CN103215386B - Isothermal amplification method for enterovirus EV nucleic acid - Google Patents
Isothermal amplification method for enterovirus EV nucleic acid Download PDFInfo
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Abstract
The invention discloses an isothermal amplification method for enterovirus EV nucleic acid. The method comprises the following specific steps of: amplifying in a reaction system, wherein the reaction system contains a specific primer pair which amplifies enterovirus EV, and the primers can amplify amplifying products corresponding to characteristic sequences of enterovirus EV from a detection sample with extremely low EV copy number. The method provided by the invention can be used for amplifying the sample to be tested containing EV RNA quickly with high specificity, high sensitivity and low pollution, and the characteristics of high detection efficiency and high accuracy as well as wide application prospect.
Description
Technical field
The present invention relates to the technical field of biological of virus, be specifically related to the primer, probe and the related kit that use in the real-time fluorescence nucleic acid constant-temperature amplification detection of the enterovirus (EV) specificity target capture technique and real-time fluorescence nucleic acid constant-temperature amplification detection technique combined.
Background technology
Hand foot mouth disease is a kind of global infectious disease, is multiplely born in infant, can cause the bleb at the position such as hand, foot, oral cavity, can cause the mortality complication such as myocarditis, pulmonary edema, meningoencephalitis in small number of patients.In China, 1981 in Shanghai Late Cambrian hand foot mouth disease; After this, there is morbidity report all parts of the country.The prevalence rate in some area is up to 10,00/,100,000.
The enterovirus of current confirmed initiation hand foot mouth disease has kind more than 20 (type), 16,4,5,9,10 types of CA group, 2,5 types of B group, Echovirus 11 and enterovirus are the more common pathogenic agent of hand foot mouth disease, wherein with coxsackie virus A 16-type (Cox A16) and enterovirus (EV71) the most common.
The main detection method of current EV has: Viral isolation method, Serological testing and RT-PCR method.Viral isolation and serological method, numerous and diverse time-consuming, process the needs of great amount of samples during cannot viral prevalence being met simultaneously.RT-PCR method needs the working cycle of experience tens temperature variation, and the amplified reaction time is long, and product is DNA, easily pollutes.Therefore, develop a kind of quick, sensitive, special and the test kit not easily polluted is very necessary.
Real-time fluorescence nucleic acid constant-temperature amplification detection technique (Simultaneous Amplification and Testing, abbreviate SAT) be a kind of method of direct rapid detection RNA, 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 at one temperature (42 DEG C), without the need to thermal cycling.Adopt M-MLV reversed transcriptive enzyme and t7 rna polymerase to carry out nucleic acid amplification, relative to other nucleic acid amplification technologies, response inhabitation thing is less, effectively can reduce false negative result.But it is different that SAT technology applies institute's problems faced in the detection of different sorts virus, the characteristic of concrete analysis virus is needed to carry out specialized designs.The research that there is no at present for the real-time fluorescence nucleic acid constant-temperature amplification detection technique of enterovirus (EV) is reported.
Summary of the invention
The object of this invention is to provide fast a kind of, split hair caccuracy, pollute and easily control, equipment is simple, the EV RNA detection method that cost is low.
A first aspect of the present invention, provide the amplification method of a kind of enterovirus EV, described amplification method comprises step: increase in reaction system, the Auele Specific Primer pair containing amplification enterovirus EV in described reaction system, and described primer pair comprises:
T7 primer: sequence is as shown in SEQ ID NO:3; With
NT7 primer: sequence is as shown in SEQ ID NO:4.
In another preference, in described reaction system, also comprise M-MLV ThermoScript II and t7 rna polymerase.
In another preference, described reaction system also comprises the capture probe of enterovirus EV.
In another preference, described reaction system also comprises the detection probes of enterovirus EV.
In another preference, one end mark fluorescent group of described capture probe, the other end mark quenching group.
In another preference, 5 ' end flag F AM fluorophor of described detection probes, and 3 ' of detection probes end mark DABCYL quenching group.
In another preference, the nucleotide sequence of described detection probes is as shown in SEQ ID NO:5.
In another preference, described capture probe can be combined with the target nucleic acids of enterovirus EV (EV RNA) sequence specific.
In another preference, described method also comprises: in another positive (contrast) reaction system or feminine gender (contrast) reaction system, carry out amplified reaction.
In another preference, in described control reaction system containing described Auele Specific Primer to mark (EV IC RNA), described capture probe and described detection probes in, EV.
In another preference, the nucleotide sequence of described capture probe is as shown in SEQ ID NO:2.
In another preference, described detection probes is used for and under t7 rna polymerase effect, copies the RNA produced according to the DNA of described EV target nucleic acids (EV RNA) copy specific combination.
In another preference, in described reaction system enterovirus EV or copy number be less than 100, being preferably 1-50 copy, is more preferably 1-10 copy.
In another preference, described method also comprises step: among amplified reaction process or afterwards, the fluorescent signal that the specific probe detecting enterovirus EV sends.
In another preference, described method is real-time fluorescence nucleic acid constant-temperature amplification method.
In another preference, also containing enterovirus to be measured or derived from described enterovirus in described reaction system.
In another preference, described determined nucleic acid is the nucleic acid from environmental sample or human excrement sample.
A second aspect of the present invention, provide the nondiagnostic detection method of a kind of enterovirus EV, it is characterized in that, described detection method comprises:
With the amplification method amplification testing sample such as described in first aspect present invention; With
Among amplified reaction process or afterwards, the fluorescent signal that the specific probe detecting enterovirus EV sends.
In another preference, described method also comprises and arranges one or more control group.
In another preference, described control group comprises: mark control group in EV positive controls, EV negative control group, EV.
A third aspect of the present invention, provide the detection kit of a kind of enterovirus EV, described test kit comprises:
(a) first container, and the Auele Specific Primer pair of amplification enterovirus being positioned at described container, described primer pair comprises:
T7 primer: sequence is as shown in SEQ ID NO:3; With
NT7 primer: sequence is as shown in SEQ ID NO:4;
And working instructions.
In another preference, described test kit is also containing one or more components being selected from lower group:
(b) capture probe;
(c) detection probes;
(d) EV interior label sequence; And/or
Mark detection probes in (e).
In another preference, in described test kit, also comprise one or more enzymes.
In another preference, described enzyme is M-MLV ThermoScript II and t7 rna polymerase.
In another preference, described M-MLV ThermoScript II and t7 rna polymerase are present in an enzyme liquid, and described capture probe is present in a nucleic acid extraction liquid, and described T7 primer, nT7 primer and EV detection probes are present in a detection liquid.
In another preference, described interior mark detection probes is present in EV and detects in liquid.
In another preference, be designated as competitive interior mark in described EV, and use same pair of primers (T7 and nT7) with EV Target nucleotides (EV RNA).
In another preference, in described test kit, also comprise EV positive control and/or EV negative control.
In another preference, described capture probe is the capture probe be combined with the target nucleic acids of enterovirus (EV RNA) sequence specific; And/or
Described detection probes copies with according to the DNA of described EV target nucleic acids (EV RNA) the EV detection probes that the RNA produced copies specific combination.
In another preference, described capture probe and/or described detection probes also can be combined with the target nucleic acids of enterovirus EV (EV RNA) sequence specific.
In another preference, described test kit also comprises the one or more features being selected from lower group:
Described detection probes comprises the nucleotide sequence as shown in SEQ ID NO:5; And/or
Described capture probe comprises the nucleotide sequence as shown in SEQ ID NO:2; And/or
Described EV interior label sequence comprises the nucleotide sequence as shown in SEQ ID NO:7; And/or
Described interior mark detection probes comprises the nucleotide sequence as shown in SEQ ID NO:6.
In another preference, described test kit comprises following component: lysate, nucleic acid extraction liquid, washings, EV reaction solution, EV detect mark in liquid, SAT enzyme liquid, EV positive control, EV negative control and EV;
Wherein,
Described lysate comprises ammonium sulfate ((NH
4)
2sO
4) and HEPES;
Described nucleic acid extraction liquid comprises capture probe and magnetic bead;
Described washings comprises NaCl and SDS;
Described EV reaction solution comprises dNTP and NTP;
Described EV detects liquid and comprises T7 primer, nT7 primer, EV detection probes and interior mark detection probes;
Described SAT enzyme liquid comprises M-MLV ThermoScript II, t7 rna polymerase;
Described EV positive control comprises the in-vitro transcription RNA dilution of the UTR gene that enterovirus 5 ' is held;
Described EV negative control is that preferably, described negative control is physiological saline not containing enterovirus target nucleic acids (EV RNA) sequence or the solution not containing enterovirus;
Mark in EV: containing marking RNA (EV IC RNA, sequence is as shown in SEQ ID NO:7 in sequence table) dilution in EV.
In another preference, described test kit comprises A box and B box, wherein,
A box is sample disposal unit, comprises described lysate, described nucleic acid extraction liquid and described washings;
B box is nucleic acid amplification detecting unit, comprises described EV reaction solution, EV detects in liquid, SAT enzyme liquid, EV positive control, EV negative control and EV and marks.
A fourth aspect of the present invention, provide the Auele Specific Primer pair of a kind of enterovirus of increasing, described primer pair comprises:
T7 primer: sequence is as shown in SEQ ID NO:3; With
NT7 primer: sequence is as shown in SEQ ID NO:4.
A fifth aspect of the present invention, provides the purposes of a kind of test kit as described in third aspect present invention or the primer pair as described in fourth aspect present invention, it is characterized in that, for detecting whether there is enterovirus EV in environmental sample or human excrement sample.
In another preference, described environmental sample comprises: the instant food such as the fishery products such as water source, shellfish, fruits and vegetables, salad etc.; Described human excrement sample comprises the vomitus, movement etc. of morbidity patient.
Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.
Accompanying drawing explanation
The primer pair of Fig. 1 comparison group and the fluoroscopic examination result of probe sequence;
The fluoroscopic examination result of the primer pair that Fig. 2 is of the present invention group and probe sequence;
Fig. 3 is the target fluoroscopic examination result that clinical throat swab sample SAT detects;
Fig. 4 is the interior mark fluoroscopic examination result that clinical throat swab sample SAT detects;
Fig. 5 is the target fluoroscopic examination result that clinical fecal sample SAT detects;
Fig. 6 is the interior mark fluoroscopic examination result that clinical fecal sample SAT detects;
Fig. 7 is for adopting the result of enterovirus detection kit (PCR-fluorescence probe method) control test clinical sample ight soil.
Embodiment
The present inventor, through long-term and deep research, through a large amount of screening and checking, develops high specific, high-sensitive primer special pair.When using the nucleic acid of this specific primer sequence to enterovirus EV to increase, have high specific, the excellent advantages such as highly sensitive, may be used for detecting enterovirus EV exactly.Based on above-mentioned discovery, contriver completes the present invention.
Primer and probe
As used herein, the term Auele Specific Primer of EV " amplification enterovirus " refer to such primer (to), the amplified production that it amplifies has the complementary strand sequence of enterovirus EV.
In design of primers, in order to meet better at present to the needs that enterovirus EV detects, the present inventor have selected EV virus 5 ' in the UTR gene held without secondary structure and high conservative section as amplified target sequence area (its nucleotide sequence is as shown in SEQ ID NO:1).Preferred primer sequence comprises the primer pair as SEQ ID NO:3 and SEQ ID NO:4 forms.
In amplification method of the present invention, except primer, in reaction system, one or more probe can also be comprised, as detection probes, capture probe etc.
As used herein, term " capture probe of enterovirus EV " refers to the nucleotide sequence that can be combined with the target nucleic acids of enterovirus EV (EV RNA) sequence specific.A kind of preferred capture probe of the present invention has the nucleotide sequence as shown in SEQ ID NO:2.More preferably, described capture probe specific combination is in target nucleic acids (EV RNA) sequence of enterovirus EV.In another preference, when described enterovirus EV contain in EV mark (EV IC RNA) time, described capture probe can also with described EV interior label sequence specific combination, for designing negative control group.
EV detection probes is molecular beacon, it is the molecular probe of a class high specific, hypersensitivity, by two ends respectively covalent labeling be made up of the single stranded nucleic acid molecule of fluorescence dye and quencher, in hair clip type or loop-stem structure, the loop section of molecular beacon and target-complementary, two becomes stem due to complementation, and molecular beacon probe is compared with linear TaqMan probe, because opening of its hairpin structure needs certain power, thus specificity is better than linear probe.A kind of preferred detection probes of the present invention has the nucleotide sequence as shown in SEQ ID NO:5.
Primer sequence of the present invention and probe sequence, according to primed probe principle of design, use DNAStar, DNAMAN software and engineer to be used for primer special and the probe sequence of real-time fluorescence nucleic acid constant-temperature amplification detection enterovirus (EV).
In primer, probe design process, create all multipair primers and probe sequence, such as T7 primer sequence: 5 ' aatttaatacgactcactatagggagaATTGTCACCATAAGCAGCCA3 ', nT7 primer sequence: 5 ' CCCCTGAATGCGGCTAATCC3', detection probes sequence is 5 ' aCGGAACCGACU ACUUUccgU3 ' (5 ' end FAM fluorescent mark, 3 ' end uses DABCYL fluorescent mark), and SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 etc.Reaction system described in above-mentioned each sequence patent 201110071284.X increased, comparison sensitivity technique also chooses comparatively the superior.
Result is as Fig. 1, Fig. 2 (Fig. 1 is comparison group, and Fig. 2 is of the present invention group).The sensitivity of comparison group can detect 10
2copies/ reacts, and the sensitivity of of the present invention group can reach 10copies/ reaction, and therefore screening obtains primer pair: SEQ ID NO:3, SEQ ID NO:4; Probe sequence: SEQ ID NO:5.
Contrast
Make amplification failure because SAT amplification is subject to various factors, test kit user of service error in judgement is got the wrong sow by the ear, therefore, contrast can also be set in test kit of the present invention, to get rid of the situation of detected result distortion.
The object of reference that can arrange in the present invention comprises: the one or more contrasts in EV positive control, EV negative control and EV in mark.
EV positive control can be the RNA for in-vitro transcription, does not have biologic activity.By detecting positive control, provable kit test method and material errorless, ensure the accuracy detected, the difference between each repeatability of detecting and stability and test kit batch can be monitored simultaneously.
In addition, critical weak sun contrast can be prepared by positive reference substance and (be mixed into diluent with physiological saline and lysate by 1:1, dilution positive control 1000 is doubly as critical weak sun contrast), the situation of checked operation when being in threshold value state can be pointed out, by critical weak sun contrast periodic detection SAT laboratory, indoor quality control can be carried out, with the situation preventing testing process from occurring undetected (false negative).
In EV, mark can be the RNA of in-vitro transcription, does not have biologic activity.Mark in the competitiveness of mark as EV RNA in EV, as object of reference, for preventing the generation of false negative result, interior target sample can be had by detecting to add, whole amplification reaction system can be understood whether suppressed, better pointing out false negative.
Negative control can get rid of false positive, under proper use of kit test method and material context, can ensure the specificity detected.
In another preference, the EV RNA of described in-vitro transcription is by preparing with following method:
(1) synthesize by chemical synthesis the UTR gene fragment (the positive segment of EV, its nucleotide sequence is as shown in SEQ ID NO:8 in sequence table) that EV5 ' holds;
(2) the UTR gene fragment of being held by EV5 ' is inserted into
in carrier, build EV positive control plasmid;
(3) EV positive control plasmid is transformed in bacillus coli DH 5 alpha, called after
bacterial strain, is stored in-70 DEG C;
(4) from
extract in bacterial strain
plasmid, carries out transcribe rna by plasmid, purifying remove DNA, and quantitatively, qualification RNA.
In another preference, the EV IC RNA of the in-vitro transcription in described EV in mark is prepared with following method:
(1) synthesize one section except probe in detecting regional sequence difference by chemical synthesis, other sequences are substantially with EV target sequence region (in EV, tap is broken, and its nucleotide sequence is as shown in SEQ ID NO:9 in sequence table);
(2) fragment is cloned into
in carrier, build mark plasmid in EV;
(3) Plastid transformation is marked in EV in bacillus coli DH 5 alpha, called after
bacterial strain, is stored in-70 DEG C;
(4) from
extract in bacterial strain
plasmid, plasmid is carried out transcribe rna purifying remove DNA, and quantitatively, qualification in mark RNA.
Detection method
Present invention also offers the detection method of enterovirus EV, comprise and with Auele Specific Primer of the present invention, testing sample being increased; With
In amplified reaction process or afterwards, detect the amplified production of enterovirus EV, such as, by fluorescent signal that detection specificity probe sends.
Described method optionally can also comprise and arranges one or more control group, as mark group in EV positive controls, EV negative control group, EV etc.
In the present invention, detection method by conventional PCR method, also can use the diverse ways such as real-time fluorescence detection method.A kind of particularly preferred method is real-time fluorescence nucleic acid constant-temperature amplification method.
Real-time fluorescence nucleic acid constant-temperature amplification technology (SAT)
Nucleic acid constant-temperature amplification real-time fluorescence detection technique, its principle is that RNA constant-temperature amplification and real-time fluorescence are detected a kind of novel nucleic acids detection technique combined.First realized by M-MLV ThermoScript II, T7RNA polymerase and optimization probe (optimal probe, patent pending) technology simultaneously.ThermoScript II is for generation of a DNA copy of target nucleic acids (RNA), T7RNA polymerase produces multiple RNA copy from DNA copy, copy specific combination with fluorescently-labeled optimization probe and these RNA, thus produce fluorescence, this fluorescent signal can be caught by detecting instrument.Assay, according to the time of occurrence of real-time fluorescent signals and intensity, judges assay in conjunction with positive control, negative control and interior mark signal.
In the present invention, enterovirus (EV) universal detection technique by using high specific and highly sensitive primer special pair, and with the use of special capture probe, can efficiently, specificity catches the RNA of EV.Nucleic acid amplification uses M-MLV ThermoScript II and t7 rna polymerase to realize simultaneously, ThermoScript II is for generation of a DNA copy of target nucleic acids RNA, t7 rna polymerase produces multiple RNA copy from DNA copy, specific combination is copied with the RNA produced after fluorescently-labeled optimum detection probe and amplification, thus generation fluorescence, this fluorescent signal can be caught by detecting instrument.
Test kit
The invention provides the detection kit of a kind of enterovirus EV, comprising:
The Auele Specific Primer pair of (a) amplification enterovirus, the amplified production that described primer amplification goes out has the complementary strand sequence of enterovirus EV;
(b) capture probe; With
(b) detection probes.
Described primer pair is a pair EV amplimer copied for the DNA producing EV target nucleic acids (EV RNA) under the effect of M-MLV ThermoScript II, in a preference of the present invention, described EV amplimer comprises: T7 primer: SEQ ID NO:3; With nT7 primer: SEQ ID NO:4.
Described capture probe is the capture probe be combined with the target nucleic acids of enterovirus (EV RNA) sequence specific; Described detection probes copies with according to the DNA of described EV target nucleic acids (EV RNA) the EV detection probes that the RNA produced copies specific combination.In another preference, described capture probe and/or described detection probes also can be combined with the target nucleic acids of enterovirus EV (EV RNA) sequence specific.
Described enterovirus EV can also be designed to containing mark (EV IC RNA) in EV, preferably, when enterovirus contains timestamp in EV, described capture probe and detection probes be designed to can also with described EV interior label sequence specific combination, form mark control group in EV.In another preference, be designated as competitive interior mark in described EV, and use same pair of primers (T7 and nT7) with EV Target nucleotides (EV RNA).
One or more enzymes can also be comprised in described test kit.As M-MLV ThermoScript II and t7 rna polymerase.In another preference, described M-MLV ThermoScript II and t7 rna polymerase are present in an enzyme liquid, and described capture probe is present in a nucleic acid extraction liquid, and described T7 primer, nT7 primer and EV detection probes are present in a detection liquid.
In another preference, described test kit comprises following component: lysate, nucleic acid extraction liquid, washings, EV reaction solution, EV detect mark in liquid, SAT enzyme liquid, EV positive control, EV negative control and EV; Wherein, described lysate comprises ammonium sulfate ((NH
4)
2sO
4) and HEPES;
Described nucleic acid extraction liquid comprises capture probe and magnetic bead;
Described washings comprises NaCl and SDS;
Described EV reaction solution comprises dNTP and NTP;
Described EV detects liquid and comprises T7 primer, nT7 primer, EV detection probes and interior mark detection probes;
Described SAT enzyme liquid comprises M-MLV ThermoScript II, t7 rna polymerase;
Described EV positive control comprises the in-vitro transcription RNA dilution of the UTR gene that enterovirus 5 ' is held;
Described EV negative control is that preferably, described negative control is physiological saline not containing enterovirus target nucleic acids (EV RNA) sequence or the solution not containing enterovirus;
Mark in EV: containing marking RNA (EV IC RNA, sequence is as shown in SEQ ID NO:7 in sequence table) dilution in EV.
In another preference, in a reacton of described test kit, 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 Μ (being preferably 5-25 μ Μ), magnetic bead 50-500mg/L (being preferably 50-250mg/L);
(3) washings: HEPES5-50mM, NaCl50-500mM, 1%SDS, EDTA1-10mM;
(4) EV reaction solution: Tris10-50mM, MgCl210-40mM, dNTP0.1-10mM (being preferably 0.5-5mM), NTP1-20mM (being preferably 1-10mM), PVP401-10%, KCl5-40mM;
(5) EV detects liquid: EV amplimer and EV detection probes are dissolved in TE solution (mixed solution of 10mM Tris and 1mM EDTA) formulated, each primer and concentration and probe concentration are reacted at 5-10pmol/; Wherein T7 primer concentration is preferably 7.5pmol/ reaction, and nT7 primer concentration is preferably 7.5pmol/ reaction, and EV 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/ reacts (being preferably 500-1500U/ reaction), t7 rna polymerase 200-2000U/ reacts (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) EV positive control; Containing 10
5-10
8the in-vitro transcription RNA dilution of the UTR gene that copy/mL enterovirus 5 ' is held;
(8) EV negative control: not containing enterovirus target nucleic acids (EV RNA) sequence or the solution not containing enterovirus;
(9) mark in EV: containing 10
5copy/mL EV IC RNA (sequence is as shown in SEQ ID NO:7 in sequence table) dilution.
In another preference, described test kit also comprises one or more container, and each above-mentioned component can lay respectively in one or more container, and in another preference, described test kit comprises A box and B box, wherein,
A box is sample disposal unit, comprises described lysate, described nucleic acid extraction liquid and described washings;
B box is nucleic acid amplification detecting unit, comprises described EV reaction solution, EV detects in liquid, SAT enzyme liquid, EV positive control, EV negative control and EV and marks.
In another preference, described detection probes comprises the nucleotide sequence as shown in SEQ ID NO:5, and/or described capture probe comprises the nucleotide sequence as shown in SEQ ID NO:2.
Utilize above test kit to carry out the detection of real-time fluorescence nucleic acid constant-temperature amplification to enterovirus, comprise the following steps:
1) with the enterovirus in lysate cracking testing sample, the lysate containing enterovirus is obtained;
2) to step 1) lysate in add nucleic acid extraction liquid and and EV IC RNA, be combined with magnetic bead again after capture probe is combined with target or interior mark nucleic acid specificity, wash with washings, remove the nucleic acid be not combined with magnetic bead, obtain nucleic acid (RNA) and the EV IC RNA of enterovirus;
3) by step 2) nucleic acid (RNA) of enterovirus that extracts and EV IC RNA add and detect in the first stage reactant that liquid forms by EV reaction solution and EV, at 60 DEG C, incubation is after 10 minutes, incubation 5 minutes at 42 DEG C again, then subordinate phase enzyme reaction thing SAT enzyme liquid is added, start thus at 42 DEG C, to continue incubation 60 minutes, with the change 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 produced according to fluorescent signal carries out qualitative detection with reference to marking detected result in EV positive control, EV negative control and EV to testing sample.
In above-mentioned detection operation, described step 1) in testing sample be throat swab or ight soil.
Compared with detecting with existing EV, the present invention has the following advantages:
(1) high specific, high purity, low stain: for the preferred capture probe of EV target nucleic acid design, can efficiently, specificity catches the RNA of EV.Meanwhile, owing to taking enclosed constant temperature amplification detection system, without the need to opening reaction system in whole reaction process, the pollution of amplicon is thus avoided.
(2) rapid detection: the amplification of nucleic acid synchronously carried out, and do not have lifting and the circulation of temperature in whole process in same closed system with detection, thus required time shortens greatly, and augmentation detection only needs 60 minutes.
(3) easily control is polluted: compared with real-time fluorescence PCR, amplified production of the present invention is that RNA, RNA very easily degrade at occurring in nature, so Environmental capacity is easier to.
(4) equipment is simple, and cost is low: compared with real-time fluorescence quantitative PCR, and the present invention's instrument used circulates without the need to heating and cooling, and thus design and production cost significantly reduce.
In sum, test kit of the present invention can detect the EV RNA in swab or ight soil, there is specificity high, highly sensitive (can reach 10 copies/reaction), pollute the feature of low (amplified production RNA is easy to degrade under physical environment) and rapid detection (completing augmentation detection in 60 minutes), play a significant role in the clinical diagnosis of enterovirus early infection, have a extensive future.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number are weight percent and parts by weight.
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 Products, and the reagent such as NTPs, dNTPs and other instruments are conventional commercially available product.
Embodiment 1 detects the primer special of enterovirus (EV) and the design of probe for real-time fluorescence nucleic acid constant-temperature amplification
The present invention selects EV virus 5 ' in the UTR gene held without secondary structure and high conservative section as amplified target sequence area (its nucleotide sequence is as shown in SEQ ID NO:1 in sequence table), according to primed probe principle of design, use DNAStar, DNAMAN software and engineer to be used for primer special and the probe sequence of real-time fluorescence nucleic acid constant-temperature amplification detection enterovirus (EV), obtain following concrete sequence:
Article (1) one, the capture probe (TCO that can be combined with the target nucleic acids of enterovirus (EV) (EV RNA) sequence specific, Target Capture Oligo), the nucleotides sequence of described capture probe is classified as: 5 ' TGCACACCGGATGGCCAATCCAATAAAAAAAAAAAAAAAAAAAAAAAA AAAAAA3 ' (SEQ ID No.:2);
(2) the EV amplimers copied for the DNA producing EV target nucleic acids (EV RNA) under the effect of M-MLV ThermoScript II for a pair, described EV amplimer is made up of T7 primer and nT7 primer, T7 primer sequence is 5 ' AATTTAATACGACTCACTATAGGGAGAATTGTCACCATAAGCAG CCA3 ' (SEQ ID No.:3), and nT7 primer sequence is 5 ' CCCCTGAATGCGGCTAATCC3 ' (in sequence table SEQ ID NO:4);
Article (3) one, for copying according to the DNA of described EV target nucleic acids (EV RNA) the EV detection probes that the RNA produced copies specific combination with under t7 rna polymerase effect, the nucleotides sequence of described EV detection probes is classified as 5 ' CCGGAACCGACUACUCCGG3 ' (in sequence table SEQ ID NO:5), 5 ' end uses FAM fluorescent mark, and 3 ' end uses DABCYL fluorescent mark.
In primer, probe design process, create all multipair primers and probe sequence, such as: T7 primer sequence: 5 ' aatttaatacgactcactatagggagaATTGTCACCATAAGCAGCCA3 ', nT7 primer sequence: 5 ' CCCCTGAATGCGGCTAATCC3 ', detection probes sequence is 5 ' aCGGAA CCGACUACUUUccgU3 ', 5 ' end uses FAM fluorescent mark, and 3 ' end uses DABCYL fluorescent mark.
With sequence 3,4,5 comparison sensitivity technique of the present invention, choose comparatively the superior.Reaction system is with reference to described in Chinese patent 201110071284.X.
Result:
Test result is as shown in Fig. 1 and Fig. 2 (Fig. 1 is comparison group, and Fig. 2 is of the present invention group).Can find out from result, the sensitivity of comparison group only can draw detected result when 100 copies/reaction, and of the present invention group that adopts primer to optimize especially, its sensitivity at least improves an order of magnitude out of a clear sky, can reach 10 copy/reactions or lower.As apparent from curve shape also can, of the present invention group is better than comparison group.
The above results shows, the primer pair shown in SEQ ID NO.:3 and SEQ ID NO.:4 is the high best primer of detection sensitivity.
For ease of carrying out interpretation of result, mark (SEQ ID NO:7) in the EV also increased in reagents box, devise interior mark detection probes, in EV, mark has identical PBR with EV Target nucleotides (EV RNA), nucleotide sequence between two primers or arrangement difference, make it can not be combined with detection probes, but can be combined with interior mark probe, in described EV, mark builds by EV target template rite-directed mutagenesis and obtains, can with capture probe specific binding, described interior mark detection probes is and EV detection probes sequence, the probe that fluorescent mark is different, the nucleotides sequence of described interior mark detection probes is classified as 5 ' CCGACAGUGAUACGAGAGTCGG3 ' (in sequence table SEQ ID NO:6), 5 ' end mark HEX fluorophor, 3 ' end mark DABCYL quenching group.
Embodiment 2 prepares the real-time fluorescence nucleic acid isothermal amplification detection kit of enterovirus (EV)
The primer special utilizing embodiment 1 to provide and probe, obtain the real-time fluorescence nucleic acid isothermal amplification detection kit of enterovirus of the present invention (EV).This test kit includes the components such as capture probe (TCO, Target Capture Oligo), T7 primer, nT7 primer, EV detection probes, interior mark detection probes, interior mark, M-MLV ThermoScript II and t7 rna polymerase.
| Capture probe | SEQ ID NO.:2 |
| T7 primer | SEQ ID NO.:3 |
| NT7 primer | SEQ ID NO.:4 |
| EV detection probes | SEQ ID NO:5 |
| Interior mark detection probes | SEQ ID NO:6 |
| Interior label sequence | SEQ ID NO:7 |
Described capture probe is present in nucleic acid extraction liquid, described T7 primer, nT7 primer and EV detection probes, interior mark detection probes is present in EV and detects in liquid, described M-MLV ThermoScript II and t7 rna polymerase are present in SAT enzyme liquid, specifically, the A box (sample disposal unit) that described test kit is divided into 2-30 DEG C the to store and-15--35 DEG C of B box (nucleic acid amplification detecting unit) stored, A box comprises lysate, nucleic acid extraction liquid and washings, B box comprises EV reaction solution, EV detects liquid, SAT enzyme liquid, EV positive control, mark in EV negative control and EV, 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 μM (being preferably 5-25 μM) and magnetic bead 50-500mg/L (being preferably 50-250mg/L);
Washings: mainly containing 1% (V/V) SDS.
B box (nucleic acid amplification detecting unit) consists of:
EV reaction solution: containing dNTP0.1-10mM (being preferably 0.5-5mM), NTP1-20mM (being preferably 1-10mM);
EV detects liquid: containing primer and probe, the concentration of each primer and probe is reacted at 5-10pmol/, wherein T7 primer concentration is preferably 7.5pmol/ reaction, nT7 primer concentration is preferably 7.5pmol/ reaction, EV detection probe concentrations is preferably 5pmol/ reaction, and interior mark detection probe concentrations is preferably 5pmol/ reaction;
SAT enzyme liquid: react (being preferably 500-1500U/ reaction) containing M-MLV ThermoScript II 400-4000U/, t7 rna polymerase 200-2000U/ reacts (being preferably 500-1000U/ reaction);
EV positive control; Containing 10
5-10
8the in-vitro transcription RNA dilution of the UTR gene that copy/mL enterovirus 5 ' is held;
EV negative control: not containing enterovirus target nucleic acids (EV RNA) sequence or the solution not containing enterovirus, as physiological saline;
Mark in EV: containing 10
5mark RNA dilution (in sequence table SEQ ID NO:7) in copy/mL EV.
The all reagent comprised in test kit all can obtain by pointing out preparation in conventional manner or business purchase obtains.
Specifically, in each reacton, the concrete assembly of described test kit all ingredients is 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 Μ (being preferably 5-25 μ Μ), magnetic bead 50-500mg/L (being preferably 50-250mg/L);
(3) washings: HEPES5-50mM, NaCl50-500mM, 1%SDS, EDTA1-10mM;
(4) EV reaction solution: Tris10-50mM, MgCl
210-40mM, dNTP0.1-10mM (being preferably 0.5-5mM), NTP1-20mM (being preferably 1-10mM), PVP401-10%, KCl5-40mM;
(5) EV detects liquid: EV amplimer and EV detection probes are dissolved in TE solution (mixed solution of 10mM Tris and 1mM EDTA) formulated, each primer and concentration and probe concentration are reacted at 5-10pmol/; Wherein T7 primer concentration is preferably 7.5pmol/ reaction, and nT7 primer concentration is preferably 7.5pmol/ reaction, and EV 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/ reacts (being preferably 500-1500U/ reaction), t7 rna polymerase 200-2000U/ reacts (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) EV positive control; Containing 10
5-10
8the in-vitro transcription RNA dilution of the UTR gene that copy/mL enterovirus (EV) 5 ' is held;
(8) EV negative control: not containing enterovirus (EV) target nucleic acids (EV RNA) sequence or the solution not containing enterovirus;
(9) mark in EV: containing 10
5mark RNA (sequence is as shown in SEQ ID NO:7 in sequence table) dilution in copy/mL EV.
The EV RNA of the in-vitro transcription in EV positive control, can prepare gained by multiple method, wherein a kind of preparation method is as follows:
(1) synthesize by chemical synthesis the UTR gene fragment (its nucleotide sequence is as shown in SEQ ID NO:8 in sequence table) that EV5 ' holds;
(2) the UTR gene fragment of being held by EV5 ' is inserted into
in carrier, build EV positive control plasmid;
(3) EV positive control plasmid is transformed in bacillus coli DH 5 alpha, called after
bacterial strain, is stored in-70 DEG C;
(4) from
extract in bacterial strain
plasmid, carries out transcribe rna by plasmid, purifying remove DNA, and quantitatively, qualification RNA.
The EV IC RNA of the in-vitro transcription in EV in mark, can prepare gained by multiple method.Wherein a kind of preparation method is as follows:
(1) synthesize one section except probe in detecting regional sequence difference by chemical synthesis, other sequences are substantially with EV target sequence region (its nucleotide sequence is as shown in SEQ ID NO:9 in sequence table);
(2) fragment is cloned into
in carrier, build mark plasmid in EV;
(3) Plastid transformation is marked in EV in bacillus coli DH 5 alpha, called after
iC bacterial strain, is stored in-70 DEG C;
(4) from
extract in IC bacterial strain
iC plasmid, carries out transcribe rna by plasmid, purifying remove DNA, and quantitatively, qualification in mark RNA.
The real-time fluorescence nucleic acid constant-temperature amplification of embodiment 3 clinical sample throat swab detects
Detect the enterovirus in clinical sample throat swab with test kit of the present invention (composition is shown in embodiment 2), concrete grammar comprises the following steps:
1, sample collection, transport and preservation
Carry out collection of specimens by clinicist according to practical situation, detection sample is throat swab, and acquisition method is: special sampling cotton swab pharynx rear wall and both sides pharyngeal tonsil part, invade in 3-5mL physiological saline by swab, sealing censorship.After sample collection, in 48 hours, Enterovirus surveillance network laboratories (or-70 DEG C of preservations are to be measured, send in a week) is delivered in 4 DEG C of preservations.
2, nucleic acid extraction
2.1 add 200 μ l lysate (HEPES50mM, (NH in sample processing tube (1.5mL centrifuge tube)
4)
2sO
435mM) He 200 μ l swab washing lotions (with the sample solution under brine, be not enough to physiological saline and supply), with the enterovirus in lysate cracking testing sample, obtain the lysate containing enterovirus.
2.2 add 100 μ l nucleic acid extraction liquid (HEPES200mM, EDTA100mM, LiCl800mM, capture probe 15 μ Μ, magnetic bead 150mg/L) and 10 μ l inner mark solutions (containing 10 in sample processing tube (1.5mL centrifuge tube)
5mark RNA in copy/mL EV), mixing, 60 DEG C are incubated 5 minutes, and room temperature places 10 minutes.
Sample processing tube is placed on magnetic bead separating device by 2.3, leaves standstill 5-10 minute.Be adsorbed in after tube wall until magnetic bead, keep sample processing tube on magnetic bead separating device, inhale and abandon liquid, retain magnetic bead.Add 1mL washings (HEPES25mM, NaCl150mM, 1%SDS, EDTA2.5mM; ) leave standstill 5-10 minute after shaken well, 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 answers 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 EV reaction solution+2.5 μ l EV detect liquid) in sample processing tube washs magnetic bead.EV reaction solution specifically comprises Tris15mM, MgCl
215mM, dNTP2.5mM, NTP3mM, PVP401%, KCl10mM; It is 7.5pmol/ reaction that EV detects T7 primer concentration in liquid, and nT7 primer concentration is 7.5pmol/ reaction, and EV detection probe concentrations is 5pmol/ reaction, and interior mark detection probe concentrations is 5pmol/ reaction.
The 3.2 above-mentioned reaction detection liquid 30 μ l getting vibration mixing add to clean micro-reaction pipe, and with 7500 type PCR instrument (American AB I Products) 60 DEG C insulation 10 minutes, 42 DEG C were incubated 5 minutes; In micro-reaction pipe, add the SAT enzyme liquid that 10 μ l have been preheated to 42 DEG C, 1200rpm vibrates and mixed 15 seconds.Containing M-MLV ThermoScript II 1500U/ reaction, t7 rna polymerase 1000U/ reaction, 10mM HEPES pH7.5,15mM N-acetyl-L-cysteine (N-acetyl-L-cysteine), 0.15mM zinc acetate (zinc acetate), 20mM trehalose (trehalose), 100mM Tris-HCl pH8.0,80mM KCl, 0.25mM EDTA, 0.5% (v/v) Triton X-100 and 30% (v/v) glycerol (glycerol) in SAT enzyme liquid.
Micro-reaction pipe is gone to constant-temperature fluorescence detector device (ABI7500 fluorescent quantitation instrument, ABI Products) by 3.3 fast, and 42 DEG C are reacted 60 minutes, set every 1 minute and detect first order fluorescence, detect 60 times altogether; Fluorescein channel selecting FAM passage (target signal detection, F1) and VIC passage (HEX and VIC wavelength is close, interior mark signal detection, F2).
4, result judges
According to the curve that SAT amplification obtains, setting threshold line, reads dt value, result of determination.
Threshold setting: with the vertex of 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 judges:
F1 passage: the sample of dt≤55 is positive; The sample suggestion of 55 < dt < 60 detects again, and the sample of detected result F1 passage: dt < 60 is positive.
2. negative findings judges: F1 passage dt is without numerical value or as 60, and F2 passage simultaneously: dt≤55 is then negative.
Quality control: each detection all arranges positive control and negative control, and result should meet positive control F1 passage simultaneously: dt≤55; Negative control F1 passage: dt is without numerical value or be 60, simultaneously F2 passage: dt≤55, otherwise this time detected result be considered as invalid.
5, result
Enterovirus throat swab clinical sample is numbered EV oropharyngeal swab specimen 1-5, separately establishes negative control (not containing enterovirus target nucleic acids (EV RNA) sequence or the solution not containing enterovirus), positive control (containing 10
7the in-vitro transcription RNA dilution of the UTR gene that copy/mL enterovirus 5 ' is held) each one.Result is as shown in Fig. 3 (F1 fluorescence channel) and Fig. 4 (F2 fluorescence channel), and according to the dt value situation of F1 passage and F2 passage, judge that sample 1,3,4 is as positive, sample 2,5 be feminine gender.This result is identical with gold standard virus culture result, show that test kit of the present invention is high for the enterovirus accuracy detected in clinical sample throat swab, but the upper machine augmentation detection time only needed to have for 60 minutes the feature of cycle short, highly sensitive, high specific, low stain and stable reaction.
The real-time fluorescence nucleic acid constant-temperature amplification of embodiment 4 clinical sample ight soil detects
This detecting pattern is another application of the invention: test kit composition is with embodiment 2, the production of reagent is carried out in GMP workshop, ight soil clinical sample carries out collection of specimens by clinicist according to practical situation, and acquisition method is: gather the stool sample that patient fell ill in 7 days.Stool collection amount 5 ~ 8g/ part, puts into aseptic urine collector after collection immediately, sealing censorship.After sample collection, in 48 hours, Enterovirus surveillance network laboratories is delivered in 4 DEG C of preservations, or-70 DEG C of preservations are to be measured, send in one week.Enterovirus ight soil clinical sample is numbered EV stool sample 1-4, separately establishes each one of negative control, positive control.Detection method is with embodiment 3.
The another synchronous Standard PCR that adopts carries out control test.
Use the detected result of test kit of the present invention as shown in Fig. 5 (F1 fluorescence channel) and Fig. 6 (F2 fluorescence channel), according to the dt value situation of F1 passage and F2 passage, judge that detected result is all the positive as sample 1-4, and Standard PCR detected result (Fig. 7) shows sample 3 for negative, there is difference.
By the SAT products as templates of sample 3, carry out pcr amplification, the rubber tapping of gained PCR primer is reclaimed, and transformed clone, order-checking, comparison EV sequence, result is consistent, proves that difference sample 3 is the positive, illustrates that this test kit has higher sensitivity.
This test kit designs for universal enterovirus, adopts real-time fluorescence constant-temperature amplification to detect RNA, easier than Standard PCR operation, and sensitivity, specificity are higher, only need within 60 minutes, can complete augmentation detection; Gene-amplification product is RNA but not DNA, easily degrades in the environment, low stain.Add in test kit competitive in mark can reduce further because system suppresses the false negative result that causes, guarantee that detected result is more accurate.
According to disclosure of the present invention, those skilled in the art too much need not test and can implement the present invention enterovirus required for protection (EV) universal real time fluorescent nucleic acid isothermal 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; or substitute preparation described here with some at the preparation that structure function chemically or on biology is relevant; 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 (12)
1. a non-diagnostic and the amplification method of the enterovirus EV of non-therapeutic, it is characterized in that, described amplification method comprises step: increase in reaction system, the Auele Specific Primer pair containing amplification enterovirus EV in described reaction system, and described primer pair comprises:
T7 primer: sequence is as shown in SEQ ID NO:3; With
NT7 primer: sequence is as shown in SEQ ID NO:4;
And the copy number of enterovirus EV is less than 100 in described reaction system.
2. the method for claim 1, is characterized in that, in described reaction system, the copy number of enterovirus EV is 1-50 copy.
3. the method for claim 1, is characterized in that, in described reaction system, the copy number of enterovirus EV is 1-10 copy.
4. the method for claim 1, is characterized in that, described reaction system also comprises the capture probe of enterovirus EV.
5. the method for claim 1, is characterized in that, described reaction system also comprises the detection probes of enterovirus EV.
6. as the method as described in arbitrary in claim 1-5, it is characterized in that, described method also comprises step: among amplified reaction process or afterwards, the fluorescent signal that the specific probe detecting enterovirus EV sends.
7. a nondiagnostic detection method of enterovirus EV, is characterized in that, described detection method comprises:
With amplification method amplification testing sample as described in claim 1; With
Among amplified reaction process or afterwards, the fluorescent signal that the specific probe detecting enterovirus EV sends.
8. a detection kit of enterovirus EV, is characterized in that, described test kit comprises:
(a) first container, and the Auele Specific Primer pair of amplification enterovirus being positioned at described container, described primer pair comprises:
T7 primer: sequence is as shown in SEQ ID NO:3; With
NT7 primer: sequence is as shown in SEQ ID NO:4;
And working instructions.
9. test kit as claimed in claim 8, is characterized in that, also containing one or more components being selected from lower group:
(b) capture probe;
The detection probes of the target nucleic acid sequence specific combination of (c) and enterovirus EV;
(d) EV interior label sequence; And/or
Mark detection probes in (e).
10. test kit as claimed in claim 9, is characterized in that, comprise the one or more features being selected from lower group:
The described nucleotide sequence comprised with the detection probes of the target nucleic acid sequence specific combination of enterovirus EV as shown in SEQ ID NO:5; And/or
Described capture probe comprises the nucleotide sequence as shown in SEQ ID NO:2; And/or
Described EV interior label sequence comprises the nucleotide sequence as shown in SEQ ID NO:7; And/or
Described interior mark detection probes comprises the nucleotide sequence as shown in SEQ ID NO:6.
The Auele Specific Primer pair of 11. 1 kinds of enteroviruses of increasing, is characterized in that, described primer pair comprises:
T7 primer: sequence is as shown in SEQ ID NO:3; With
NT7 primer: sequence is as shown in SEQ ID NO:4.
The purposes of 12. test kits as claimed in claim 8 or primer pair according to claim 11, is characterized in that, for non-diagnostic and non-therapeutic detect whether there is enterovirus EV in environmental sample.
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| CN101333564B (en) * | 2007-06-27 | 2011-02-23 | 上海仁度生物科技有限公司 | Method for extracting and purifying target nucleic acid and use thereof |
| CN101407846B (en) * | 2008-10-13 | 2011-05-11 | 浙江省疾病预防控制中心 | Fluorescent quantitative RT-PCR detection kit for enterovirus |
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