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.
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.