Reagent is formed	Liquid reagent	Solid reagent
			Mode one	Component A, B component	Component C, protective material
Mode two	Component A	B component, component C, protective material
			Mode three	Do not have	Component A, B component, component C, protective material

Embodiment 6: dry method

PCR detection of nucleic acids with Pestivirus suis is example, illustrates that liquid and the solid reagent of normal temperature preservation nucleic acid amplification reagent formed, and concrete grammar comprises:

One, be target gene with Pestivirus suis (GenBank ID:HQ380231) sequence, the design primer:

Shown in the F:AAACGGAGGACTAGCCGT(SEQ ID NO.18)

Shown in the B:TGCCATGTACAGCAGAGA(SEQ ID NO.19)

Two, normal temperature is preserved the composed as follows of nucleic acid amplification reagent:

Liquid reagent: Tris-HCl(PH8.5), 0.03M, 0.04M KCl, 0.2M MgCl₂, volume percent is 0.1%Triton X-100, totally 22 μ l;

Solid reagent: primers F 0.1 μ M, primer R0.1 μ M, dNTP1mM, TaqDNA polysaccharase 8U, MMLV enzyme 5U, volume ratio is 0.001% sodium azide;

Three, Gan Zao method is as follows:

The preparation of the solid reagent of Pestivirus suis nucleic acid amplification reagent is carried out according to three kinds of technologies in the table 2:

Table 2 drying process

Embodiment 7: the stability of the nucleic acid amplification reagent that normal temperature is preserved

PCR detection of nucleic acids with Pestivirus suis is example, and the stability of the nucleic acid amplification reagent that normal temperature is preserved is described, concrete grammar comprises:

Shown in the F:AAACGGAGGACTAGCCGT(SEQ ID NO.20)

Shown in the B:TGCCATGTACAGCAGAGA(SEQ ID NO.21)

Two, the swine fever nucleic acid amplification reagent of normal temperature preservation

Described inembodiment 1, prepare three kinds of swine fever nucleic acid amplification reagent of forming the normal temperature preservation of mode,

The swine fever amplifing reagent that table 3 normal temperature is preserved

B component: primers F 0.1 μ M, primer R0.1 μ M, dNTP1mM, totally 22 μ l;

Component C:TaqDNA polysaccharase 8U, AMV enzyme 5U;

Protective material: 0.5M BSA, 0.6M casein, 0.5M trehalose, 2% arginine of volume ratio and 0.001% Proclin300;

The drying means of solid reagent carries out according to three kinds of methods among theembodiment 2 respectively, prepares the swine fever nucleic acid amplification reagent that nine kinds of normal temperature are preserved altogether.

Three, the stability of the nucleic acid amplification reagent of normal temperature preservation

Extract the RNA in the Pestivirus suis living vaccine, gradient dilution to 400,200,100,50,25copy/ml, with not containing of new preparation of protectant swine fever nucleic acid amplification PCR reagent detection, minimum detectability is: 50copy/ml.The RNA template of 50copy/ml is regarded standard substance, estimate the normal temperature of preparation and preserve swine fever nucleic acid amplification reagent, and set up negative control.Nine kinds of reagent carry out the heat collapse acceleration under 37 ℃, negative control is set up, positive findings is as shown in table 4: the thermostability of three kinds of drying meanss does not have difference, and the thermal stability difference of different dry composition modes is remarkable, mode one reagent of dried ingredients C, heat accelerate back 3 days stable, 7 days instabilities, while dried ingredients B and the mode two of C and the reagent of mode three, heat accelerate to 7 days and stablize.

Table 4

Nine kinds of reagent carry out the long-term stable experiment of amplifing reagent at normal temperatures, negative control is set up, positive findings is as shown in table 5: the thermostability of three kinds of drying meanss does not have difference, and the thermal stability difference of different dry composition modes is remarkable, mode one reagent of dried ingredients C, normal temperature is preserved and was stablized in 3 months, and unstable after 4 months, the reagent of the mode two of dried ingredients B and C and mode three can be stablized preservation 6 months at normal temperatures simultaneously.

Table 5

Embodiment 8 protective materials are to the influence of amplifing reagent stability

PCR detection of nucleic acids with Pestivirus suis is example, according to the method forembodiment 3, protective material is described to the influence of amplifing reagent stability, and concrete grammar comprises:

According to the method forembodiment 3, in component, do not add protective material and carry out drying, nine kinds of reagent can only amplify 200copy/ml RNA template after heat is accelerated 3 days, and amplification efficiency is serious.

Table 6 protective material is to the influence of amplifing reagent stability

Embodiment 9 drying protectants are to the interference of PCR reaction

PCR detection of nucleic acids with Pestivirus suis is example, protective material is described to the interference of pcr amplification, and concrete grammar comprises:

Shown in the F:AAACGGAGGACTAGCCGT(SEQ ID NO.22)

Shown in the B:TGCCATGTACAGCAGAGA(SEQ ID NO.23)

Two, the component of amplifing reagent is as follows:

Tris-HCl(PH8.5), 0.02M, 0.05M KCl, 0.15M MgCl₂, volume percent is 0.2%Triton X-100, totally 22 μ l; Primers F 0.1 μ M, primer R0.1 μ M, dNTP1mM, totally 50 μ l;

Three, protective material is to the interference of pcr amplification

The protective material of finite concentration scope is added in the conventional liq PCR reaction system, the minimum detectability of before relatively adding and interpolation back RNA template, and set up negative control.Presentation of results after the protective material of interpolation certain limit concentration, under the situation that negative control is set up, does not disturb the lowest detectable limit of Pestivirus suis pcr amplification.

Table 7 different concns protective material is to the interference of pcr amplification

Additive	BSA	Casein	Calf serum	Sucrose	Trehalose
						Concentration range	1%，5%，10%	1%，5%，10%	1%，5%，10%	1%，5%，10%	1%，5%，10%
Minimum detectability	50copy/ml	50copy/ml	50copy/ml	50copy/ml	50copy/ml
						Additive	Arginine	Tween 20	Triton-100	Sodium azide	Proclin300
Concentration range	0.1%，3%	0.05%，0.1%，1%	0.05%，0.1%，1%	0.001%，0.1%	0.001%，0.1%
						Minimum detectability	50copy/ml	50copy/ml	50copy/ml	50copy/ml	50copy/ml

The detection of embodiment 10:RNA virus

(Pig Reproductive and Respiratory Syndrome Virus PRRSV) is example, with the inventive method pathogenic agent is carried out LAMP and detects, and concrete grammar comprises the steps: with high-pathogenicity porcine reproduction and breathing syndrome virus

One, the extraction of PRRSV viral RNA

1, the component of extraction reagent is:

1) component of lysate is: 0.2M Tris-HCl(pH8.0), and 4M guanidinium isothiocyanate, 2M CTAB, 0.02M EDTA;

2) component of washings A is: volume percent is 100% Virahol;

3) component of washings B is: volume percent is 50% Virahol;

4) component of lysate is: deionized water no RNAase(RNA enzyme).

2, extraction step

Positive and the negative lobe of the lung of PRRSV, get the glass homogenizer that it is good that 30mg places dry sterilization and be ground to no tangible tissue particles, with homogenate behind the centrifugal 2min of 8000rpm, get 100 μ L supernatants and add 300 μ L lysates, the concussion mixing also is statically placed in room temperature 1min, add 400 μ L washings A again, 12000rpm abandoned supernatant in centrifugal 5 minutes behind the concussion mixing, the washings B that adds 800 μ L precoolings in the precipitation, behind the mixing in the centrifugal 1min of 12000rpm, supernatant is absorbed the clean drying at room temperature 3min that places, standby with 8 μ L lysate dissolution precipitations.

Two, amplification

1, with ORF5(GenBank ID:JF906888) sequence is target gene, design primer and probe sequence:

Shown in the F3:TGCGAAGAACTGCATGTCC(SEQ ID NO.1)

Shown in the B3:TGTTCCGCTGAAACTCTGG(SEQ ID NO.2)

The FIP:Bio(vitamin H)-CCGCCAACGATAGAGTCTGCCTGGCGCTACTCTTGTACCA(SEQ ID NO.3 shown in)

Shown in the BIP:CCGTCATTGTGGAGAAAGGGGGCGGAACCATCAAGCACAACT(SEQ ID NO.4)

Shown in the LOOP F:ATACGAGTTGCGCGGAA(SEQ ID NO.16)

Shown in the LOOP B:GCCAAGCACAACTTACG(SEQ ID NO.17)

Probe: the FITC(fluorescein isothiocyanate)-AAGGTTGAGGTCGAAGGT(SEQ ID NO.5 shown in)

2, the preparation of amplifing reagent and normal temperature are preserved

(1) component of amplifing reagent:

1) liquid reagent: 0.02M Tris-HCl (pH8.0), 0.04M (NH4) 2SO4,0.02M NaCl, 0.02M MgSO4, volume percent is 0.2%Triton X-100, trimethyl-glycine 0.8M, totally 22 μ l.

2) solid reagent: outer primer F30.2 μ M, outer primer B30.2 μ M, inner primer FIP0.4 μ M, inner primer BIP0.4 μ M, each 1.0 μ M of ring primer LOOP F and LOOP B, aim sequence probe 0.4 μ M, dNTP1.4mM, ThermoScript II AMV0.8U, Bst archaeal dna polymerase 10U, 0.6M BSA, 0.5M casein, 1M trehalose.

(2) preparation and normal temperature are preserved step:

Step 1, at first prepare liquid reagent, step is as follows:

Step 11: each component concentration that calculates 25 μ l system amplifying nucleic acid amplification liquid reagent

Step 12: in deionized water, final volume to 22 μ l is stored in the Eppendorf pipe of 200 μ l with these components dissolved.

Step 13: the nucleic acid amplification reagent for preparing stores at normal temperatures and gets final product prolonged preservation.

Step 2, prepare solid reagent further, step is as follows:

Step 21: protective material is solidified in preparation, with 0.5～1M BSA, and 0.5～1M casein, 0.5～1M trehalose is dissolved in the deionized water.

Step 22: each component concentration that calculates 25 μ l system amplifying nucleic acid amplification solid reagent.

Step 23: the component of nucleic acid amplification solid reagent is dissolved in the drying protectant, and final volume is 10ul.

Step 24: the above-mentioned solution conduit that the liquid solid reagent of 10 μ l is housed is positioned in the vacuum drying oven with the form of uncapping.

Step 25: the chamber door of vacuum drying oven is closed, and the setting drying parameter is: vacuum tightness is 10～20%, and humidity is: 20～30, and temperature is 0～4 ℃, the time is 1～2 hour.

Step 26: open vacuum drying oven, the solution conduit of solid reagent is equipped with in sealing.

Step 27: the solid reagent that drying finishes stores at normal temperatures and gets final product prolonged preservation.

3, nucleic acid amplification

The liquid reagent of 22 μ l is added in the solid reagent, and fully dissolving then, adds the RNA that 2 μ l extract, and places 64 ℃ of reaction 60min.

Three, the result detects

1, the primary structure of colloidal gold strip

Conjugate pad: the colloid gold particle that uses 40nm.The colloid gold particle mark of detection line is anti--antibody of vitamin H, and concentration is 5OD; The colloid gold particle mark rabbit igg antibody of nature controlling line, concentration is 5OD.

Nitrocellulose filter: the M135 type film that uses Merck ﹠ Co., Inc..The detection line bag is by anti--FITC antibody of 2mg/ml; The nature controlling line bag is by the goat anti-rabbit igg of 5mg/ml.

2, detect step

The pipe connecting of closed chromatographic apparatus is connected with the test paper seal of tube, rotation discharged the reaction solution of finishing the LAMP constant-temperature amplification after the solution conduit that finishes of will increasing was again inserted the pipe connecting of proofing unit, the question response liquid layer is analysed nitrocellulose filter and is picked up counting, observations in the time of 5 minutes:

If the nature controlling line C of yin and yang attribute sample has band clearly, the detection line T(embedding of positive sample anti--FITC monoclonal antibody) have intensity to be ++ condition, PRRSV detected result positive (seeing Figure 15 B) is described.The detection line T of negative sample does not have band (seeing Figure 15 A).

The detection of embodiment 11:DNA virus

(Canine Parvovirus CPV) is example, with the inventive method pathogenic agent is detected, and concrete grammar comprises the steps: with canine parvovirus

One, the extraction of CPV viral DNA

1, the component of extraction reagent is:

1) component of lysate is: 0.1M Tris-HCl(pH8.0), and 3M guanidinium isothiocyanate, 1M CTAB, 0.05M EDTA;

2) component of washings A is: volume percent is 90% Virahol; 3) component of washings B is: volume percent is 45% Virahol;

4) component of lysate is: deionized water no RNAase(RNA enzyme).

2, the extraction of DNA

Get the positive and the negative serum sample of 100 μ L canine parvovirus, add 300 μ L lysates, the concussion mixing also is statically placed in room temperature 1min, add 400 μ L washings A again, the concussion mixing after 12000rpm abandoned supernatant in centrifugal 5 minutes, toward the precipitation in the adding 800 μ L precoolings washings B, behind the mixing in the centrifugal 1min of 12000rpm, supernatant is absorbed the clean drying at room temperature 3min that places, standby with 8 μ L lysate dissolution precipitations.

Two, amplification

1, with CPV NS1(GenBank ID:HQ694567.2) sequence is target gene, design PCR primer and probe sequence:

The F:BIO(vitamin H)-CTTTTTTTGTTTGCTTATGTCTG(SEQ ID NO.6 shown in)

Shown in the R:TTTATTCAACATGAATGGGGAA(SEQ ID NO.7)

Probe: the FITC(fluorescein isothiocyanate)-GCTTGTTGTAAGTTCTTA(SEQ ID NO.10 shown in)

2, the preparation of amplifing reagent and normal temperature are preserved

(1) component of amplifing reagent:

1) liquid reagent:

Tris-HCl(PH8.5), 0.02M, 0.05M KCl, 0.15M MgCl₂, volume percent is 0.2%Triton X-100, totally 22 μ l.

2) solid reagent: primers F 0.1 μ M, primer R0.1 μ M, dNTP1mM, TaqDNA polysaccharase 8U, 0.5M BSA, 0.6M casein, 0.5M trehalose, totally 22 μ l.

(2) preparation and normal temperature are preserved step:

Step 1, at first prepare liquid reagent, step is as follows:

Step 2, prepare solid reagent further, step is as follows:

3, nucleic acid amplification

The liquid reagent of 22 μ l is added in the solid reagent, and fully dissolving then, adds the DNA that 2 μ l extract, amplification totally 35 circulations: 94 ℃ of 1min of sex change condition, 55 ℃ of 30S of annealing conditions, 72 ℃ of 1min of extension condition.

Three, the result detects

1. the primary structure of latex test strip comprises:

1) sample pad: formed by the glass fibre cutting;

2) conjugate pad: physical material is glass fibre, and the saturated immersion of latex solution or the quantitative back drying of spraying are formed; Described latex solution is that diameter is the particle of 100～200nm, and buying is from Merck ﹠ Co., Inc.; Comprise two kinds of latex particles in the latex solution: be marked with antibody or the small molecules antigen of anti--antigen on the latex particle of detection line, comprise anti--FITC, anti--digoxin, Streptavidin; Be marked with antigen or antibody on the latex particle of nature controlling line, comprise: rabbit igg, mouse IgG; The detection line latex particle of different small molecules antigen-antibodies that can comprise several marks in the same latex solution; The concentration of latex particle is 3～5OD;

3) nitrocellulose filter: the embedding of detection line position anti--antibody or the small molecules antigen of antigen, comprise anti--FITC, anti--digoxin, Streptavidin; In the embedding of nature controlling line position antigen or antibody, comprise sheep anti-mouse igg, goat anti-rabbit igg, anti-double-strandednucleic acid antibody and anti-single-chain nucleic acid antibody; The detection line of several different small molecules antigen-antibodies of embedding on the described nitrocellulose filter; The concentration of embedding albumen is 2～5mg/ml;

4) thieving paper: formed by the filter paper cutting that possesses syphonic effect;

On several different detection lines of latex test strip, catch different " latex particle-nucleic acid amplification product " simultaneously, form several different macroscopic bands.

Adopt conjugate pad, nitrocellulose filter specific as follows in the present embodiment:

Conjugate pad: the colloid gold particle that uses 30nm.The colloid gold particle mark of detection line is anti--antibody of vitamin H, and concentration is 5OD; The colloid gold particle mark rabbit igg antibody of nature controlling line, concentration is 5OD.

Nitrocellulose filter: the M135 type film that uses Merck ﹠ Co., Inc..The detection line bag is by anti--FITC antibody of 3mg/ml; The nature controlling line bag is by the goat anti-rabbit igg of 4mg/ml.

2. detection step

The pipe connecting of closed chromatography card is connected with the test paper seal of tube, rotation discharged the reaction solution of finishing amplification after the solution conduit that finishes of will increasing was again inserted the pipe connecting of proofing unit, the question response liquid layer is analysed nitrocellulose filter and is picked up counting, observations in the time of 5 minutes:

Nature controlling line C as the yin and yang attribute sample has band clearly, the detection line T(embedding of positive sample anti--FITC monoclonal antibody) have intensity to be +++band, the detected result positive (seeing Figure 15 B) of CPV is described, the detection line T of negative sample does not have band (seeing Figure 15 A).

The double check of embodiment 12:PRRSV and Pestivirus suis

With high-pathogenicity porcine reproduction and breathing syndrome virus (Pig Reproductive and Respiratory Syndrome Virus, PRRSV) and swine fever (Classical Swine Fever Virus, CSFV) be example, with the inventive method pathogenic agent is carried out dual LAMP and detect, concrete grammar comprises the steps:

One, the extraction of PRRSV and CSFV viral RNA

The component of extracting reagent is:

1) lysate, its component is: 0.15M pH8.0Tris-HCl, 5M guanidinium isothiocyanate, 1.5M CTAB(cetyl trimethylammonium bromide), 0.04M EDTA(ethylenediamine tetraacetic acid (EDTA));

2) washings A, its component is: volume percent is 95% Virahol;

3) washings B, its component is: volume percent is 48% Virahol;

4) lysate, its component is: deionized water no RNAase(RNA enzyme).

The RNA that carries out PRRSV and the CSFV positive and negative sample according to the method forembodiment 1 respectively extracts, with two kinds of positive sample RNA equal proportion mixing for standby use.

Two, amplification

1, amplimer and probe design

The PRRSV primer is identical withembodiment 1 with probe design.

CSFV primer and probe design: with 5'UTR(GenBank ID:KC149990.1) sequence is target gene, design primer and probe sequence, as follows respectively:

Shown in the F3:AGCTCCCTGGGTGGTCTA(SEQ ID NO.11)

Shown in the B3:TGTTAGTGGGCCTCTGCA(SEQ ID NO.12)

The FIP:Bio(vitamin H)-CATAGCATCTCGAGGTGGGCTC-CCTGAGTACAGGACAGTCGT(SEQ ID NO.13 shown in)

Shown in the BIP:CGAGGGCATGCCCAAGACAC-AGGTCGTACCCCCATCAC(SEQ ID NO.14)

Probe: the Digo(digoxin)-ACCTTAACCCTGGCGG(SEQ ID NO.15 shown in)

2, the preparation of amplifing reagent and normal temperature are preserved

(1) component of amplifing reagent:

1) liquid reagent:

0.04M Tris-HCl (pH8.0), 0.06M (NH4) 2SO4,0.04M NaCl, 0.02M MgSO4, volume percent is 0.3%TritonX-100, trimethyl-glycine 1.5M, totally 22 μ l.

2) solid reagent: ThermoScript II AMV2U, Bst archaeal dna polymerase 5U, dNTP2mM, each 0.3 μ M of the outer primer F3 of PRRSV and B3, each 0.6 μ M of the interior FIP of PRRSV and BIP, the probe 0.4 μ M of PRRSV, the outer F3 of the primer of CSFV and each 0.2 μ M of B3, each 0.4 μ M of the interior FIP of CSFV and BIP, the probe 0.4 μ M of CSFV, 1M BSA, 1M casein, 0.8M trehalose.

(2) preparation and normal temperature are preserved step:

Step 1, at first prepare liquid reagent, step is as follows:

Step 2, prepare solid reagent further, step is as follows:

3, nucleic acid amplification

22 μ l liquid reagents are added in the solid reagent, and fully dissolving then, adds PRRV RNA that 2 μ l extract and the mixed solution of CSFV RNA, places 63 ℃ of reaction 90min.

Three, the result detects

1. the primary structure of colloidal gold strip

Conjugate pad: the colloid gold particle that uses 35nm.The colloid gold particle mark of detection line is anti--antibody of vitamin H, and concentration is 3OD; The colloid gold particle mark rabbit igg antibody of nature controlling line, concentration is 4OD.

Nitrocellulose filter: the M135 type film that uses Merck ﹠ Co., Inc..Detection line 1 bag is by anti--FITC antibody of 2mg/ml,detection line 2 bags by 2mg/ml anti--monoclonal antibody of Digo; The nature controlling line bag is by the goat anti-rabbit igg of 3mg/ml.

2. detection step

The pipe connecting of closed chromatography card is connected with the test paper seal of tube, rotation discharged the reaction solution of finishing the LAMP constant-temperature amplification after the solution conduit that finishes of will increasing was again inserted the pipe connecting of proofing unit, the question response liquid layer is analysed nitrocellulose filter and is picked up counting, observations in the time of 5 minutes:

If the nature controlling line C of yin and yang attribute sample has band clearly, the detection line 1T1(embedding of positive sample anti-FITC monoclonal antibody) have band intensity to be ++, illustrate that the PRRSV positive (sees Figure 12 B, be respectively nature controlling line C from top to bottom, detection line 1T1, detection line 2T2); Detection line 2T2(embedding anti--Digo monoclonal antibody) have band intensity to be ++, the swine fever CSFV positive (seeing Figure 16 B) is described.The detection line 1T1 of negative sample and detection line 2T2 all do not have band (seeing Figure 16 A).

The decontamination method ofembodiment 13 amplified productions

Add an amount of 2mg/ml nanometer titanium dioxide titanium solution in an opened type container, amount of solution is advisable for 2-3 millimeters to cover closed proofing unit.With embodiment 10, orembodiment 11, or the result is judged to be positive closed chromatographic test paper proofing unit fracture atannular recesses 34 places of test paper pipe 3 (seeing Figure 14) among theembodiment 12, cut channel sheet (123) roof pressure with solution conduit lid counterbore shape cavity bottom breaks simultaneously, place all components above-mentioned nanometer titanium dioxide titanium solution broken then, open ultraviolet lamp, 260nm wavelength uv irradiating 24 hours, amplified production in the abundant degraded immuno-chromatographic test paper strip, false positive results is avoided in effectively decontamination.

Collect and to handle as stated above and untreated sample 500 μ L respectively, use the phenol-chloroform extracting, the method for isopropanol precipitating is carried out the nucleic acid extracting, and sample of nucleic acid dissolves with 8 μ L pure water, increases and detects according to

embodiment

10,11,12 described methods.Undressed sample results shows positive (seeing Figure 17 B), and the sample results of handling through the titania solution uv irradiating shows negative (seeing Figure 17 A).

Claims

1. a normal temperature is preserved the method for nucleic acid amplification reagent, it is characterized in that, with the part reagent in the nucleic acid amplification reagent or whole reagent, change into solid-state by curing process, thereby realization nucleic acid amplification reagent can be stablized preservation at normal temperatures, and then realizes kit for detecting nucleic acid stable preservation the at normal temperatures.

2. method according to claim 1 is characterized in that described normal temperature refers to that in 9～45 ℃ temperature range after described stable preservation referred to preserve 3 months at least, performance variation coefficient CV such as the biology of each reagent, physics and chemistry were less than 10%.

3. the method for claim 1 is characterized in that, described nucleic acid amplification reagent comprises following component:

Component A: can comprise: water, Tris-HCl, MgSO in the liquid and solid-state down stable component of preserving of normal temperature in the nucleic acid amplification reagent₄, trimethyl-glycine, (NH₄)₂SO₄, NaCl, MgCl₂, Triton;

B component: not necessarily stable and in the solid-state component that can stablize preservation down of normal temperature under the normal temperature liquid state in the nucleic acid amplification reagent, comprise: deoxynucleoside triphosphate, primer, probe or other nucleotide sequences of primer, probe or other nucleotide sequences and band small molecules mark;

Component C: unstable and in solid-state stable component down, comprising: Taq archaeal dna polymerase, Tth archaeal dna polymerase, Vent archaeal dna polymerase, Pwo archaeal dna polymerase, Pfu archaeal dna polymerase, Bst archaeal dna polymerase, t7 rna polymerase, AMV reversed transcriptive enzyme, MMLV reversed transcriptive enzyme under the normal temperature liquid state in the nucleic acid amplification reagent.

4. method according to claim 3 is characterized in that, realizes stable preservation the under the normal temperature of nucleic acid amplification reagent, needs to solidify described component C at least, with described component A and described B component with the liquid form stably stored; Perhaps, simultaneously B component and component C are solidified, A preserves with liquid form with component; Perhaps, simultaneously component A, B component and component C are all solidified, whole nucleic acid amplification reagent is preserved at ambient stable with solid form.

5. the method for claim 1 is characterized in that, described curing process refers to liquid nucleic acid amplification reagent component in the presence of curing is protectant, and the technology of dewatering by physical dryness becomes solid; The technology of described physical dryness dehydration, comprising: vacuum is drained technology, vacuum freezedrying technology and stoving process.

6. method as claimed in claim 5 is characterized in that, described curing protective material comprises sugar, albumen, tensio-active agent, antioxidant and sanitas; Described sugar comprises disaccharide and polysaccharide, and its final concentration that uses is 0.5～20% of volume ratio; Described albumen comprises animal serum and animal and plant albumen, and its final concentration that uses is 0.1～15% of volume ratio; Described tensio-active agent comprises ion and non-ionic tensio-active agent, and its final concentration that uses is 0.01～5% of volume ratio; Described antioxidant comprises VITAMIN, superoxide-dismutase, and its final concentration that uses is 0.01～5% of volume ratio; Described sanitas comprises sodium azide, Thiomersalate, Proclin300, and its final concentration that uses is 0.001～0.1% of volume ratio.

7. the closed nucleic acid chromatographic test paper detection kit that normal temperature is preserved is characterized in that: comprise the nucleic acid extracting reagent of sample, the nucleic acid amplification reagent that can preserve at normal temperatures and closed chromatographic test paper proofing unit; Described closed nucleic acid chromatographic test paper detection kit refers to that in closed chromatographic test paper proofing unit the amplified production solution of nucleic acid amplification reagent that normal temperature is preserved realizes rapid detection by immune chromatography test paper;

Described nucleic acid amplification reagent adopts each described method of claim 1-6 to carry out normal temperature and preserves;

Described closed chromatographic test paper proofing unit comprises: immune chromatography test paper and closed proofing unit; Wherein immune chromatography test paper is fixed in the closed proofing unit, and detected sample solution can not be with the contact of device outside atmosphere.

8. test kit as claimed in claim 7 is characterized in that, the detected target object of described nucleic acid amplification reagent comprises: RNA and DNA; Described nucleic acid amplification reagent is used for from the amplification of DNA or RNA template startup nucleotide sequence, final produce far above the template initial concentration and with RNA or the dna fragmentation product of template complementation or identical sequence, the nucleic acid amplification reaction method comprises: polymerase chain reaction, reverse transcriptase polymerase chain reaction, chain substitute amplification, rolling circle amplification, transcriptive intermediate amplification, the real time fluorescent quantitative loop-mediated isothermal amplification of amplification, ligase chain reaction (LCR), dependence nucleotide sequence.

9. test kit as claimed in claim 7, it is characterized in that: described closed chromatographic test paper proofing unit is by solution conduit, and pipe connecting and test paper pipe assembly:

10. test kit as claimed in claim 9, it is characterized in that: the lid of the solution conduit of described closed chromatographic test paper proofing unit is provided with a counterbore shape cavity, in this counterbore shape cavity, can insert another solution conduit lower tube body and form the stack sleeve structure, and discharge the product solution of nucleic acid amplification reaction, realize detecting simultaneously the reaction solution that the multitube amplification is finished; The counterbore shape cavity bottom of described solution conduit lid is provided with the solution releasing structure, and solution releasing structure bottom is provided with solution channel; Described solution body bottom outer circumference surface is provided with exterior groove; Solution conduit inner lid face near cap top is provided with the raised seals ring, and the exterior groove of another solution conduit that this raised seals ring is interior with inserting lid counterbore shape cavity cooperates the back to form reverse barb sealed structure.

11. test kit as claimed in claim 9 is characterized in that: described test paper pipe distal portion is provided with annular recesses, and when firmly acquiring a certain degree, from then on the annular recesses test paper pipe that partly fractures is convenient to that test sample is carried out abatement of pollution and is handled.

12. a method of utilizing test kit as claimed in claim 7 to carry out detection of nucleic acids is characterized in that, comprises the steps:

(1) adopt nucleic acid extracting reagent to carry out nucleic acid extraction;

A, get the liquid sample of 100～1000 μ l, add 300～4000 μ l lysates, room temperature leaves standstill 1～2min behind the concussion mixing;

B, adding 350～4000 μ l washings A with the centrifugal 3～5min of 10000～16000rpm, abandon supernatant behind the concussion mixing;

The washings B of c, adding 350～1000 μ l, the concussion washing makes the floss dissolving, with the centrifugal 1～2min of 10000～16000rpm, abandons supernatant subsequently, gets precipitation;

D, be deposited in drying at room temperature, add the lysate of 1-10 μ l with resolution of precipitate;

(2) adopt nucleic acid amplification reagent to carry out DNA or RNA amplification;

Liquid reagent in the nucleic acid amplification reagent is moved to the solution conduit of solid reagent with liquid-transfering gun or dropper, and fully dissolving forms nucleic acid amplification reaction liquid; The DNA that 1～10 μ l nucleic acid extracting reagent is extracted or RNA add in the nucleic acid amplification reaction liquid of getting ready then, carry out amplified reaction according to different nucleic acid amplification methods, recover room temperature subsequently and stop nucleic acid amplification;

(3) utilize the closed proofing unit of nucleic acid to carry out DNA or RNA detection:

A. the related immuno-chromatographic test paper strip of test paper pipe is injected in the interface of pipe connecting and its coupling, sealing can not be extracted;

The solution conduit that b. will finish amplification is inserted in the pipe connecting of closed chromatographic apparatus of horizontal positioned by the rotation solution conduit, solution conduit bottom protrusion breaking releasing, continue to insert fully solution conduit, the solution conduit exterior groove is cooperated with the first pipe connecting raised seals ring form overhead structure, this solution conduit will oppositely be withstood to be and can not extract and sealed state by this overhead structure;

C. the nucleic acid amplification reaction liquid that has increased is flow on the immuno-chromatographic test paper strip by the cut channel sheet of solution conduit bottom and pipe connecting inside in opened condition, the colloidal gold chromatographic immunology detection of beginning nucleic acid;

D. appearing at nitrocellulose filter from liquid picks up counting, immunochromatography finishes result of determination behind the 3-15min, have at nature controlling line under the prerequisite of bands visible, it is positive that band explanation detected result appears in detection line, and detection line does not have band explanation detected result negative.

13. method as claimed in claim 12, it is characterized in that, increase following nucleic acid amplification product harmless treatment step afterwards in step (3): the closed chromatographic test paper proofing unit after will finishing using fractures at the annular recesses place of test paper pipe, cut channel sheet roof pressure with solution conduit lid counterbore shape cavity bottom breaks simultaneously, then all components is immersed in the nanometer titanium dioxide titanium solution, then the nucleic acid amplification product in the irradiation decline deblocking enclosed chromatographic test paper device of UV-light.

14. the method described in claim 13 is characterized in that, described nano titanium oxide comprises: rutile-type, sharp ore deposit type, and the concentration of nanometer titanium dioxide titanium solution is: 0.5～5mg/ml; Described ultraviolet wavelength scope is 200～420nm, and irradiation time is 2～48 hours.