7 rna polymerase, described M-MLV ThermoScript II and t7 rna polymerase are present in a SAT enzyme liquid, described capture probe is present in a nucleic acid extraction liquid, and described T7 primer, nT7 primer and VC detection probes are present in a VC and detect in liquid.
Further described test kit also includes mark and interior mark detection probes in VC again; In described VC, be designated as competitive in mark, can with capture probe specific binding, and use same pair of primers (T7 and nT7) with VC target Nucleotide (VC RNA), in VC, mark is by the VC IC RNA shown in sequence in sequence table 7; The nucleotide sequence of described interior mark detection probes is as shown in sequence in sequence table 6, and 5 ' holds mark HEX fluorophor, 3 ' end mark DABCYL quenching group, and described interior mark detection probes is present in VC detection liquid.
Further, described test kit comprises mark in lysate, nucleic acid extraction liquid, washings, VC reaction solution, VC detection liquid, SAT enzyme liquid, VC positive control, VC negative control and VC, wherein:
Lysate: liquid containing ammonium sulfate ((NH 4) 2sO 4) and HEPES;
Nucleic acid extraction liquid: containing capture probe and magnetic bead;
Washings: containing NaC1 and SDS;
VC reaction solution: containing dNTP and NTP;
VC detects liquid: containing T7 primer, nT7 primer, VC detection probes and interior mark detection probes;
SAT enzyme liquid: containing M-MLV ThermoScript II, T7 RNA polymerase;
VC positive control; Containing the in-vitro transcription RNA dilution of vibrio cholerae (VC) toxR gene;
VC negative control: do not contain vibrio cholerae (VC) target nucleic acids (VC RNA) sequence or do not contain the solution of vibrio cholerae, as physiological saline;
Mark in VC: containing mark RNA (sequence is as shown in sequence in sequence table 7) dilution in VC.
Concrete, in described test kit, in a reacton, above-mentioned all ingredients is composed as follows:
(1) lysate: HEPES 25-250mM, (NH 4) 2sO 45-50mM;
(2) nucleic acid extraction liquid: HEPES 50-400mM, EDTA 40-200mM, LiCl 400-2000mM, capture probe 1-50 μ M (being preferably 5-25 μ M), magnetic bead 50-500mg/L (being preferably 50-250mg/L);
(3) washings: HEPES 5-50mM, NaCl 50-500Mm, 1%SDS, EDTA 1-10mM;
(4) VC reaction solution: Tris 10-50mM, MgCl 210-40mM, dNTP 0.1-10mM (being preferably 0.5-5mM), NTP 1-20mM (being preferably 1-10mM), PVP40 1-10%, KCl 5-40mM;
(5) VC detects liquid: VC is detected to primer and VC detection probes and be dissolved in TE solution (mixed solution of 10mM Tris and 1mMEDTA) formulatedly, each primer and concentration and probe concentration all can in 5-15pmol/ reaction; Wherein T7 primer concentration is preferably 12.5pmol/ reaction, and nT7 primer concentration is preferably 12.5pmol/ reaction, and VC detection probe concentrations is preferably 5pmol/ reaction, and interior mark detection probe concentrations is preferably 5pmol/ reaction;
(6) SAT enzyme liquid: M-MLV ThermoScript II 400-4000U/ reaction (being preferably 500-1500U/ reaction), T7 RNA polymerase 200-2000U/ reaction (being preferably 500-1000U/ reaction), 2-10mM HEPES pH7.5, 10-100mMN-acetyl-L-cysteine, 0.04-0.4mM zinc acetate, 10-100mM trehalose, 40-200mMTris-HCl pH8.0, 40-200mM KC1, 0.01-0.5mM EDTA, 0.1-1% (v/v) Triton X-100 and 20-50% (v/v) glycerol,
(7) VC positive control; Containing 10 5-10 8the in-vitro transcription RNA dilution of copy/mL vibrio cholerae (VC) toxR;
(8) VC negative control: do not contain vibrio cholerae (VC) target nucleic acids (VC RNA) sequence or do not contain the solution of vibrio cholerae;
(9) mark in VC: containing 10 5-10 8copy/mL VC IC RNA (sequence is as shown in sequence in sequence table 7) in-vitro transcription RNA dilution.
Another kind is form more specifically, described test kit is that sample disposal unit and B box are that nucleic acid amplification detecting unit forms by A box, wherein A box is packed described lysate, described nucleic acid extraction liquid and described washings, and B box is packed described VC reaction solution, VC detects mark in liquid, SAT enzyme liquid, VC positive control, VC negative control and VC.
The VC RNA of the in-vitro transcription in described VC positive control is prepared with following method:
1) with in the synthetic VC toxR of chemical synthesis without secondary structure and high conservative section as amplified target sequence area (its nucleotide sequence is as shown in sequence in sequence table 8);
2) fragment is cloned into in-T carrier, build VC positive control plasmid;
3) VC positive control plasmid is transformed in bacillus coli DH 5 alpha, called after -T-VC bacterial strain, is stored in-70 DEG C:
4) from in-T-VC bacterial strain, extract -T-VC plasmid, carries out transcribe rna by plasmid, and purifying is removed DNA, and quantitative, qualification RNA.
The VC IC RNA of the in-vitro transcription in described VC in mark is prepared with following method:
1) remove probe in detecting regional sequence difference by synthetic one section of chemical synthesis, other sequences are substantially with VC target sequence region (its nucleotide sequence is as shown in sequence in sequence table 9);
2) fragment is cloned into in-T carrier, build mark plasmid in VC;
3) in VC, mark plasmid is transformed in bacillus coli DH 5 alpha, called after -T-VC IC bacterial strain, is stored in-70 DEG C;
4) from in-T-VC IC bacterial strain, extract -T-VC IC plasmid, carries out transcribe rna by plasmid, and purifying is removed DNA, and quantitative, the interior mark of qualification RNA.
Special agent in described vibrio cholerae (VC) real-time fluorescence nucleic acid constant-temperature amplification detection kit is one of following material representing:
(1) capture probe (TC0 that can target nucleic acids (VC RNA) sequence specific of the vibrio cholerae shown in sequence 1 (VC) is combined in sequence table, Target Capture Oligo), the nucleotide sequence of described capture probe is as shown in sequence in sequence table 2;
(2) the VC detection primer T7 and the nT7 that copy for produce the DNA of VC target nucleic acids (VC RNA) under the effect of M-MLV ThermoScript II, T7 primer sequence is as shown in sequence in sequence table 3, and nT7 primer sequence is as shown in sequence in sequence table 4;
(3) for the VC detection probes with the RNA copy specific combination producing according to the DNA copy of described VC target nucleic acids (VC RNA) under t7 rna polymerase effect, the nucleotide sequence of described VC detection probes is as shown in sequence in sequence table 5,5 ' end flag F AM fluorophor, 3 ' end mark DABCYL quenching group;
(4) mark and interior mark detection probes in, inside be designated as the interior mark of competitiveness of VC nucleotide sequence (VC RNA), can with capture probe specific binding, and use same pair of primers (T7 and nT7 primer), interior target nucleotide sequence is VC IC RNA as shown in sequence in sequence table 7, the nucleotide sequence of interior mark detection probes is as shown in sequence in sequence table 6, and 5 ' holds mark HEX fluorophor, 3 ' end mark DABCYL quenching group.
The using method of described test kit, detects for the real-time fluorescence nucleic acid constant-temperature amplification of vibrio cholerae (VC), comprises following operation:
1) with the vibrio cholerae (VC) in lysate cracking testing sample, obtain the lysate that contains vibrio cholerae (VC) nucleic acid;
2) to step 1) lysate in add nucleic acid extraction liquid, in test kit, there is timestamp in VC, add mark in VC simultaneously, after being combined with target or interior mark nucleic acid specificity, capture probe is combined with magnetic bead again, wash with washings, remove the nucleic acid of not being combined with magnetic bead, obtain nucleic acid (RNA) and the VC IC RNA of vibrio cholerae (VC);
3) by step 2) nucleic acid (RNA) of vibrio cholerae (VC) and the VC IC RNA that extract add in the first stage reactant being made up of VC reaction solution and VC detection liquid, at 60 DEG C, incubation is after 10 minutes, incubation 5 minutes at 42 DEG C again, then add subordinate phase enzyme reaction thing SAT enzyme liquid, start thus at 42 DEG C, to continue incubation 50 minutes, with the variation of detector synchronous recording fluorescent signal; The volume ratio of described first stage reactant and subordinate phase enzyme reaction thing is 3: 1;
4) time and intensity producing according to fluorescent signal carries out qualitative detection with reference to marking detected result in VC positive control, VC negative control and VC to testing sample.
The real-time fluorescence nucleic acid constant-temperature amplification detection kit that the invention provides a kind of vibrio cholerae (VC), is used this test kit to detect, and compared with detecting, has the following advantages with existing VC:
(1) high specific, high purity, low pollution: for the preferred capture probe of VC target nucleic acid design, can be efficiently, specificity catches the RNA of VC.Meanwhile, owing to taking enclosed constant temperature amplification detection system, in whole process, without opening reaction system, thereby avoided the pollution of amplicon.
(2) rapid detection: the amplification of nucleic acid is synchronizeed and carried out with detecting in same closed system, and there is no lifting and the circulation of temperature in whole process, thereby required time shortens greatly, augmentation detection only needs 50 minutes.
(3) pollute easily control: compared with real-time fluorescence PCR, amplified production of the present invention is RNA, and RNA very easily degrades at occurring in nature, be easier to so pollute to control.
(4) equipment is simple, and cost is low: compared with real-time fluorescence quantitative PCR, the present invention's instrument used circulates without heating and cooling, thereby design and production cost significantly reduce.
In sum, test kit of the present invention can detect the VC RNA in food, there is specificity high, highly sensitive (can reach 100CFU/ml), pollute the feature of low (amplified production RNA be easy to degraded) and rapid detection (completing augmentation detection for 50 minutes) under physical environment, to in vibrio cholerae rapid detection, play a significant role, have a extensive future.
Below in conjunction with specific embodiment, the present invention is described in further details.
Brief description of the drawings
The detected result of Fig. 1 display sensitivity
Fig. 2 shows the detected result of negative specificity reference material
Fig. 3 is the target fluoroscopic examination result of food samples
Fig. 4 is the interior mark fluoroscopic examination result of food samples
Embodiment
Vibrio cholerae of the present invention (VC) detection technique, is combined specificity target capture technique and form with real-time fluorescence nucleic acid constant-temperature amplification (SAT) technology.
Because the hypotype that vibrio cholerae comprises is more, as common 01,0139, so cover so many hypotype, in the selection of target, just must consider the versatility that test kit detects each hypotype detecting, therefore must be chosen to be at gene that between each hypotype, conservative property is stronger as detecting target.The gene that between the each hypotype of vibrio cholerae, conservative property is strong is toxR, and therefore the present inventor has determined that by researching and analysing sequence that toxR the preceding paragraph conservative property is strong is as detecting target.
The present invention is by the capture probe of design specialized, the RNA efficient, specificity is caught VC; Nucleic acid amplification is realized with M-MLV ThermoScript II and T7 RNA polymerase simultaneously, ThermoScript II is for generation of a DNA copy of target nucleic acids RNA, T7 RNA polymerase produces multiple RNA copies from DNA copy, with the RNA copy specific combination producing after fluorescently-labeled optimum detection probe and amplification, thereby generation fluorescence, this fluorescent signal can be caught by detecting instrument.
Primer special and probe in the present invention comprise:
(1) capture probe: one can target nucleic acids (VC RNA) sequence specific of the vibrio cholerae shown in sequence 1 (VC) be combined in sequence table capture probe (TCO, Target Capture Oligo), in the time having in VC mark (VC IC RNA), its also can with this VC interior label sequence specific combination; The nucleotide sequence of described capture probe is as shown in sequence in sequence table 2.
(2) VC detects primer: a pair of VC detection primer copying for produce the DNA of VC target nucleic acids (VC RNA) under the effect of M-MLV ThermoScript II, described VC detects primer and is made up of T7 primer and nT7 primer, T7 primer sequence is as shown in sequence in sequence table 3, and nT7 primer sequence is as shown in sequence in sequence table 4;
(3) VC detection probes: for the VC detection probes with the RNA copy specific combination that copies to produce according to the DNA of described VC target nucleic acids (VCRNA) under t7 rna polymerase effect, the nucleotide sequence of described VC detection probes is as shown in sequence in sequence table 5,5 ' end FAM fluorescent mark, 3 ' end DABCYL fluorescent mark.
For ease of carrying out interpretation of result, also comprise: in (4) one, mark mark in detection probes and VC, interior mark detection probes is using the interior mark detection probes that timestamp and this interior mark are used in conjunction with in VC, its nucleotide sequence is as shown in sequence in sequence table 6,5 ' end mark HEX fluorophor, 3 ' end mark DABCYL quenching group; In VC, be designated as the interior mark of competitiveness of VC nucleotide sequence (VC RNA), while and described capture probe specific binding, and use same pair of primers (T7 and nT7 primer).Target nucleotide sequence is as shown in sequence in sequence table 7 in VC, called after VC IC RNA (IC implication is interior mark).
Based on above design, the present invention further provides the real-time fluorescence nucleic acid constant-temperature amplification detection kit of a kind of vibrio cholerae (VC).
This test kit, at least comprises described capture probe (sequence 2), a pair of described T7 primer (sequence 3) and nT7 primer (sequence 4), and a described VC detection probes (sequence 5).
Further, described test kit also can include M-MLV ThermoScript II and t7 rna polymerase, described M-MLV ThermoScript II and t7 rna polymerase are present in SAT enzyme liquid, described capture probe is present in nucleic acid extraction liquid, and described T7 primer, nT7 primer and VC detection probes are present in VC and detect in liquid.
Further again, described test kit also can comprise mark (sequence 7) and interior mark detection probes (sequence 6) in competitive VC, and described interior mark detection probes is present in VC and detects in liquid.
More specifically, described test kit comprises mark in lysate, nucleic acid extraction liquid, washings, VC reaction solution, VC detection liquid, SAT enzyme liquid, VC positive control, VC negative control and VC, and each components description is as follows:
(1) lysate: for the vibrio cholerae (VC) of cracking and preservation testing sample, for containing the solution of washing agent and HEPES damping fluid, washing agent is mainly ammonium sulfate ((NH4) 2SO4, is preferably 5-50mM);
(2) nucleic acid extraction liquid: for extracting and purifying VC thalli RNA, be the aqueous solution containing capture probe 1-50 μ M (being preferably 5-25 μ M) and magnetic bead 50-500mg/L (being preferably 50-250mg/L);
(3) washings: cleaning for magnetic bead, is the aqueous solution containing 1wt%SDS.
(4) VC reaction solution: the SAT required component that increases, containing the aqueous solution of dNTP 0.1-10mM (being preferably 0.5-5mM) and NTP1-20mM (being preferably 1-10mM);
(5) VC detects liquid: containing the increase aqueous solution of required primer and probe of SAT, the concentration of each primer or probe all can in 5-15pmol/ reaction, wherein T7 primer concentration is preferably 12.5pmol/ reaction, nT7 primer concentration is preferably 12.5pmol/ reaction, VC detection probe concentrations is preferably 5pmol/ reaction, and interior mark detection probe concentrations is preferably 5pmol/ reaction;
(6) SAT enzyme liquid: the SAT required multienzymatic reaction system that increases, mainly containing M-MLV ThermoScript II 400-4000U/ reaction (being preferably 500-1500U/ reaction), t7 rna polymerase 200-2000U/ reaction (being preferably 500-1000U/ reaction);
(7) VC positive control; Containing 10 5-10 8the in-vitro transcription RNA dilution of copy/mL vibrio cholerae (VC) toxR;
(8) VC negative control: do not contain vibrio cholerae (VC) target nucleic acids (VC RNA) sequence or do not contain the solution of vibrio cholerae, as physiological saline;
(9) mark in VC: containing 10 5-10 8mark RNA (sequence 7) in copy/mL VC, is the interior mark of competitiveness of VC nucleotide sequence (VC RNA), is in-vitro transcription RNA (VC IC RNA) dilution.
Further illustrating of respectively forming in the above test kit is as follows:
The main effective constituent of lysate is washing agent, and the existence of high density washing agent can make the rapid inactivation of RNase, effectively preserves RNA.Nucleic acid extraction liquid is to have utilized magnetic bead partition method to carry out nucleic acid extraction, and its main component is magnetic-particle and capture probe.Capture probe one end and target complementation; one end is connected with magnetic-particle is complementary; in nucleic acid extraction process; magnetic-particle specific combination in the nucleic acid that bacteria lysis discharges and nucleic acid extraction liquid; in the case of not needing traditional centrifugally operated, clean magnetic-particle by washings and obtain pure bacterial target nucleic acid (RNA).The extraction of bacteria RNA realizes by specific adsorption principle.
It is molecular beacon that VC detects VC detection probes in liquid, it is the molecular probe of a class high specific, hypersensitivity, by two ends respectively covalent labeling formed by the single stranded nucleic acid molecule of fluorescence dye and quencher, be hair clip type or loop-stem structure, the loop section of molecular beacon and target complementation, two is due to the complementary stem that becomes, and molecular beacon probe is compared with linear TaqMan probe, because opening of its hairpin structure needs certain power, thereby specificity is better than linear probe.
Make to increase unsuccessfully because SAT amplification is subject to various factors, test kit user of service error in judgement is got the wrong sow by the ear, in test kit of the present invention, be provided with mark in VC positive control, VC negative control and VC.Wherein, in IVA positive control and VC, be designated as the RNA of in-vitro transcription, do not there is biologic activity.
By detecting positive control, provable kit test method and material are errorless, ensure the accuracy that detects, can monitor the difference between the repeatability of each detection and stability and test kit batch simultaneously.In VC, in the competitiveness of mark as VC RNA, mark, its topmost effect is exactly the generation of controlling false negative result, is added and is had interior target sample by detection, whether suppressedly can understand whole amplification reaction system, better points out false negative.Negative control can be got rid of false positive, uses in kit test method and material situation correct, can ensure the specificity detecting.
Utilize above test kit to carry out the detection of real-time fluorescence nucleic acid constant-temperature amplification to vibrio cholerae (VC), comprise the following steps:
(1) with the vibrio cholerae (VC) in lysate cracking testing sample, obtain the lysate that contains vibrio cholerae (VC) nucleic acid;
(2) to step 1) lysate in add nucleic acid extraction liquid, in test kit, there is timestamp in VC, add mark in VC simultaneously, after being combined with target or interior mark nucleic acid specificity, capture probe is combined with magnetic bead again, wash with washings, remove the nucleic acid of not being combined with magnetic bead, obtain nucleic acid (RNA) and the VC IC RNA of vibrio cholerae (VC);
(3) by step 2) nucleic acid (RNA) of vibrio cholerae (VC) and the VC IC RNA that extract add in the first stage reactant being made up of VC reaction solution and VC detection liquid, at 60 DEG C, incubation is after 10 minutes, incubation 5 minutes at 42 DEG C again, then add subordinate phase enzyme reaction thing SAT enzyme liquid, start thus at 42 DEG C, to continue incubation 50 minutes, with the variation of detector synchronous recording fluorescent signal; The volume ratio of described first stage reactant and subordinate phase enzyme reaction thing is 3: 1;
(4) time and intensity producing according to fluorescent signal carries out qualitative detection with reference to marking detected result in VC positive control, VC negative control and VC to testing sample.
Described step 4) in VC positive control for containing 10 5-10 8the in-vitro transcription RNA dilution of copy/mL vibrio cholerae (VC) toxR; VC negative control is the solution that does not contain vibrio cholerae (VC) target nucleic acids (VC RNA) sequence or do not contain vibrio cholerae; In VC, be designated as containing 10 5-10 8mark RNA (sequence 7) dilution in copy/mL VC.
Embodiment implements under taking technical solution of the present invention as prerequisite, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
In following embodiment, method therefor is ordinary method if no special instructions.In embodiment, main raw material SAT enzyme liquid used, positive control and interior target in-vitro transcription RNA are provided by RD Biosciences company of the U.S., 7500 type PCR instrument are American AB I company product, MX3005 fluorescent quantitation instrument is Stratagene company product, and the reagent such as NTPs, dNTPs and other instruments are conventional commercially available product.
Embodiment 1, for real-time fluorescence nucleic acid constant-temperature amplification detect vibrio cholerae (VC) primer special and the design of probe
The present invention selects in VC bacterium toxR without secondary structure and high conservative section as amplified target sequence area (its nucleotide sequence is as shown in sequence in sequence table 1), according to primer probe design principle, use DNA ATAR, DNAman software and be manually designed for real-time fluorescence nucleic acid constant-temperature amplification the primer special and the probe sequence that detect vibrio cholerae (VC), obtain following concrete sequence:
Article (1) one, the capture probe (TCO that can be combined with target nucleic acids (VC RNA) sequence specific of the vibrio cholerae (VC) as shown in sequence in sequence table 1, Target Capture Oligo), the nucleotides sequence of described capture probe is classified 5 '-GCCAGCUGGUAUCUUCGACUGACUUCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3 ' (sequence 2 in sequence table) as;
(2) a pair of VC detection primer copying for produce the DNA of VC target nucleic acids (VC RNA) under the effect of M-MLV ThermoScript II, described VC detects primer and is made up of T7 primer and nT7 primer, T7 primer sequence is 5 '-AATTTAATACGACTCACTATAGGGAGAATCACCTCGTTTGGACGTTGGGCCAGCA-3 ' (sequence 3 in sequence table), and nT7 primer sequence is 5 '-TCCCCTAAGCAATACTCTGA3 ' (sequence 4 in sequence table);
Article (3) one, for the VC detection probes with the RNA copy specific combination producing according to the DNA copy of described VC target nucleic acids (VC RNA) under t7 rna polymerase effect, the nucleotides sequence of described VC detection probes is classified 5 '-CGCAUAGUGAAGAGAUCAUUCGAGUGCG-3 ' (sequence 5 in sequence table) as, 5 ' end FAM fluorescent mark, 3 ' end DABCYL fluorescent mark.
(4) for ease of carrying out interpretation of result, mark (sequence 7) in the competitive VC also increasing in reagents box, design competitive interior mark detection probes, in VC, mark has identical PBR with VC target Nucleotide (VC RNA), nucleotide sequence between two primers or arrangement are different, it can not be combined with detection probes, but can be combined with interior mark probe, in described VC, mark can be built and be obtained by VC target template rite-directed mutagenesis, can with capture probe specific binding, described interior mark detection probes is and VC detection probes sequence, fluorescent mark difference, but the probe that base number is consistent, the nucleotides sequence of described interior mark detection probes is classified 5 '-CGCUGGAGCGUGGUGAGCAGCG-3 ' (sequence 6 in sequence table) as, 5 ' end mark HEX fluorophor, 3 ' end mark DABCYL quenching group.
The real-time fluorescence nucleic acid constant-temperature amplification detection kit of embodiment 2, preparation vibrio cholerae (VC)
The primer special and the probe that utilize embodiment 1 to provide, obtain the real-time fluorescence nucleic acid constant-temperature amplification detection kit of vibrio cholerae of the present invention (VC).This test kit includes capture probe (TCO, Target Capture Oligo), T7 primer, nT7 primer, VC detection probes, interior mark detection probes, M-MLV ThermoScript II and t7 rna polymerase.
Described capture probe is present in nucleic acid extraction liquid, described T7 primer, nT7 primer and VC detection probes, interior mark detection probes is present in VC and detects in liquid, described M-MLV ThermoScript II and t7 rna polymerase are present in SAT enzyme liquid, specifically, described test kit is divided into the A box (sample disposal unit) of 2-30 DEG C of storage and the B box (nucleic acid amplification detecting unit) of-15--35 DEG C of storages, A box comprises lysate, nucleic acid extraction liquid and washings, B box comprises VC reaction solution, VC detects liquid, SAT enzyme liquid, VC positive control, VC negative control, if mark in existing, B box also comprises mark in VC, main component is as follows:
A box (sample disposal unit) consists of:
Lysate; Liquid containing ammonium sulfate ((NH 4) 2sO 4) and HEPES;
Nucleic acid extraction liquid: containing capture probe 1-50 μ M (being preferably 5-25 μ M) and magnetic bead 50-500mg/L (being preferably 50-250mg/L);
Washings: mainly containing 1wt%SDS.
B box (nucleic acid amplification detecting unit) consists of:
VC reaction solution: containing dNTP 0.1-10mM (being preferably 0.5-5mM), NTP 1-20mM (being preferably 1-10mM);
VC detects liquid: containing primer and probe, the concentration of each primer and probe all can in 5-15pmol/ reaction, wherein T7 primer concentration is preferably 12.5pmol/ reaction, nT7 primer concentration is preferably 12.5pmol/ reaction, VC detection probe concentrations is preferably 5pmol/ reaction, and interior mark detection probe concentrations is preferably 5pmol/ reaction;
SAT enzyme liquid: containing M-MLV ThermoScript II 400-4000U/ reaction (being preferably 500-1500U/ reaction), t7 rna polymerase 200-2000U/ reaction (being preferably 500-1000U/ reaction);
VC positive control; Containing 10 5-10 8the in-vitro transcription RNA dilution of copy/mL vibrio cholerae (VC) toxR;
VC negative control: do not contain vibrio cholerae (VC) target nucleic acids (VC RNA) sequence or do not contain the solution of vibrio cholerae, as physiological saline;
If mark in existing, mark in VC: containing 10 5-10 8copy/mL VC IC RNA dilution (sequence 7 in sequence table).
The all reagent that comprise in test kit all can obtain with ordinary method preparation by prompting or business purchase obtains.
Specifically,, in each reacton, the concrete assembly of described test kit all ingredients is as follows:
(1) lysate: be the vibrio cholerae (VC) in cracking and preservation sample, the solution that contains ammonium sulfate and HEPES damping fluid, specifically comprises HEPES 25-250mM, (NH 4) 2sO 45-50mM;
(2) nucleic acid extraction liquid: for extracting the solution of the one section of RNA sequence that contains the coated magnetic bead of oligo (dT) and specific combination target nucleic acids (VC RNA) of vibrio cholerae (VC) RNA, specifically comprise HEPES 50-400mM, EDTA40-200mM, LiCl 400-2000mM, capture probe 1-50 μ M (being preferably 5-25u M), magnetic bead 50-500mg/L (being preferably 50-250mg/L);
(3) washings: for the solution containing SDS, NaCl, specifically comprise HEPES 5-50mM, NaCl 50-500Mm, 1%SDS 1-10mM, EDTA 1-10mM;
(4) VC reaction solution: for the required component that increases containing dNTPs and NTPs, specifically comprise Tris 10-50mM, MgCl 210-40mM, dNTP 0.1-10mM (being preferably 0.5-5mM), NT P1-20mM (being preferably 1-10mM), PVP4O1-10%, KCl 5-40mM;
(5) VC detects liquid: VC detection primer and VC detection probes essential during by constant-temperature amplification are dissolved in TE solution (mixed solution of 10mM Tris and 1mM EDTA) formulated, primer and concentration and probe concentration all can in 5-15pmol/ reaction, wherein T7 primer concentration is preferably 12.5pmol/ reaction, nT7 primer concentration is preferably 12.5pmol/ reaction, VC detection probe concentrations is preferably 5pmol/ reaction, and interior mark detection probe concentrations is preferably 5pmol/ reaction;
(6) SAT enzyme liquid: essential multienzyme components system during for constant-temperature amplification, containing M-MLV ThermoScript II 400-4000U/ reaction (being preferably 500-1500U/ reaction), t7 rna polymerase 200-2000U/ reaction (being preferably 500-1000U/ reaction), 2-10mM HEPES pH7.5, 10-100mM N-acetyl-L-cysteine, 0.04-0.4mM zinc acetate, 10-100mM trehalose, 40-200mM Tris-HCl pH8.0, 40-200mM KCl, 0.01-0.5mM EDTA, 0.1-1% (v/v) Triton X-100 and 20-50% (v/v) glycerol,
(7) VC positive control; Containing 10 5-10 8the in-vitro transcription RNA dilution of copy/mL vibrio cholerae (VC) toxR;
(8) VC negative control: do not contain vibrio cholerae (VC) target nucleic acids (VC RNA) sequence or do not contain the solution of vibrio cholerae, as physiological saline;
(9) if marked in existing, mark in VC: containing 10 5-10 8mark RNA (sequence 7 in sequence table) in copy/mL VC.
The VC RNA of the in-vitro transcription in VC positive control, can prepare gained by several different methods, and wherein a kind of preparation method is as follows:
1) with in the synthetic VC toxR of chemical synthesis without secondary structure and high conservative section as amplified target sequence area (its nucleotide sequence is as shown in sequence in sequence table 8);
2) fragment is cloned into in-T carrier, build VC positive control plasmid;
3) VC positive control plasmid is transformed in bacillus coli DH 5 alpha, called after -T-VC bacterial strain, is stored in-70 DEG C;
4) from in-T-VC bacterial strain, extract -T-VC plasmid, carries out transcribe rna by plasmid, and purifying is removed DNA, and quantitative, qualification RNA.
The VC IC RNA of the in-vitro transcription in VC in mark, can prepare gained by several different methods, and wherein a kind of preparation method is as follows:
1) remove probe in detecting regional sequence difference by synthetic one section of chemical synthesis, other sequences are substantially with VC target sequence region (its nucleotide sequence is as shown in sequence in sequence table 9);
2) fragment is cloned into in-T carrier, build mark plasmid in VC;
3) in VC, mark plasmid is transformed in bacillus coli DH 5 alpha, called after -T-VC IC bacterial strain, is stored in-70 DEG C;
4) from in-T-VCIC bacterial strain, extract -T-VC IC plasmid, carries out transcribe rna by plasmid, and purifying is removed DNA, and quantitative, the interior mark of qualification RNA.
The real-time fluorescence nucleic acid constant-temperature amplification detection sensitivity of embodiment 3, vibrio cholerae
With the vibrio cholerae (VC) in test kit of the present invention (composition is shown in embodiment 2, does not have the interior mark of VC and detect the interior mark detection probes in liquid in test kit) detection food samples, concrete grammar comprises the following steps:
(1) bacterium liquid dilution
Measuring concentration is 1 × 10 5the vibrio cholerae culture of CFU/mL, 10 times of gradient dilutions to 10cFU/mL as the linear sensitivity reference material of vibrio cholerae.
(2) nucleic acid extraction
2.1 add 200 μ l lysates (to contain HEPES 35mM, (NH in sample processing tube (1.5mL centrifuge tube) 4) 2sO 420mM), 200 μ l bacterium liquid, with the vibrio cholerae (VC) in lysate cracking testing sample, obtain the lysate that contains vibrio cholerae (VC) nucleic acid.
2.2 add 100 μ l nucleic acid extraction liquid (to contain HEPES 100mM, EDTA 50mM, LiCl 500mM in sample processing tube (1.5mL centrifuge tube), capture probe 10 μ M, magnetic bead 250mg/L), mix, 60 DEG C are incubated 5 minutes, and room temperature is placed 10 minutes.
2.3 are placed in sample processing tube on magnetic bead separating device, leave standstill 5-10 minute.After magnetic bead is adsorbed in tube wall, keep sample processing tube on magnetic bead separating device, inhale and abandon liquid, retain magnetic bead.After adding 1mL washings (containing HEPES50mM, NaCl 100mM, 1%SDS, EDTA 5mM) vibration evenly, leave standstill 5-10 minute, abandon liquid, retain magnetic bead, 2 times repeatedly.
Sample processing tube is moved apart magnetic bead separating device by 2.4, Guan Zhongwei magnetic bead-nucleic acid complexes, (this step is answered high-visible magnetic bead) for subsequent use.
(3) SAT nucleic acid amplification detects
3.1 add 40 μ l reaction detection liquid (40 μ l VC reaction solution+2.5 μ l VC detect liquid) washing magnetic bead in sample processing tube.VC reaction solution specifically comprises Tris 30mM, MgCl 210mM, dNTP 1mm, NTP 2mM, PVP4O5%, KCl25mM; It is 12.5pmol that VC detects T7 primer concentration in liquid, and nT7 primer concentration is 12.5pmol, and VC detection probe concentrations is 5pmol.
3.2 get the above-mentioned reaction detection liquid 30 μ l that mix that vibrate adds to clean micro-reaction pipe, and 60 DEG C are incubated 10 minutes, and 42 DEG C are incubated 5 minutes; In micro-reaction pipe, add 10 μ l to be preheated to the SAT enzyme liquid of 42 DEG C, 1200rpm vibrates and mixed 15 seconds, carries out real-time fluorescence detection with 7500 type PCR instrument (American AB I company product).In SAT enzyme liquid, contain M-MLV ThermoScript II 750U, t7 rna polymerase 500U/ reaction, 10mM HEPES pH7.5,20mMN-acety1-L-cysteine (N-acetyl-L-cysteine), 0.1mM zinc acetate (zinc acetate), 10mMtrehalose (trehalose), 100mM Tris-HCl pH8.0,100mM KC1,0.1mm EDTA, 0.8% (v/v) Triton X-100 and 40% (v/v) glycerol (glycerol);
Micro-reaction pipe is gone to fast constant-temperature fluorescence detector device (ABI7500 fluorescent quantitation instrument, ABI instrument is optional VIC passage only, but VIC is close with HEX wavelength) by 3.3, and 42 DEG C of reactions 50 minutes, set every 1 minute and detect first order fluorescence, detect altogether 50 times.
(4) result is judged
The curve obtaining according to pcr amplification result, sets threshold line, reads dt value, result of determination.
Threshold setting: the vertex with threshold line just above normal negative control amplification curve.Dt represents the X-coordinate reading (similar with the ct value of general real-time fluorescence PCR experimental result) of sample curve and threshold line intersection point
1. positive findings is judged:
FAM passage: the sample of dt≤45 is positive; The sample suggestion of 45 < dt < 50 detects again, detects knot
Really: the sample of dt < 50 is positive.
2. negative findings is judged: VIC passage: dt is without numerical value or be 50, negative.
(5) result
The detection figure of Fig. 1 display sensitivity is 1 × 10 by concentration 5the positive reference material of CFU/mL, detects by 10 times of gradient dilutions.In the time that the concentration of positive reference material is 100CFU/mL, detecting dt value is 25, detects lower limit sensitivity and can reach 100CFU/mL.
The real-time fluorescence nucleic acid constant-temperature amplification detection specificity of embodiment 4, vibrio cholerae
This detecting pattern is another application of the invention: test kit forms with embodiment 2, and the production of reagent is carried out in GMP workshop; The method of specificity reference material processing is as follows: 6 routine negative reference materials comprise streptococcus aureus (SA), Shigellae (SH), colon bacillus 0157 (0157), Listeria Monocytogenes (Lm), Salmonellas (Sal.spp.), Vibrio parahaemolyticus (VP), separately establishes each one of negative control, positive control.Detection method is with embodiment 3, and in detection, agents useful for same amount is with embodiment 3.
Use the detected result of test kit of the present invention as Fig. 2, the amplification curve of 6 negative reference materials is straight and with baseline without intersecting, can clearly be judged to be feminine gender.6 routine negative reference materials comprise streptococcus aureus (SA), Shigellae (SH), colon bacillus 0157 (0157), Listeria Monocytogenes (Lm), Salmonellas (Sal.spp.), Vibrio parahaemolyticus (VP).
The real-time fluorescence nucleic acid constant-temperature amplification of embodiment 5, actual food product sample detects
With the vibrio cholerae VC in test kit of the present invention (composition is shown in embodiment 2, and test kit contains mark in VC, detects liquid and contains interior mark probe) detection food samples, concrete grammar comprises the following steps:
(1) sample process
Take 25g (mL) sample and pack in the aseptic homogenizing bag that fills 225mL nutrient broth, pat 1min~2min with slap type homogenizer.If sample is liquid, do not need homogeneous, concussion mixes.As be frozen prods, should be no more than below 15min at 45 DEG C, or 2 DEG C~5 DEG C are no more than 18h and thaw.Vibrio cholerae sample number is VC food sample 1-10, separately establishes each one of negative control, positive control.
(2) nucleic acid extraction
2.1 add 200 μ l lysates (to contain HEPES 35mM, (NH in sample processing tube (1.5mL centrifuge tube) 4) 2sO 420mM), 200 μ l bacterium liquid, with the vibrio cholerae (VC) in lysate cracking testing sample, obtain the lysate that contains vibrio cholerae (VC) nucleic acid.
2.2 add 100 μ l nucleic acid extraction liquid (containing HEPES 100mM, EDTA 150mM, LiCl 1500mM, capture probe 20 μ M, magnetic bead 250mg/L) in sample processing tube (1.5mL centrifuge tube), and 10 μ l inner mark solutions are (containing 1 × 10 8mark RNA in copy/mL VC), mix, 60 DEG C are incubated 5 minutes, and room temperature is placed 10 minutes.
2.3 are placed in sample processing tube on magnetic bead separating device, leave standstill 5-10 minute.After magnetic bead is adsorbed in tube wall, keep sample processing tube on magnetic bead separating device, inhale and abandon liquid, retain magnetic bead.After adding 1mL washings (containing HEPES50mM, NaCl 100mM, 1%SDS, EDTA 5mM) vibration evenly, leave standstill 5-10 minute, abandon liquid, retain magnetic bead, 2 times repeatedly.
Sample processing tube is moved apart magnetic bead separating device by 2.4, Guan Zhongwei magnetic bead-nucleic acid complexes, (this step is answered high-visible magnetic bead) for subsequent use.
(3) SAT nucleic acid amplification detects
3.1 add 40 μ l reaction detection liquid (40 μ l VC reaction solution+2.5 μ l VC detect liquid) washing magnetic bead in sample processing tube.VC reaction solution specifically comprises Tris 50mM, MgCl 220mM, dNTP 5mM, NTP 10mM, PVP4O5%, KCl 40mM; It is 12.5pmol that VC detects T7 primer concentration in liquid, and nT7 primer concentration is 12.5pmol, and VC detection probe concentrations is 5pmol, and interior mark detection probe concentrations is 5pmol.
3.2 get the above-mentioned reaction detection liquid 30 μ l that mix that vibrate adds to clean micro-reaction pipe, and 60 DEG C are incubated 10 minutes, and 42 DEG C are incubated 5 minutes; In micro-reaction pipe, add 10 μ l to be preheated to the SAT enzyme liquid of 42 DEG C, 1200rpm vibrates and mixed 15 seconds, carries out real-time fluorescence detection with 7500 type PCR instrument (American AB I company product).In SAT enzyme liquid, contain M-MLV ThermoScript II 1500U, t7 rna polymerase 1000U/ reaction, 10mM HEPES pH7.5,50mM N-acetyl-L-cysteine (N-acetyl-L-cysteine), 0.4mM zinc acetate (zinc acetate), 60mM trehalose (trehalose), 100mM Tris-HCl pH8.0,200mM KCl, 0.3mM EDTA, 0.8% (v/v) Triton X-100 and 40% (v/v) glycerol (glycerol).
Micro-reaction pipe is gone to fast constant-temperature fluorescence detector device by 3.3, and (ABI instrument is optional VIC passage only for ABI7500 fluorescent quantitation instrument, ABI company product, but VIC is close with HEX wavelength), 42 DEG C are reacted 50 minutes, set every 1 minute and detect first order fluorescence, detect altogether 50 times.
(4) result is judged
The curve obtaining according to pcr amplification result, sets threshold line, reads dt value, result of determination.
Threshold setting: the vertex with threshold line just above normal negative control amplification curve.Dt represents the X-coordinate reading (similar with the ct value of general real-time fluorescence PCR experimental result) of sample curve and threshold line intersection point
3. positive findings is judged:
F1 passage: the sample of dt≤45 is positive; The sample suggestion of 45 < dt < 50 detects again, detected result
F1 passage: the sample of dt < 50 is positive.
4. negative findings is judged: F1 passage dt is without numerical value or be 50, simultaneously F2 passage: dt≤45, and negative.
Quality control: each detection all arranges positive control and negative control, and result should meet positive control F1 passage simultaneously: dt≤45; Negative control F1 passage: dt is without numerical value or be 50, simultaneously F2 passage: dt≤45, otherwise this time detected result be considered as invalid.
(5) result
According to the dt value situation of F1 passage (Fig. 3) and F2 passage (Fig. 4), judge sample 3,7 test positive, sample 1,2,4,5,6,8,9,10 is negative, and (sample 1,2,4,5,6,8,9,10 Fig. 3 are shown as feminine gender, Fig. 4 shows signal, just illustrating that interior mark can detect, reaction system does not have affected by environment, gets rid of false negative).
According to disclosure of the present invention, those skilled in the art need not too much test and can implement the present invention vibrio cholerae required for protection (VC) real-time fluorescence nucleic acid constant-temperature amplification detection kit, and produce a desired effect.Embodiment disclosed by the invention only describes the present invention, but also not enough formation limits the present invention.Those skilled in the art are with apparent similar surrogate or transformation; with some chemically or the preparation that on biology, structure function is relevant substitute preparation described here; or associated viscera of the present invention is changed; but do not exceed spirit of the present invention, scope and thought, all fall into the scope of protection of present invention.

Claims (8)

1. the real-time fluorescence nucleic acid constant-temperature amplification detection kit of a vibrio cholerae (VC), the target nucleic acids that includes the vibrio cholerae VC shown in sequence 1 in a sequence table is the capture probe of VC RNA, a pair of VC detection primer T7 and the nT7 copying for produce the DNA of VC target nucleic acids VC RNA under the effect of M-MLV ThermoScript II, article one, for the VC detection probes with the RNA copy specific combination producing according to the DNA copy of described VC target nucleic acids VC RNA under t7 rna polymerase effect, article one, for competing each other with VC nucleotide sequence, with capture probe specific binding and to use with pair of primers be that to mark Nucleotide in the VC of T7 and nT7 be VC IC RNA, with one with VC in mark in the VC of the RNA copy specific binding that the DNA copy of Nucleotide produces and mark detection probes,
The nucleotide sequence of described capture probe is as shown in sequence in sequence table 2; Described VC detects primer and is made up of T7 primer and nT7 primer, and T7 primer sequence is as shown in sequence in sequence table 3, and nT7 primer sequence is as shown in sequence in sequence table 4; The nucleotide sequence of described VC detection probes is as shown in sequence in sequence table 5, and 5 ' holds flag F AM fluorophor, 3 ' end mark DABCYL quenching group; In described VC, the nucleotide sequence of mark detection probes is as shown in sequence in sequence table 6, and 5 ' holds mark HEX fluorophor, 3 ' end mark DABCYL quenching group.
2. test kit according to claim 1, it is characterized in that: described test kit also includes M-MLV ThermoScript II and t7 rna polymerase, described M-MLV ThermoScript II and t7 rna polymerase are present in a SAT enzyme liquid, described capture probe is present in a nucleic acid extraction liquid, and in described T7 primer, nT7 primer, VC detection probes and VC, mark detection probes is present in a VC detection liquid.
3. according to the arbitrary described test kit of claim 1 to 2, it is characterized in that: described test kit comprises mark in lysate, nucleic acid extraction liquid, washings, VC reaction solution, VC detection liquid, SAT enzyme liquid, VC positive control, VC negative control and VC, wherein:
Lysate: liquid containing ammonium sulfate ((NH 4) 2sO 4) and HEPES;
Nucleic acid extraction liquid: containing capture probe and magnetic bead;
Washings: containing NaCl and SDS;
VC reaction solution: containing dNTP and NTP;
VC detects liquid: containing T7 primer, nT7 primer, VC detection probes and interior mark detection probes;
SAT enzyme liquid: containing M-MLV ThermoScript II, t7 rna polymerase;
VC positive control; Containing the in-vitro transcription RNA dilution of vibrio cholerae toxR;
VC negative control: do not contain vibrio cholerae target nucleic acids sequence or do not contain the solution of vibrio cholerae;
Mark in VC: VC IC RNA, sequence is as shown in sequence in sequence table 7.
4. test kit according to claim 3, is characterized in that: described VC negative control is physiological saline.
5. test kit according to claim 3, is characterized in that: in described test kit, in a reacton, all ingredients is composed as follows:
(1) lysate: HEPES25-250mM, (NH 4) 2sO 45-50mM;
(2) nucleic acid extraction liquid: HEPES50-400mM, EDTA40-200mM, LiCl400-2000mM, capture probe 1-50 μ M, magnetic bead 50-500mg/L;
(3) washings: HEPES5-50mM, NaCl50-500Mm, 1%SDS, EDTA1-10mM;
(4) VC reaction solution: Tris10-50mM, MgCl 210-40mM, dNTP0.1-10mM, NTP1-20mM, PVP401-10%, KCl5-40mM;
(5) VC detects liquid: VC is detected to primer and VC detection probes is dissolved in TE solution, formulated in the mixed solution of 10mM Tris and 1mMEDTA, each primer and concentration and probe concentration are reacted at 5-15pmol/.
(6) SAT enzyme liquid: M-MLV ThermoScript II 400-4000U/ reaction, t7 rna polymerase 200-2000U/ reaction, 2-10mM HEPES pH7.5,10-100mM N-acetyl-L-cysteine, 0.04-0.4mM zinc acetate, 10-100mM trehalose, 40-200mM Tris-HCl pH8.0,40-200mM KCl, 0.01-0.5mM EDTA, volume percent 0.1-1%Triton X-100 and concentration expressed in percentage by volume 20-50%glycerol;
(7) VC positive control; Containing 10 5-10 8the in-vitro transcription RNA dilution of copy/mL vibrio cholerae (VC) toxR;
(8) VC negative control: do not contain vibrio cholerae target nucleic acids sequence or do not contain the solution of vibrio cholerae;
(9) mark in VC: containing 10 5-10 8copy/mL VC IC RNA in-vitro transcription RNA dilution.
6. test kit according to claim 5, is characterized in that: in reagent (2) nucleic acid extraction liquid, and capture probe 5-25 μ M, magnetic bead 50-250mg/L; In reagent (4) VC reaction solution, dNTP concentration is 0.5-5mM, and NTP concentration is 1-10mM; Reagent (5) VC detects in liquid, and T7 primer concentration is 12.5pmol/ reaction, and nT7 primer concentration is 12.5pmol/ reaction, and VC detection probe concentrations is 5pmol/ reaction, and interior mark detection probe concentrations is 5pmol/ reaction; In reagent (6) SAT enzyme liquid, M-MLV ThermoScript II is 500-1500U/ reaction, and t7 rna polymerase is 500-1000U/ reaction.
7. according to the test kit described in claim 5 or 6, it is characterized in that: be that sample disposal unit and B box are that nucleic acid amplification detecting unit forms by A box, wherein A box is packed described lysate, described nucleic acid extraction liquid and described washings, and B box is packed described VC reaction solution, VC detects mark in liquid, SAT enzyme liquid, VC positive control, VC negative control and VC.
8. according to arbitrary described test kit in claim 5 to 7, it is characterized in that: the VC RNA of the in-vitro transcription in described VC positive control is prepared with following method:
(1) with in the synthetic VC toxR of chemical synthesis without secondary structure and high conservative section the fragment as amplified target sequence area, its nucleotide sequence is as shown in sequence in sequence table 8;
(2) fragment is cloned into in carrier, build VC positive control plasmid;
(3) VC positive control plasmid is transformed in bacillus coli DH 5 alpha, called after bacterial strain, is stored in-70 DEG C;
(4) purifying is removed DNA, and quantitative, qualification RNA;
Have timestamp in VC, the VC IC RNA of in-vitro transcription is prepared with following method:
(1) remove probe in detecting regional sequence difference by synthetic one section of chemical synthesis, other sequences are substantially with the fragment in VC target sequence region, and its nucleotide sequence is as shown in sequence in sequence table 9;
(2) fragment is cloned into in carrier, build mark plasmid in VC;
(3) in VC, mark plasmid is transformed in bacillus coli DH 5 alpha, called after iC bacterial strain, is stored in-70 DEG C;
(4) purifying is removed DNA, and quantitative, the interior mark of qualification RNA.
CN201210203302.XA 2012-06-19 2012-06-19 Simultaneous amplification and testing reagent kit for VC (vibrio cholerae) Active CN102719542B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201210203302.XA CN102719542B (en) 2012-06-19 2012-06-19 Simultaneous amplification and testing reagent kit for VC (vibrio cholerae)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201210203302.XA CN102719542B (en) 2012-06-19 2012-06-19 Simultaneous amplification and testing reagent kit for VC (vibrio cholerae)

Publications (2)

Publication Number Publication Date
CN102719542A CN102719542A (en) 2012-10-10
CN102719542B true CN102719542B (en) 2014-10-01

Family

ID=46945407

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201210203302.XA Active CN102719542B (en) 2012-06-19 2012-06-19 Simultaneous amplification and testing reagent kit for VC (vibrio cholerae)

Country Status (1)

Country Link
CN (1) CN102719542B (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108611431A (en) * 2018-05-11 2018-10-02 重庆出入境检验检疫局检验检疫技术中心 The sandwich DNA hybridization of comma bacillus, which quickly detects, uses probe, kit and detection method

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104928300B (en) * 2014-10-31 2018-12-21 上海市计量测试技术研究院 A kind of polynucleotides, method and kit for the detection of comma bacillus real-time fluorescence quantitative PCR
CN108265102A (en) * 2016-12-30 2018-07-10 上海仁度生物科技有限公司 A kind of real-time fluorescence nucleic acid isothermal amplification detection kit of Cronobacter Enterobacter sakazakii
CN107385057B (en) * 2017-08-10 2020-11-13 广州海关技术中心 RPA-IAC primer and method for detecting vibrio cholerae
CN110734988B (en) * 2018-07-20 2024-06-18 上海仁度生物科技股份有限公司 Constant-temperature amplification method for methicillin-resistant staphylococcus aureus (MRSA) nucleic acid
CN112646908A (en) * 2020-12-31 2021-04-13 广州赛哲生物科技股份有限公司 Vibrio vulnificus isothermal amplification primer, probe, kit and detection method

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
TaqMan荧光PCR技术在霍乱弧菌毒力基因检测中的应用;曲梅 等;《中国预防医学杂志》;20080630;第9卷(第6期);546-549 *
实时荧光核酸恒温扩增技术检测尿液中***的分析;高志华 等;《国际检验医学杂志》;20120229;第33卷(第4期);463-464 *
曲梅 等.TaqMan荧光PCR技术在霍乱弧菌毒力基因检测中的应用.《中国预防医学杂志》.2008,第9卷(第6期),546-549.
高志华 等.实时荧光核酸恒温扩增技术检测尿液中***的分析.《国际检验医学杂志》.2012,第33卷(第4期),463-464.

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108611431A (en) * 2018-05-11 2018-10-02 重庆出入境检验检疫局检验检疫技术中心 The sandwich DNA hybridization of comma bacillus, which quickly detects, uses probe, kit and detection method

Also Published As

Publication number Publication date
CN102719542A (en) 2012-10-10

Similar Documents

Publication Publication Date Title
CN102719542B (en) Simultaneous amplification and testing reagent kit for VC (vibrio cholerae)
Ding et al. A multiplex RT-PCR assay for S. aureus, L. monocytogenes, and Salmonella spp. detection in raw milk with pre-enrichment
US5587286A (en) Methods and kits for detection of cells in food materials
CN103320434B (en) Salmonella LAMP (loop-mediated isothermal amplification) primer group and kit and detection method
Köster et al. Analytical methods for microbiological water quality testing
CN101570783A (en) Detection kit and detection method for 9 species of pathogenic organisms in marine products
CN102703603B (en) Real-time fluorescence nucleic acid constant temperature amplification detection kit of general influenza a virus (IAV)
CN103421896B (en) For the RNA constant-temperature amplification kit for detecting nucleic acid of Escherichia coli O 157
CN113462795A (en) Combined detection method for rapidly detecting Listeria monocytogenes, system and application thereof
EP3902929A1 (en) Fast and portable microfluidic detection system as an alternative to salmonella&#39;s classical culture method
CN103388033A (en) Enterovirus type 71 (EV71) real-time fluorescent nucleic acid isothermal amplification detection kit
Zeng et al. A polymerase chain reaction based lateral flow test strip with propidium monoazide for detection of viable Vibrio parahaemolyticus in codfish
CN103421897B (en) RNA isothermal amplification nucleic acid detection kit aiming at Shigella (SH)
CN104342487B (en) Mycoplasma nucleic acid constant-temperature amplification method
Kou et al. Simultaneous detection of norovirus and rotavirus in oysters by multiplex RT–PCR
CN108265125A (en) A kind of swine fever virus(CSFV)Real-time fluorescence nucleic acid isothermal amplification detection kit
CN103388032A (en) Coxsackie virus type A16 (CA16) real-time fluorescent nucleic acid isothermal amplification detection kit
CN103571942B (en) A kind of vibrio parahaemolytious VP nucleic acid constant-temperature amplification method
CN104450930A (en) Molecular detection method of vibrio parahaemolyticus and application thereof
CN103215386A (en) Isothermal amplification method for enterovirus EV nucleic acid
CN105200044B (en) The nucleotides special to vibrio fluvialis O1, O6, O7, O8 and O9 and its application
CN104032000B (en) The detection method of a kind of bacillus cereus and test kit
Lee et al. Sample preparation for PCR
CN104531861A (en) Molecular detection method of enterobacter sakazakii and application of molecular detection method
CN105200045A (en) Nucleotides specific to vibrio fluvialis O11, O14, O16 and O17 as well as application of nucleotides

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
EE01 Entry into force of recordation of patent licensing contract
EE01 Entry into force of recordation of patent licensing contract

Application publication date: 20121010

Assignee: Zhangjiakou Jian Yuan Food Safety Technology Co., Ltd.

Assignor: Shanghai Rendu Biotechnology Co., Ltd.

Contract record no.: 2018310000002

Denomination of invention: Simultaneous amplification and testing reagent kit for VC (vibrio cholerae)

Granted publication date: 20141001

License type: Common License

Record date: 20180110

EC01 Cancellation of recordation of patent licensing contract
EC01 Cancellation of recordation of patent licensing contract

Assignee: Zhangjiakou Jian Yuan Food Safety Technology Co.,Ltd.

Assignor: SHANGHAI RENDU BIOTECHNOLOGY Co.,Ltd.

Contract record no.: 2018310000002

Date of cancellation: 20210108

CP03 Change of name, title or address
CP03 Change of name, title or address

Address after: Room B, building 15, No. 528 Ruiqing Road, East District, Zhangjiang hi tech park, Pudong New Area, Shanghai, 201201

Patentee after: Shanghai Rendu Biotechnology Co., Ltd

Address before: 3 / F, building 7, No. 590 Ruiqing Road, East District, Zhangjiang High Tech Park, Shanghai, 201201

Patentee before: SHANGHAI RENDU BIOTECHNOLOGY Co.,Ltd.