CN103525945B

CN103525945B - H1N1virus (2009) real-time fluorescence nucleic acid isothermal amplification detection kit

Info

Publication number: CN103525945B
Application number: CN201210225251.0A
Authority: CN
Inventors: 方亮; 冯金梦; 于明辉; 居金良
Original assignee: SHANGHAI RENDU BIOTECHNOLOGY CO Ltd
Current assignee: Shanghai Rendu Biotechnology Co., Ltd
Priority date: 2012-07-02
Filing date: 2012-07-02
Publication date: 2018-08-31
Anticipated expiration: 2032-07-02
Also published as: CN103525945A

Abstract

The invention discloses a kind of H1N1virus (2009) real-time fluorescence nucleic acid isothermal amplification detection kits.Including：The reagents such as capture probe, H1N1 (2009) detection primer T7 primers and nT7 primers, H1N1 (2009) detection probe, M MLV reverse transcriptase and T7 RNA polymerases.Kit of the present invention can detect H1N1 (2009) RNA in swab, have the characteristics that specific high, high sensitivity (up to 100copies/ reactions), pollute low (amplified production RNA is easy to degrade in a natural environment) and quickly detection (completion detection in conventional 50 minutes), will play a significant role in the clinical diagnosis of Influenza A H1N1 early infection.

Description

H1N1virus (2009) real-time fluorescence nucleic acid isothermal amplification detection kit

Technical field

The present invention relates to the Measurements for Biotechnique of virus, and in particular to by specific target capture technique and real-time fluorescence core Make in the real-time fluorescence nucleic acid constant-temperature amplification detection for the H1N1virus (2009) that sour constant-temperature amplification detection technique combines Primer, probe and related kit.

Background technology

Influenza virus is divided into first, second, the third three types according to nucleoprotein (NP) and stromatin (M) difference.Influenza A virus Genome is made of 8 negative strand RNA segments, is divided into 16 HA according to the difference of surface hemagglutinin (HA) and neuraminidase (NA) Hypotype and 9 NA hypotypes.

Influenza A virus is common influenza virus, is easily made a variation, most attack.It can cause the influenza of people and cause sometimes Pneumonia and other complication mainly the necrosis of trachea and bronchus ciliated columnar epithelial cells and fall off, some Strain energy Natural infection birds and mammal.With breaking out, spread rapidly, the characteristics of being widely current.Worldwide influenza Be very popular it is total occur 4 times, wherein 1918-1919 pandemics cause 20,000,000 people death.Since Chinese nearly half a century Influenza pandemic is total to be occurred 18 times, wherein being for 3 times to be very popular.

H1N1virus (2009) belongs to orthomyxovirus section influenza A virus category, and typical virus is in spherical. In March, 2009, Mexico break out H1N1 epidemic disease tides, hundred people are caused to infect.Epidemic situation travels to rapidly the whole world.World Health Organization's handle The Global influenza warning level that is very popular is increased to the 5th grade.The influenza is the porcine respiratory infectious disease caused by influenza A, Strain includes swine flu, the DNA genetic fragment of three kinds of influenza viruses of bird flu and human influenza, while possessing Asia Continent swine flu and African swine influenza virus feature.Although having found H1N1virus (2009) in pig body, at present still No evidence shows that animal is the infection sources.A small number of case disease progressions are rapid, and it is incomplete or decline respiratory failure, multi-organ function occur It exhausts, or even dead.H1N1virus directly can be transmitted to people from pig, also may occur in which person-to-person propagation, cause worldwide It is very popular, is a kind of major disease for endangering human health.

The laboratory examination method of currently used H1N1virus includes：1) immunological detection method, it is such as fast Fast influenza antigens detection, immunofluorescence technique (or IFA of assembly), this method is more demanding to operator；2) cell biology detects Method, it is common to have egg inoculation method and cell culture method.This method is more sensitive to isolation of virus, but needs 2-3 weeks, can not Realize getting up early quick diagnosis.3) molecular biology for detection.There is reverse transcriptase-PCR (Reverse at present Transcriptase-PCR, abbreviation RT-PCR) and Fluorescent quantitative PCR (Real Time Fluorescent Quantified PCR, abbreviation FQ PCR), RT-PCR uses the operation of " gene magnification+amplicon detection " Pattern easily causes the pollution of amplified matter, often results in the false positive or false negative phenomenon of experimental result.Though real-time fluorescence quantitative PCR Technically solves the above problem, sensitivity and accuracy are higher, but complicated for operation, and testing cost is relatively expensive, hinder It is used in the large-scale promotion of developing country.

Real-time fluorescence nucleic acid constant-temperature amplification detection technique (Simultaneous Amplification and Testing, Abbreviation SAT) it is that a kind of method directly quickly detecting RNA, nucleic acid amplification and detection are polymerize using M-MLV reverse transcriptase, T7RNA Enzyme and optimization probe technique are come while being realized.Reverse transcriptase is used to generate a DNA copy of target nucleic acids (RNA), T7 RNA Polymerase generates multiple RNA copies from DNA copy, and optimization probe and these RNA with fluorescent marker copy specific bond, To generate fluorescence, which can be captured by detecting instrument.Compared with the real-time fluorescence PCR of detection DNA, difference, The step of the former detection architecture more reverse transcription reaction, nucleic acid amplification carries out (42 DEG C) at one temperature, is followed without heat Ring.Nucleic acid amplification is carried out using M-MLV reverse transcriptases and T7 RNA polymerases, relative to other nucleic acid amplification technologies, reaction suppression Object processed is less, can effectively reduce false negative result.This SAT technology is applied for a patent by applicant and authorizes (ZL early period 200810111479.0), it is combined the method (ZL of magnetic bead-RNA beneficiation technologies extraction purification target RNAs 200810111478.6), inventor is prepared for the detection examination of H1N1virus (2009) real-time fluorescence nucleic acid constant-temperature amplification Agent box.

Invention content

A kind of the present invention provides detection cycles short, highly sensitive, high specific, low stain, stable reaction and it is easy to The real-time fluorescence nucleic acid constant-temperature amplification detection technique of the H1N1virus (2009) of popularization and application, including primer special, Probe, kit and its use.

H1N1virus (2009) real-time fluorescence nucleic acid isothermal amplification detection kit provided by the present invention, packet Containing a capture probe, a pair under the effect of M-MLV reverse transcriptase for generating H1N1 (2009) target nucleic acids (H1N1 (2009) RNA) DNA copy H1N1 (2009) detection primer T7 and nT7 and one for T7 RNA polymerases act on The lower RNA generated according to the DNA copy of the H1N1 (2009) target nucleic acids (H1N1 (2009) RNA) copies specific bond H1N1 (2009) detection probe.

The capture probe can be with the target nucleic acids of the H1N1virus (2009) as shown in sequence 1 in sequence table (H1N1 (2009) RNA) sequence specific combines, and when there is H1N1 (2009) internal standard (H1N1 (2009) IC RNA), preferably may be used also With H1N1 (2009) the interior label sequence specific bond, in the nucleotide sequence such as sequence table of the capture probe shown in sequence 2；Institute It states H1N1 (2009) detection primers to be made of T7 primers and nT7 primers, in T7 primer sequences such as sequence table shown in sequence 3, nT7 draws In object sequence such as sequence table shown in sequence 4；5 institute of sequence in the nucleotide sequence such as sequence table of H1N1 (2009) detection probe Show, 5 ' end flag F AM fluorophors, 3 ' end label DABCYL quenching groups.

Further, the kit also includes M-MLV reverse transcriptase and T7 RNA polymerases, the M-MLV reverse transcriptions Enzyme and T7 RNA polymerases are present in a SAT enzyme solutions, and the capture probe is present in a nucleic acid extraction liquid, and the T7 draws Object, nT7 primers and H1N1 (2009) detection probe are present in a H1N1 (2009) detection liquid.

Further the kit also includes H1N1 (2009) internal standards and internal standard detection probe；The H1N1 (2009) Inside be designated as competitive internal standard, can be specifically bound with capture probe, and with H1N1 (2009) Target nucleotides (H1N1 (2009) RNA) using same pair of primers (T7 and nT7), H1N1 (2009) internal standards H1N1 (2009) IC shown in sequence in sequence table 7 RNA；In the nucleotide sequence such as sequence table of the internal standard detection probe shown in sequence 6,5 ' end label HEX fluorophors, 3 ' ends DABCYL quenching groups are marked, and the internal standard detection probe is present in H1N1 (2009) detection liquid.

Further, the kit includes lysate, nucleic acid extraction liquid, cleaning solution, H1N1 (2009) reaction solution, H1N1 (2009) liquid, SAT enzyme solutions, H1N1 (2009) positive control, H1N1 (2009) negative controls and H1N1 (2009) internal standard are detected, In：

Lysate：Liquid containing ammonium sulfate ((NH₄)₂SO₄) and HEPES；

Nucleic acid extraction liquid：Containing capture probe and magnetic bead；

Cleaning solution：Containing NaCl and SDS；

H1N1 (2009) reaction solution：Containing dNTP and NTP；

H1N1 (2009) detects liquid：Primer containing T7, nT7 primers, H1N1 (2009) detection probes and internal standard detection probe；

SAT enzyme solutions：Reverse transcriptase containing M-MLV, T7 RNA polymerases；

H1N1 (2009) positive control；In-vitro transcription RNA dilutions containing H1N1virus (2009) HA genes；

H1N1 (2009) negative control：Without containing H1N1virus (2009) target nucleic acids (H1N1 (2009) RNA) sequence or the solution without containing influenza A virus, such as physiological saline；

H1N1 (2009) internal standard：Containing H1N1 (2009) internal standard RNA (in sequence such as sequence table shown in sequence 7) dilution.

Specifically, above-mentioned various reagents composition is as follows in a reacton in the kit：

(1) lysate：HEPES 25-250mM, (NH₄)₂SO₄5-50mM；

(2) nucleic acid extraction liquid：HEPES 50-400mM, EDTA 40-200mM, LiCl 400-2000mM, capture probe 1- 50 μM (preferably 5-25 μM), magnetic bead 50-500mg/L (preferably 50-250mg/L)；

(3) cleaning solution：HEPES 5-50mM, NaCl 50-500Mm, 1% SDS, EDTA 1-10mM；

(4) H1N1 (2009) reaction solution：Tris 10-50mM, MgCl₂10-40mM, dNTP 0.1-10mM are (preferably 0.5-5mM), NTP 1-20mM (preferably 1-10mM), PVP40 1-10%, KCl 5-40mM；

(5) H1N1 (2009) detects liquid：H1N1 (2009) detection primers and H1N1 (2009) detection probe are dissolved in TE It is formulated in solution (mixed liquor of 10mM Tris and 1mM EDTA), each primer and probe concentration is equal in 5-15pmol/ reactions It can；Wherein T7 primer concentrations are preferably 10pmol/ reactions, and nT7 primer concentrations are preferably 10pmol/ reactions, H1N1 (2009) inspections It is preferably 7pmol/ reactions to survey concentration and probe concentration, and internal standard detection probe concentrations are preferably 7pmol/ reactions；

(6) SAT enzyme solutions：M-MLV reverse transcriptase 400-4000U/ reactions (preferably 500-1500U/ reactions), T7 RNA are poly- Synthase 200-2000U/ reactions (preferably 500-1000U/ reactions), 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) H1N1 (2009) positive control；Containing 10²-10⁵The body of copy/mL H1N1viruses (2009) HA genes Outer transcription RNA dilutions；

(8) H1N1 (2009) negative control：Without containing H1N1virus (2009) target nucleic acids (H1N1 (2009) RNA) sequence or the solution without containing influenza A virus；

(9) H1N1 (2009) internal standard：Containing 10²-10⁵Copy/mL H1N1 (2009) IC RNA (sequence in sequence such as sequence table Shown in 7) in-vitro transcription RNA dilutions.

Another more specific form, the kit is by A boxes, that is, sample disposal unit and B boxes, that is, nucleic acid amplification detection unit Composition, wherein A boxes pack the lysate, the nucleic acid extraction liquid and the cleaning solution, and it is anti-that B boxes pack the H1N1 (2009) Answer liquid, H1N1 (2009) detections liquid, SAT enzyme solutions, H1N1 (2009) positive control, H1N1 (2009) negative controls and H1N1 (2009) internal standard.

H1N1 (2009) RNA of in-vitro transcription in H1N1 (2009) positive control is prepared with following methods：

1) chemical synthesis is used to synthesize in H1N1 (2009) HA genes without secondary structure and highly conserved section as expanding Target sequence region (in its nucleotide sequence such as sequence table shown in sequence 8)；

2) segment is cloned intoIn carrier, H1N1 (2009) positive control plasmid is built；

3) H1N1 (2009) positive control plasmid is transformed into bacillus coli DH 5 alpha, is named as-T-H1N1 (2009) bacterial strain is stored in -70 DEG C；

4) fromIt is extracted in-T-H1N1 (2009) bacterial strain- T-H1N1 (2009) plasmid, by plasmid into Row transcription RNA, purifying removal DNA, and quantitative, identification RNA.

H1N1 (2009) IC RNA of in-vitro transcription in H1N1 (2009) internal standard are prepared with following methods：

1) it synthesizes one section with chemical synthesis and removes probe in detecting regional sequence difference, the substantially same H1N1 (2009) of other sequences Target sequence region (in its nucleotide sequence such as sequence table shown in sequence 9)；

2) segment is cloned intoIn carrier, H1N1 (2009) internal standard plasmid is built；

3) H1N1 (2009) internal standard plasmid is transformed into bacillus coli DH 5 alpha, is named as-T-H1N1(2009) IC bacterial strains, are stored in -70 DEG C；

4) fromIt is extracted in-T-H1N1 (2009) IC bacterial strains- T-H1N1 (2009) IC plasmids, by matter Grain carries out transcription RNA, purifying removal DNA, and quantitative, identification internal standard RNA.

Special agent in H1N1virus (2009) the real-time fluorescence nucleic acid isothermal amplification detection kit, For one of the substance of following presentation：

It (1) can be with the target nucleic acids (H1N1 (2009) of H1N1virus (2009) shown in sequence in sequence table 1 RNA) the capture probe (TCO, Target Capture Oligo) that sequence specific combines, the nucleotide sequence of the capture probe As shown in sequence 2 in sequence table；

(2) it is used to generate H1N1 (2009) target nucleic acids (H1N1 (2009) RNA) under the effect of M-MLV reverse transcriptase H1N1 (2009) detection primer T7 and nT7 of DNA copy, in T7 primer sequences such as sequence table shown in sequence 3, nT7 primer sequences As shown in sequence 4 in sequence table；

(3) be used for T7 RNA polymerases effect under according to the H1N1 (2009) target nucleic acids (H1N1 (2009) RNA H1N1 (2009) detection probe for the RNA copy specific bonds that DNA copy) generates, H1N1 (2009) detection probe Nucleotide sequence such as sequence table in shown in sequence 5,5 ' end flag F AM fluorophors, 3 ' end label DABCYL quenching groups；

(4) internal standard and internal standard detection probe are inside designated as the competition of H1N1 (2009) nucleotide sequence (H1N1 (2009) RNA) Property internal standard, can be specifically bound with capture probe, and using same pair of primers (T7 and nT7 primers), interior target nucleotide sequence H1N1 (2009) the IC RNA as shown in sequence 7 in sequence table, 6 institute of sequence in the nucleotide sequence such as sequence table of internal standard detection probe Show, 5 ' end label HEX fluorophors, 3 ' end label DABCYL quenching groups.

The application method of the kit is used for the real-time fluorescence nucleic acid constant-temperature amplification of H1N1virus (2009) Detection, including following operation：

1) H1N1virus (2009) in sample to be tested is cracked with lysate, obtained containing Influenza A H1N1 The lysate of viral (2009) nucleic acid；

2) nucleic acid extraction liquid is added into the lysate of step 1), there are when H1N1 (2009) internal standard in kit, simultaneously H1N1 (2009) IC RNA are added, are combined again with magnetic bead after so that capture probe is combined with target or internal standard nucleic acid specificity, with washing Liquid washs, and removes the nucleic acid not combined with magnetic bead, obtains the nucleic acid (RNA) and H1N1 of H1N1virus (2009) (2009)IC RNA；

3) nucleic acid (RNA) of the H1N1virus (2009) of step 2) extraction and H1N1 (2009) ICRNA are added Enter in the first stage reactant being made of H1N1 (2009) reaction solutions and H1N1 (2009) detection liquid, 10 points are incubated at 60 DEG C Zhong Hou, then incubated at 42 DEG C 5 minutes, second stage enzyme reaction object SAT enzyme solutions are then added, thus start to continue at 42 DEG C It incubates 50 minutes, with the variation of detector synchronous recording fluorescence signal；The first stage reactant and second stage enzyme reaction The volume ratio of object is 3: 1；

4) time and intensity generated according to fluorescence signal is negative right with reference to H1N1 (2009) positive control, H1N1 (2009) Qualitative detection is carried out to sample to be tested according to H1N1 (2009) internal standard testing result.

The present invention provides a kind of H1N1virus (2009) real-time fluorescence nucleic acid isothermal amplification detection kit, It is detected using the kit, compared with existing H1N1 (2009) is detected, is had the following advantages：

(1) high specific, high-purity, low stain：It, can be high for the preferred capture probe of H1N1 (2009) target nucleic acid design The RNA of effect, specificity capture H1N1 (2009).Simultaneously as enclosed constant temperature amplification detection system is taken, in whole process Without opening reaction system, thus avoid the pollution of amplicon.

(2) quickly detection：By the amplification of nucleic acid synchronous progress in same closed system with detection, and in whole process There is no the lifting of temperature and cycle, thus required time greatly shortens, augmentation detection only needs 50 minutes.

(3) it pollutes easily-controllable：Compared with real-time fluorescence PCR, amplified production of the invention is RNA, RNA in nature easily Degradation, so contamination control is easier to.

(4) equipment is simple, at low cost：Compared with real-time fluorescence quantitative PCR, the instrument used in the present invention is followed without heating and cooling Ring, thus design and produce cost and be greatly reduced.

In conclusion kit of the present invention can detect H1N1 (2009) RNA in swab, have specificity high, sensitive Degree high (being reacted up to 100copies/) pollutes low (amplified production RNA is easy to degrade in a natural environment) and quickly detection (50 Minute completes augmentation detection) the characteristics of, it will play a significant role in the clinical diagnosis of H1N1virus early infection.

The present invention is described in further details with reference to specific embodiment.

Description of the drawings

The fluoroscopic examination result of Fig. 1 sensitivity

Fig. 2 specificity fluorescent testing results

The target fluoroscopic examination result of Fig. 3 clinic throat swab samples

The internal standard fluoroscopic examination result of Fig. 4 clinic throat swab samples

The testing result of Fig. 5 H1N1viruses (2009) RNA detection kits (fluorescent PCR method)

Specific implementation mode

H1N1virus (2009) detection technique of the present invention, by specific target capture technique and real-time fluorescence core Sour constant-temperature amplification (SAT) technology in conjunction with and formed.

The present invention chooses Influenza virus H1N1 HA genes and guards section, by the capture probe of design specialized, efficiently, The RNA of specificity capture H1N1 (2009)；Nucleic acid amplification is come while being realized using M-MLV reverse transcriptase and T7 RNA polymerases, Reverse transcriptase is used to generate a DNA copy of target nucleic acids RNA, and T7 RNA polymerases generate multiple RNA from DNA copy and copy The RNA copy specific bonds generated after shellfish, optimizing detection probe with fluorescent marker and amplification, to generate fluorescence, this is glimmering Optical signal can be captured by detecting instrument.

Primer special and probe in the present invention include：

(1) capture probe：One can be with the target core of H1N1virus (2009) shown in sequence in sequence table 1 The capture probe (TCO, Target Capture Oligo) that sour (H1N1 (2009) RNA) sequence specific combines, is there is H1N1 It (2009), can also be with H1N1 (2009) the interior label sequence specific bond when internal standard (H1N1 (2009) IC RNA)；The capture In the nucleotide sequence of probe such as sequence table shown in sequence 2.

(2) H1N1 (2009) detection primer：A pair under the effect of M-MLV reverse transcriptase for generating H1N1 (2009) target H1N1 (2009) detection primer of the DNA copy of nucleic acid (H1N1 (2009) RNA), H1N1 (2009) detection primer are drawn by T7 Object and nT7 primers form, in T7 primer sequences such as sequence table shown in sequence 3, in nT7 primer sequences such as sequence table shown in sequence 4；

(3) H1N1 (2009) detection probe：One for T7 RNA polymerases effect under according to the H1N1 (2009) H1N1 (2009) detections for the RNA copy specific bonds that the DNA copy of target nucleic acids (H1N1 (2009) RNA) generates are visited Needle, in the nucleotide sequence such as sequence table of H1N1 (2009) detection probe shown in sequence 5,5 ' end FAM fluorescent markers, 3 ' End DABCYL fluorescent markers.

For ease of carrying out interpretation of result, further include：(4) internal standard detection probes and H1N1 (2009) internal standard, internal standard inspection Probing needle is the internal standard detection probe being used cooperatively with the internal standard when using H1N1 (2009) internal standard, nucleotide sequence such as sequence In list shown in sequence 6,5 ' end label HEX fluorophors, 3 ' end label DABCYL quenching groups；It is designated as in H1N1 (2009) The competitive internal standard of H1N1 (2009) nucleotide sequence (H1N1 (2009) RNA), while being specifically bound with the capture probe, And use same pair of primers (T7 and nT7 primers).The interior target nucleotide sequences of H1N1 (2009) as shown in sequence 7 in sequence table, It is named as H1N1 (2009) IC RNA (IC meanings are internal standard).

Based on the above design, the present invention further provides a kind of H1N1virus (2009) real-time fluorescence nucleic acid perseverances Warm amplification detection kit.

The kit includes at least the capture probe (sequence 2), a pair of T7 primers (sequence 3) and nT7 primers (sequence 4) and H1N1 (2009) detection probe (sequence 5).

Further, the kit also may include thering is M-MLV reverse transcriptase and T7 RNA polymerases, the M-MLV reversions Record enzyme and T7 RNA polymerases are present in SAT enzyme solutions, and the capture probe is present in nucleic acid extraction liquid, the T7 primers, NT7 primers and H1N1 (2009) detection probe are present in H1N1 (2009) detection liquid.

Further, the kit also may include competitive H1N1 (2009) internal standard (sequence 7) and internal standard detection probe (sequence 6), the internal standard detection probe are present in H1N1 (2009) detection liquid.

More specifically, the kit include lysate, nucleic acid extraction liquid, cleaning solution, H1N1 (2009) reaction solution, H1N1 (2009) is detected in liquid, SAT enzyme solutions, H1N1 (2009) positive control, H1N1 (2009) negative controls and H1N1 (2009) Mark, each component are described as follows：

(1) lysate：For cracking and preserving the influenza A virus in sample to be tested (H1N1 (2009)), to contain The solution of dirty agent and HEPES buffer solution, detergent are mainly ammonium sulfate ((NH₄)₂SO₄, preferably 5-50mM)；

(2) nucleic acid extraction liquid：For extracting and purifying H1N1 (2009) viral RNA, to contain 1-50 μM of capture probe (preferably Be 5-25 μM) and magnetic bead 50-500mg/L (preferably 50-250mg/L) aqueous solution；

(3) cleaning solution：It is cleaned for magnetic bead, for the aqueous solution of the SDS containing 1wt%.

(4) H1N1 (2009) reaction solution：Component needed for SAT amplifications, 0.1-10mM containing dNTP (be preferably 0.5-5mM) and The aqueous solution of NTP1-20mM (preferably 1-10mM)；

(5) H1N1 (2009) detects liquid：Containing SAT expand needed for primer and probe aqueous solution, each primer or probe it is dense Degree is reacted in 5-15pmol/, and wherein T7 primer concentrations are preferably 10pmol/ reactions, and nT7 primer concentrations are preferably 10pmol/ reacts, and H1N1 (2009) detection probe concentrations are preferably 7pmol/ reactions, and internal standard detection probe concentrations are preferably 7pmol/ reacts；

(6) SAT enzyme solutions：Multienzymatic reaction system needed for SAT amplifications, the main 400-4000U/ of reverse transcriptase containing M-MLV reactions (preferably 500-1500U/ reactions), T7 RNA polymerases 200-2000U/ reactions (preferably 500-1000U/ reactions)；

(8) H1N1 (2009) negative control：Without containing H1N1virus (2009) target nucleic acids (H1N1 (2009) RNA) sequence or the solution without containing influenza A virus, such as physiological saline；

(9) H1N1 (2009) internal standard：Containing 10²-10⁵Copy/mL H1N1 (2009) internal standard RNA (sequence 7), is H1N1 (2009) the competitive internal standard of nucleotide sequence (H1N1 (2009) RNA) is that in-vitro transcription RNA (H1N1 (2009) IC RNA) is dilute Release object.

What is respectively formed in kit described above is further described below：

The principle active component of lysate is detergent, and the presence of high concentration detergent can make RNase fast deactivations, have Effect preserves RNA.Nucleic acid extraction liquid is that Beads enrichment method is utilized to carry out nucleic acid extraction, and main component is magnetic-particle and capture Probe.Capture probe one end and target-complementary, one end are connect with magnetic-particle complementation, during nucleic acid extraction, bacteria lysis The nucleic acid released and the magnetic-particle specific bond in nucleic acid extraction liquid, in the case where not needing traditional centrifugally operated, Magnetic-particle is cleaned by cleaning solution by obtain pure viral targets nucleic acid (RNA).The extraction of viral RNA is inhaled by specificity Attached principle is realized.

It is molecular beacon that H1N1 (2009), which detects H1N1 (2009) detection probe in liquid, is a kind of high specific, high sensitive Property molecular probe, by both ends, covalent labeling is made of the single stranded nucleic acid molecule of fluorescent dye and quencher respectively, be in hair clip type Or loop-stem structure, the loop section and target-complementary of molecular beacon, both ends become stem due to complementation, molecular beacon probe with it is linear TaqMan probe compare, because the opening of its hairpin structure needs certain power, thus specificity is better than linear probe.

So that amplification is failed since SAT amplifications are easily affected by many factors, kit user of service's error in judgement is made to obtain The conclusion of mistake is provided with H1N1 (2009) positive control, H1N1 (2009) negative controls and H1N1 in kit of the invention (2009) internal standard.Wherein, H1N1 (2009) positive controls and the interior RNA for being designated as in-vitro transcription of H1N1 (2009), do not have biology Learn activity.

By detecting positive control, provable kit test method and material are errorless, ensure the accuracy of detection, simultaneously The difference between the repeatability detected every time and stability and kit batch can be monitored.In addition, can be made by positive reference substance Standby critical weak sun control (is mixed into dilution, dilution 1000 times of conducts of positive control are faced with physiological saline and lysate by 1: 1 The weak sun control in boundary), checked operation situation when can prompt in critical state of value passes through critical weak sun control periodic detection The laboratories SAT can carry out indoor quality control, to prevent detection process from the case where missing inspection (false negative) occur.H1N1(2009) Competitive internal standard of the internal standard as H1N1 (2009) RNA, most important effect is exactly the generation for controlling false negative result, is passed through Detection, which is added, interior target sample, it may be appreciated that whether entire amplification reaction system is suppressed, preferably prompts false negative.It is negative right According to false positive can be excluded, under proper use of kit test method and material context, it is ensured that the specificity of detection.

Real-time fluorescence nucleic acid constant-temperature amplification detection, packet are carried out to H1N1virus (2009) using the above kit Include following steps：

In above-mentioned detection operation, the sample to be tested in the step 1) is throat swab or nose swab.

H1N1 (2009) positive control in the step 4) is containing 10²-10⁵Copy/mL H1N1viruses (2009) the in-vitro transcription RNA dilutions of HA genes；H1N1 (2009) negative control is without containing H1N1virus (2009) target nucleic acids (H1N1 (2009) RNA) sequence or the solution without containing influenza A virus；It is designated as in H1N1 (2009) Containing 10²-10⁵Copy/mL H1N1 (2009) internal standard RNA (sequence 7) dilution.

Embodiment is being implemented down based on the technical solution of the present invention, gives detailed embodiment and specific Operating process, but protection scope of the present invention is not limited to following embodiments.

Method therefor is conventional method unless otherwise instructed in following embodiments.Primary raw material SAT used in embodiment Enzyme solution, positive control and interior target in-vitro transcription RNA are provided by RD Biosciences companies of the U.S., and 7500 type PCR instruments are U.S. State's ABI Products, the reagents such as NTPs, dNTPs and other instruments are conventional commercially available product.

Embodiment 1, the primer special that H1N1virus (2009) is detected for real-time fluorescence nucleic acid constant-temperature amplification With the design of probe

The present invention selects in H1N1 (2009) virus HA gene without secondary structure and highly conserved section as amplification target sequence Column region (in its nucleotide sequence such as sequence table shown in sequence 1), according to primed probe design principle, using DNAATAR, DNAman softwares and engineer are for the special of real-time fluorescence nucleic acid constant-temperature amplification detection H1N1virus (2009) Primer and probe sequence obtains following particular sequence：

(1) one can be with the target nucleic acids (H1N1 of the H1N1virus (2009) as shown in sequence 1 in sequence table (2009) RNA) sequence specific combine capture probe (TCO, Target Capture Oligo), the nucleosides of the capture probe Acid sequence is 5 '-CCAUUGUCUGAACUAGAUGUUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3 ' (sequences in sequence table Row 2)；

(2) a pair of for generating H1N1 (2009) target nucleic acids (H1N1 (2009) RNA) under the effect of M-MLV reverse transcriptase DNA copy H1N1 (2009) detection primer, H1N1 (2009) detection primers are made of T7 primers and nT7 primers, T7 Primer sequence is 5 '-aatttaatacgactcactatagggagaACCAGATCCAGCATTTCTTTCCATTG-3 ' (sequence tables Middle sequence 3), nT7 primer sequences are 5 '-AATAACATTCGAAGCAACTGG-3 ' (sequence 4 in sequence table)；

(3) one for T7 RNA polymerases effect under according to the H1N1 (2009) target nucleic acids (H1N1 (2009) RNA) H1N1 (2009) detection probe of RNA copy specific bonds for generating of DNA copy, the H1N1 (2009) inspection The nucleotides sequence of probing needle is classified as 5 '-cacGGUACCGAGAUAUGCccgug-3 ' (sequence 5 in sequence table), and 5 ' ends are glimmering with FAM Signal, 3 ' end DABCYL fluorescent markers.

(4) for ease of carrying out interpretation of result, increased competitiveness H1N1 (2009) internal standard (sequence in reagents box is gone back 7) competitive internal standard detection probe, H1N1 (2009) internal standards and H1N1 (2009) Target nucleotides (H1N1 (2009), are devised RNA) possess identical primer binding zone, nucleic acid sequence between two primers or arrangement are different, prevent its from detection probe knot It closes, but can be combined with internal standard probe, H1N1 (2009) internal standard can be built by H1N1 (2009) target template rite-directed mutagenesis It obtains, can be specifically bound with capture probe, the internal standard detection probe is and H1N1 (2009) detection probe sequence, fluorescence mark Note is different, but the probe that base number is consistent, the nucleotides sequence of the internal standard detection probe are classified as 5 '- CCAGGGUAAUUCGGCACGUCCUGG-3 ' (sequence 6 in sequence table), 5 ' end label HEX fluorophors, 3 ' end label DABCYL Quenching group.

Embodiment 2 prepares H1N1virus (2009) real-time fluorescence nucleic acid isothermal amplification detection kit

It is real to obtain H1N1virus of the present invention (2009) for the primer special and probe provided using embodiment 1 When fluorescence nucleic acid isothermal amplification detection kit.The kit includes capture probe (TCO, Target Capture Oligo), T7 primers, nT7 primers, H1N1 (2009) detection probe, M-MLV reverse transcriptase and T7 RNA polymerases；Kit is deposited Further include internal standard detection probe in internal standard.

The capture probe is present in nucleic acid extraction liquid, and the T7 primers, nT7 primers and H1N1 (2009) detections are visited Needle, internal standard detection probe are present in H1N1 (2009) detection liquid, and the M-MLV reverse transcriptase and T7 RNA polymerases are present in In SAT enzyme solutions, specifically, the kit is divided into the A boxes (sample disposal unit) that 2-30 DEG C stores and -15~-35 DEG C of storages The B boxes (nucleic acid amplification detection unit) deposited, A boxes include lysate, nucleic acid extraction liquid and cleaning solution, and B boxes include (2009) H1N1 Reaction solution, H1N1 (2009) detections liquid, SAT enzyme solutions, H1N1 (2009) positive control, H1N1 (2009) negative control, if deposited In internal standard, B boxes further include H1N1 (2009) internal standard, and main component is as follows：

A boxes (sample disposal unit) group becomes：

Lysate；Liquid containing ammonium sulfate ((NH₄)₂SO₄) and HEPES；

Nucleic acid extraction liquid：1-50 containing capture probe μM (preferably 5-25 μM) and magnetic bead 50-500mg/L (preferably 50- 250mg/L)；

Cleaning solution：Main SDS containing 1wt%.

B boxes (nucleic acid amplification detection unit) group becomes：

H1N1 (2009) reaction solution：0.1-10mM containing dNTP (preferably 0.5-5mM), NTP 1-20mM (preferably 1- 10mM)；

H1N1 (2009) detects liquid：Containing primer and probe, the concentration of each primer and probe is reacted in 5-15pmol/, Wherein T7 primer concentrations are preferably 10pmol/ reactions, and nT7 primer concentrations are preferably 10pmol/ reactions, and H1N1 (2009) detections are visited Needle concentration is preferably 7pmol/ reactions, and internal standard detection probe concentrations are preferably 7pmol/ reactions；

SAT enzyme solutions：The 400-4000U/ of reverse transcriptase containing M-MLV reactions (preferably 500-1500U/ reactions), T7 RNA are poly- Synthase 200-2000U/ reactions (preferably 500-1000U/ reactions)；

H1N1 (2009) positive control；Containing 10²-10⁵Copy/mL H1N1viruses (2009) HA genes it is external Transcribe RNA dilutions；

H1N1 (2009) internal standard：Containing 10²-10⁵Copy/mL H1N1 (2009) IC RNA dilutions (sequence 7 in sequence table).

All reagents included in kit can be prepared acquirement in conventional manner or are commercially available by prompt.

Specifically, in each reacton, specifically assembling for the kit various reagents is as follows：

(1) lysate：To crack and H1N1virus (2009) (H1N1 in depositary's throat swab and nose swab (2009)), the solution containing ammonium sulfate and HEPES buffer solution includes specifically HEPES25-250mM, (NH₄)₂SO₄5-50mM；

(2) nucleic acid extraction liquid：Contain oligo for extraction H1N1virus (2009) (H1N1 (2009)) RNA (dT) solution of coated magnetic bead and one section of RNA sequence of specific bond target nucleic acids (H1N1 (2009) RNA) includes specifically HEPES 50-400mM, EDTA 40-200mM, LiCl 400-2000mM, 1-50 μM of capture probe (preferably 5-25 μM), magnetic Pearl 50-500mg/L (preferably 50-250mg/L)；

(3) cleaning solution：For the solution containing SDS, NaCl, specifically comprising HEPES 5-50mM, NaCl 50-500Mm, 1% SDS 1-10mM, EDTA 1-10mM；

(4) H1N1 (2009) reaction solution：Include specifically Tris10-50mM to contain component needed for dNTPs and NTPs amplifications, MgCl₂10-40mM, dNTP 0.1-10mM (preferably 0.5-5mM), NTP 1-20mM (preferably 1-10mM), PVP40 1- 10%, KCl 5-40mM；

(5) H1N1 (2009) detects liquid：Required H1N1 (2009) detection primers and H1N1 (2009) when by constant-temperature amplification Detection probe is dissolved in TE solution (mixed liquor of 10mM Tris and 1mM EDTA) and is formulated, and primer and probe concentration exists 5-15pmol/ reacts, and wherein T7 primer concentrations are preferably 10pmol/ reactions, and nT7 primer concentrations are preferably 10pmol/ anti- It answers, H1N1 (2009) detection probe concentrations are preferably 7pmol/ reactions, and internal standard detection probe concentrations are preferably 7pmol/ reactions；

(6) SAT enzyme solutions：For constant-temperature amplification when required multienzyme components system, the 400-4000U/ of reverse transcriptase containing M-MLV is anti- Answer (preferably 500-1500U/ reactions), T7 RNA polymerases 200-2000U/ reactions (preferably 500-1000U/ reactions), 2- 10mM HEPES pH7.5、10-100mM N-acetyl-L-cysteine、0.04-0.4mMzinc 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；

(9) if there is internal standard, H1N1 (2009) internal standard：Containing 10²-10⁵Copy/mL H1N1 (2009) internal standard RNA (sequences Sequence 7 in table).

H1N1 (2009) RNA of in-vitro transcription in H1N1 (2009) positive control can prepare institute by a variety of methods , one of which preparation method is as follows：

H1N1 (2009) IC RNA of in-vitro transcription in H1N1 (2009) internal standard can prepare institute by a variety of methods , one of which preparation method is as follows：

Embodiment 3, the real-time fluorescence nucleic acid constant-temperature amplification detection sensitivity of H1N1virus (2009)

With kit of the present invention, (group prejudice embodiment 2, kit is interior to be not present H1N1 (2009) internal standard, detects in liquid There is no internal standard detection probes) measured concentration be 1 × 10⁴Copies/ reactions, 1 × 10³Copies/ reactions, 1 × 10²copies/ H1N1virus (2009) positive control RNA of reaction, 10copies/ reactions, determines lower sensitivity limit.Specific steps It is as follows：

(1) it dilutes

By 1 × 10⁴H1N1virus (2009) positive reference product RNA of copies/ reactions, 10 times of gradient dilutions To 1 × 10³Copies/ reactions, 1 × 10²Copies/ reactions, 10copies/ reactions.

(2) nucleic acid extraction

2.1 are added 200 μ l lysates (35mM containing HEPES, (NH in each sample processing tube (1.5mL centrifuge tubes)₄)₂SO₄20mM), 200 μ l influenza virus positive reference product dilutions crack H1N1virus (2009) (mould with lysate Quasi- sample process), obtain the lysate containing H1N1virus (2009) nucleic acid.

2.2 are added 100 μ l nucleic acid extractions liquid (100mM containing HEPES, EDTA in sample processing tube (1.5mL centrifuge tubes) 50mM, LiCl 500mM, 20 μM of capture probe, magnetic bead 200mg/L) mixing, 60 DEG C keep the temperature 5 minutes, are placed at room temperature for 10 minutes.

2.3 are placed in sample processing tube on magnetic bead separating device, stand 5-10 minutes.After magnetic bead is adsorbed in tube wall, protect Sample processing tube is held on magnetic bead separating device, liquid is abandoned in suction, retains magnetic bead.1mL cleaning solutions are added and (contain HEPES50mM, NaCl 100mM, 1% SDS, EDTA 5mM) 5-10 minutes are stood after shaken well, liquid is abandoned, retains magnetic bead, 2 times repeatedly.

Sample processing tube is moved away from magnetic bead separating device by 2.4, and Guan Zhongwei magnetic beads-nucleic acid complexes, spare (this step should be clear It can be seen that magnetic bead).

(3) SAT nucleic acid amplifications detect

3.1 40 μ l reaction detections liquid (+2.5 μ lH1N1 of 40 μ l H1N1 (2009) reaction solution are added into sample processing tube (2009) liquid is detected) washing magnetic bead.H1N1 (2009) reaction solution includes specifically Tris 30mM, MgCl₂20mM, dNTP 5mM, NTP 8mM, PVP40 5%, KCl 25mM；It is 10pmol that H1N1 (2009), which detects T7 primer concentrations in liquid, and nT7 primer concentrations are 10pmol, H1N1 (2009) detection probe concentrations are 7pmol, and internal standard detection probe concentrations are 7pmol.

3.2 take the 30 μ l of above-mentioned reaction detection liquid of oscillation mixing to add to clean micro-reaction pipe, (beautiful with 7500 type PCR instruments State's ABI Products) 60 DEG C of heat preservations 10 minutes, 42 DEG C keep the temperature 5 minutes；10 μ 1 are added into micro-reaction pipe and are pre-heated to 42 DEG C SAT enzyme solutions, 1200rpm vibrate 15 seconds mixings.The 1200U of reverse transcriptase containing M-MLV in SAT enzyme solutions, T7 RNA polymerases 800U/ reacts, 10mM HEPES pH7.5,20mMN-acetyl-L-cysteine (N-acetyl-L-cysteine), 0.25mM Zinc acetate (zinc acetate), 30mMtrehalose (trehalose), 100mM Tris-HCl pH8.0,200mM KCl, 0.1mM EDTA, 0.8% (v/v) Triton X-100 and 40% (v/v) glycerol (glycerine)；

Micro-reaction pipe is rapidly turned to constant-temperature fluorescence detector device by 3.3, and 42 DEG C are reacted 50 minutes, is set every 1 minute and is examined First order fluorescence is surveyed, is detected 50 times altogether.

(4) result judgement

According to the curve that PCR amplification result obtains, threshold line is set, reads dt values, judges result.

Threshold value is set：The peak of normal negative control amplification curve is just above with threshold line.Dt indicates sample curve (similar with the general ct values of real-time fluorescence PCR experimental result) is read with the abscissa of threshold line intersection point

1. positive findings judge：

The channels FAX：The sample of dt≤45 is the positive；The sample suggestion of 45 ＜ dt ＜ 50 detects again, testing result：Dt ＜ 50 sample is the positive.

2. negative findings judge：

Negative findings judge：The channels FAX：Dt is without numerical value or is 50, then is feminine gender.

(5) result

The detection figure of Fig. 1 display sensitivities, to ensure that the repeatability of testing result, each positive reference product repeat to examine It surveys 2 times, as a result shows：When the concentration of positive reference product is when 10copies/ reacts, can not detect simultaneously.And concentration exists 100copies/ reactions are above to be, repeatable detection.H1N1virus (2009) real-time fluorescence nucleic acid constant-temperature amplification The sensitivity of detection kit is reacted up to 100copies/.

Embodiment 4, the real-time fluorescence nucleic acid constant-temperature amplification detection specificity of H1N1virus (2009)

This detection pattern is another application of the invention：Kit forms choose H1N1 (2009) sample with embodiment 2 With 3 negative samples：16 type of Coxsack viral (CA16), enterovirns type 71 (EV71), mycoplasma pneumoniae (MP), separately set feminine gender Control, each one of positive control are detected.Detection method and specific steps are the same as embodiment 3.

Testing result such as Fig. 2, positive control and H1N1 (2009) sample dt values are respectively 10.2,13.8, and are expanded Curve can determine that as the positive；And the amplification curve of 3 negative samples and negative control it is straight and with baseline without intersecting, can define It is determined as feminine gender；3 negative samples are 16 type of Coxsack viral (CA16), enterovirns type 71 (EV71), mycoplasma pneumoniae (MP)。

The real-time fluorescence nucleic acid constant-temperature amplification detection of embodiment 5, clinical sample throat swab

This detection pattern is another application of the invention：With embodiment 2, (kit contains 1 × 10 to kit forms⁵It copies Shellfish/mL H1N1 (2009) internal standard, the interior internal standard probe containing 7pmol/ reactions of detection liquid), to number 1-5 Influenza A H1N1s Viral (2009) oropharyngeal swab specimen, and respectively set a negative control and positive control is detected, specifically include following detection Step：

(1) sample collection

Collection of specimens is carried out according to actual conditions by clinician, detection sample is throat swab, and acquisition method is：It is special to adopt Sample cotton swab swallows rear wall and both sides pharyngeal tonsils part, and swab is invaded in 3-5mL physiological saline, seals inspection.Sample is adopted 4 DEG C of preservations are sent to influenza surveillance network laboratory (or -70 DEG C of preservations are to be measured, are sent in one week) in 48 hours after collection.

(2) nucleic acid extraction

Include H1N1 (2009) internal standard in kit, therefore in nucleic acid processing, 100 μ 1 are added in each sample processing tube 10 μ, 1 inner mark solutions, then mixing need to be added in nucleic acid extraction liquid simultaneously.Other concrete operation steps are the same as embodiment 2.

(3) SAT augmentation detections

With embodiment 3.

(4) result judgement：

3. positive findings judge：

The channels F1 (channels FAX)：The sample of dt≤45 is the positive；The sample suggestion of 45 ＜ dt ＜ 50 detects again,

The channels testing result F1：The sample of dt ＜ 50 is the positive.

4. negative findings judge：The channels F1 dt is without numerical value or is 50, while (channels VIC, ABI instrument are only optional in the channels F2 The channels VIC, but VIC is close with HEX wavelength)：Dt≤45 are then feminine gender.

Quality control：Detection is respectively provided with positive control and negative control every time, and result should meet positive control F1 simultaneously Channel：dt≤45；The channels negative control F1：Dt is without numerical value or is 50, while the channels F2：Dt≤45, otherwise this time testing result It is invalid to be considered as.

Another synchronous progress control test：

H1N1virus (2009) the RNA detection kits produced with Shanghai Zhijiang Biological Science Co., Ltd (fluorescent PCR method) (state eat medicine supervise tool (standard) word 2009 the 3401022nd) and with reference to kit specification to the first in throat swab Type H1N1 influenza viruses (2009) RNA is detected, and specific method includes the following steps (throat swab sample collection is same as above)：

(1) the nucleic acid cleavage processing of sample

It takes 100 μ l samples to be placed in 1.5ml centrifuge tubes, 300 μ l lysates (Trizol) is added, add 100 μ l chloroforms, Mixing 5sec or reverse mixings are vibrated on vortex mixer 15 times.13,000rpm centrifugations, 15min draw supernatant liquid, are added isometric Isopropanol overturns mixing.13,000mm centrifugation 15min, gently remove supernatant：75% ethyl alcohol of 700ul is added, overturns washing. 13,000rpm centrifugation 10min, are inverted on blotting paper, abandon most liquid.4,000rpm centrifugation 5sec, by the residual solution on tube wall Body is thrown to bottom of the tube, and liquid is blotted as possible with micro sample adding appliance, and 20 μ lDEPC-H2O are added and dissolve RNA.

(2) PCR is detected

2.1 preparation of reagents：Take n × 19 μ l H1N1 (2009) nucleic acid fluorescent PCR detection mixed liquors and the μ lRT-PCR enzymes of n × 1 (n is reaction tube number), Zhen Dang Kun even several seconds, 3000rpm centrifuges the several seconds.

2.2 sample-adding：Take 20 μ l of above-mentioned mixed liquor to be placed in PCR pipe, then by sample RNA, DEPC-H2O (negative control), Each 5 μ l of positive reference substance are separately added into PCR pipe, are covered pipe lid, are carried out pcr amplification reaction immediately.

2.3PCR amplification：Reaction tube is placed on quantitative fluorescence PCR instrument, and fluorescence channel detection is selected：The channels FAM recommend to follow Ring parameter setting：45℃×10min；95℃×15min；95 DEG C × 15sec-60 DEG C × 60sec is pressed again, is recycled 40 times；Single-point At 60 DEG C, reaction system is 25 μ l for fluoroscopic examination.

(3) judge result

Baseline and threshold value setting baseline adjustment takes 6-15 recycle fluorescence signal, threshold value setting principle with threshold line just More than the peak that DEPC-H2O detects fluorescence curve.Result should be judged in conjunction with the form of Ct values and typical case's S type curves.

1. amplification curve is S-type, and the columns Cycle threshold show≤38 (ABI7500), then are determined as the positive；

2. if Ct values are higher and when there is irregular curve, it is judged as feminine gender；

If the result display field of instrument is shown between 38~40, replication is needed：If result still between 38~40, It is negative to be then judged to Flu-A H1N1 viral (2009).

Shown in testing result such as Fig. 3 (F1 fluorescence channels) and Fig. 4 (F2 fluorescence channels) using kit of the present invention, fluorescence Plain channel F1 is FAM, and F2 VIC, 42 DEG C are reacted 50 minutes, sets every 1 minute detection first order fluorescence, detects 50 times altogether.According to The dt value situations in the channels F1 and the channels F2, judgement sample 2,4 are detected as feminine gender, and sample 1,3,5 is that (2,4 Fig. 3 of sample is shown the positive For feminine gender, Fig. 4 shows signal, is just illustrating that internal standard can detect that, reaction system does not have affected by environment, exclusion false negative), As a result (see Fig. 5, Ct values are meant that with kit testing result has been listed at present：Fluorescence signal in each reaction tube reaches The recurring number undergone when the thresholding of setting, the Ct in the kit of the Zhijiang River refer in 95 DEG C of 15sec, the process of 60 DEG C of 60sec In recycled 40 times, acquire 40 fluorescence.It heats up and cools down involved in this process, 1 recycles about 2~3 minutes, therefore whole A pcr amplification reaction at least 100 minutes) it is identical.

Kit of the present invention is used to detect H1N1virus (2009) (H1N1 in clinical sample throat swab (2009)) accuracy is high, and the augmentation detection time only needs 50 minutes, and this kit amplified production is RNA, is easily dropped in the environment Solution, low stain.Therefore this kit has the characteristics that short period, highly sensitive, high specific, low stain and stable reaction.

Disclosure according to the present invention, those skilled in the art experiment can excessively need not want the present invention It asks H1N1virus (2009) real-time fluorescence nucleic acid isothermal amplification detection kit of protection to be implemented, and reaches pre- Phase effect.Embodiment disclosed by the invention is only that the present invention will be described in detail, but simultaneously insufficient constitute limits the present invention.This The those of skill in the art in field with obvious similar substitute or transformation, or with it is certain in chemistry or biology goes up structure work( The relevant preparation of energy substitutes preparation described here, or is changed to related content of the present invention, but without departing from the essence of the present invention God, range and thought, each fall within the scope of protection of present invention.

Claims

1. a kind of H1N1virus (2009) real-time fluorescence nucleic acid isothermal amplification detection kit, which is characterized in that packet Contain：

The capture probe of the target nucleic acids of H1N1virus (2009) shown in sequence 1, the target in one sequence table Mark nucleic acid is H1N1 (2009) RNA,

The H1N1 (2009) of DNA copy of a pair for generating H1N1 (2009) target nucleic acids under the effect of M-MLV reverse transcriptase Detection primer T7 and nT7,

One for T7 RNA polymerases effect under according to the DNA copy of the H1N1 (2009) target nucleic acids generate RNA copies H1N1 (2009) detection probes and the emulative internal standard of H1N1 (2009) nucleotide sequence and interior of specific bond Mark detection probe；

Wherein, in the nucleotide sequence of the capture probe such as sequence table shown in sequence 2：

CCAUU GUCUG AACUA GAUGU UAAAA AAAAA AAAAA AAAAA AAAAA AAAAA AA；

H1N1 (2009) detection primers are made of T7 primers and nT7 primers, 3 institute of sequence in T7 primer sequences such as sequence table Show, in nT7 primer sequences such as sequence table shown in sequence 4；

In the nucleotide sequence such as sequence table of H1N1 (2009) detection probe shown in sequence 5：

CACGG UACCG AGAUA UGCCC GUG, and 5 ' the end flag F AM fluorophors of H1N1 (2009) detection probe, 3 ' end label DABCYL quenching groups；

The competitiveness internal standard can specifically bind with capture probe, and use T7 and nT7 primers, the competitive internal standard be by H1N1 (2009) IC RNA shown in sequence 7 in sequence table；

In the nucleotide sequence such as sequence table of the internal standard detection probe shown in sequence 6,5 ' end label HEX fluorophors, 3 ' ends DABCYL quenching groups, the internal standard detection probe is marked to be present in H1N1 (2009) detection liquid；

Also, the kit contains the enzyme being made of M-MLV reverse transcriptase and T7 RNA polymerases.

2. kit according to claim 1, it is characterised in that：The M-MLV reverse transcriptase and T7 RNA polymerases are deposited It is in a SAT enzyme solutions, the capture probe is present in a nucleic acid extraction liquid, the T7 primers, nT7 primers and H1N1 (2009) detection probe is present in a H1N1 (2009) detection liquid.

3. kit according to claim 2, it is characterised in that：The kit includes lysate, nucleic acid extraction liquid, washes Wash liquid, H1N1 (2009) reaction solution, H1N1 (2009) detections liquid, SAT enzyme solutions, H1N1 (2009) positive control, H1N1 (2009) Negative control and H1N1 (2009) internal standard, wherein：

Lysate：Liquid containing ammonium sulfate and HEPES；

Nucleic acid extraction liquid：Containing capture probe and magnetic bead；

Cleaning solution：Containing NaCl and SDS；

H1N1 (2009) reaction solution：Containing dNTP and NTP；

SAT enzyme solutions：Reverse transcriptase containing M-MLV and T7 RNA polymerases this two kinds of enzymes；

H1N1 (2009) negative control：Without containing H1N1virus (2009) target nucleic acids（H1N1(2009) RNA）Sequence Row or the solution without containing influenza A virus；

H1N1 (2009) internal standard：H1N1 (2009) IC RNA, in sequence such as sequence table shown in sequence 7.

4. kit according to claim 3, it is characterised in that：H1N1 (2009) negative control is physiological saline.

5. kit according to claim 3, it is characterised in that：Various reagents in a reacton in the kit Composition is as follows：

（1）Lysate：HEPES 25-250mM, (NH₄)₂SO₄5-50mM；

（2）Nucleic acid extraction liquid：HEPES 50-400mM, EDTA 40-200mM, LiCl 400-2000mM, capture probe 1-50 μ Μ, magnetic bead 50-500mg/L；

（3）Cleaning solution：HEPES 5-50mM, NaCl 50-500 mM, 1% SDS, EDTA 1-10mM；

（4）H1N1 (2009) reaction solution：Tris 10-50mM, MgCl₂10-40mM, dNTP 0.1-10mM, NTP 1-20mM, PVP40 1-10%, KCl 5-40mM；

（5）H1N1 (2009) detects liquid：H1N1 (2009) detection primers and H1N1 (2009) detection probe are dissolved in 10mM It is formulated in the mixed liquor of Tris and 1 mM EDTA, each primer and probe concentration is reacted in 5-15pmol/；

（6）SAT enzyme solutions：M-MLV reverse transcriptase 400-4000U/ reactions, T7 RNA polymerases 200-2000U/ reactions, 2-10mM HEPES pH7.5,10-100 mM n-acetyl-L-cysteine, 0.04-0.4 mM zinc acetates, 10-100 mM seaweed Sugar, 40-200 mM Tris-HCl pH 8.0,40-200mM KCl, 0.01-0.5mM EDTA, volume ratio are 0.1-1%'s Triton X-100 and the glycerine that volume ratio is 20-50%；

（7）H1N1 (2009) positive control；Containing 10²-10⁵Copy/mL H1N1viruses (2009) HA genes it is external Transcribe RNA dilutions；

（8）H1N1 (2009) negative control：Without containing H1N1virus (2009) target nucleic acid sequence or first is not contained The solution of type influenza virus；

（9）H1N1 (2009) internal standard：, in-vitro transcription RNA dilutions shown in sequence 7, contain 10 in sequence such as sequence table²-10⁵Copy/ mL H1N1(2009) IC RNA。

6. kit according to claim 5, it is that 10pmol/ is anti-that the H1N1 (2009), which detects T7 primer concentrations in liquid, It answers, nT7 primer concentrations react for 10pmol/, and H1N1 (2009) detection probe concentrations are reacted for 7pmol/, and internal standard detection probe is dense Degree is that 7pmol/ reacts.

7. kit according to claim 6, it is characterised in that：Reagent（2）In nucleic acid extraction liquid, the concentration of capture probe For 5-25 μ Μ, magnetic bead dosage is 50-250mg/L；Reagent（4）In H1N1 (2009) reaction solution, a concentration of 0.5-5mM of dNTP, A concentration of 1-10mM of NTP；Reagent（5）H1N1 (2009) is detected in liquid, and T7 primer concentrations react for 10pmol/, nT7 primer concentrations It is reacted for 10pmol/, H1N1 (2009) detection probe concentrations are reacted for 7pmol/, and internal standard detection probe concentrations are that 7pmol/ is anti- It answers；Reagent（6）In SAT enzyme solutions, the 500-1500U/ reactions of M-MLV reverse transcriptase, the 500-1000U/ reactions of T7 RNA polymerases.

8. kit according to claim 7, it is characterised in that：It is made of A boxes and B boxes, wherein the A boxes are at sample Unit is managed, the B boxes are nucleic acid amplification detection unit, and A boxes pack the lysate, the nucleic acid extraction liquid and the washing Liquid, B boxes pack H1N1 (2009) reaction solution, H1N1 (2009) detections liquid, SAT enzyme solutions, H1N1 (2009) positive control, H1N1 (2009) negative controls and H1N1 (2009) internal standard.

9. kit according to claim 8, it is characterised in that：In-vitro transcription in H1N1 (2009) positive control H1N1 (2009) RNA be to be prepared to following methods：

1) nucleotide sequence shown in sequence 8 in chemical synthesis composition sequence table is used；

2) segment is cloned into pGEM-carrier T, builds H1N1 (2009) positive control plasmid；

3) H1N1 (2009) positive control plasmid is transformed into bacillus coli DH 5 alpha, is named as pGEM-T-H1N1 (2009) bacterium Strain, is stored in -70 DEG C；

4) purifying removal DNA, and quantitative, identification RNA.

10. kit according to claim 9, it is characterised in that：There are H1N1 (2009) internal standard in the kit,

H1N1 (2009) IC RNA of in-vitro transcription are prepared with following methods：

（1）The nucleotide sequence shown in sequence 9 in chemical synthesis composition sequence table；

（2) segment is cloned into pGEM-carrier T, builds H1N1 (2009) internal standard plasmid；

（3) H1N1 (2009) internal standard plasmid is transformed into bacillus coli DH 5 alpha, is named as pGEM-T-H1N1 (2009) IC bacterium Strain, is stored in -70 DEG C；

（4）Purifying removal DNA, and quantitative, identification internal standard RNA.