CN102146451B - PCR (Polymerase Chain Reaction) product qualitative and semiquantitative detecting kit - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to a PCR (Polymerase Chain Reaction) product qualitative and semiquantitative detecting kit (color PCR kit). The kit comprises a plurality of reagents, such as inosine monophosphate, xanthine oxidase, iodine chloride nitro-tetrazole, hypoxanthineguanine phosphoribesyltransferase, pyrophosphoric acid, and the like. The invention can be used for qualitatively or semiquantitatively detecting a PCR product.

Description

Qualitative and half-quantitative detection test kit of PCR product and uses thereof

Technical field

The present invention relates to biological technical field, be specifically related to the qualitative and half-quantitative detection test kit (colored PCR test kit) of a kind of PCR product.

Background technology

The detection method of polymerase chain reaction (PCR) product has qualitative and quantitative (comprising semiquantitative method) two kinds; Qualitative method is accomplished through gel electrophoresis usually, and quantitatively (sxemiquantitative) method is accomplished the mensuration of PCR product through real-time (real time) fluorescent mark PCR method.The determination object of two kinds of methods all is the nucleic acid moiety of polymerase chain reaction (PCR) product.Now technology can be with reference to following document:

(1)Tagiri-Endo?M，A?colorimetric?assay?for?inorganic?pyrophosphate?that?is?alsouseful?for?measuring?product?accumulation?in?polymerase?chain?reactions.Anal.Biochem.，2003，315(2)：170-4.

(2)R.Hisada，T.Yagi，1-Methoxy-5-Methylphenazinium?Methyl?Sulfate?APhotochemically?Stable?Electron?Mediator?between?NADH?and?Various?Electron?Acceptors，J.Biochem.，1977，82(5)，1469-1473.

(3) Yagi spark gap reaches a man of virtue and ability, 1-メ ト キ シ PMS, and Dojin News, 1979,14,1.

(4)S.Nakamura，K.Arimura，K.Ogawa，T.Yagi，Use?of?1-Methoxy-5-methylphenaziniumMethyl?Sulfate(1-Methoxypms)in?the?Assay?of?Some?Enzymes?of?Diagnosticimportance，Clin.Chim.Acta，1980，101，321-323.

(5)R.Hisada，W.Shinkai，T.Yagi，Photochemical?Stabilities?and?BiochemicalReactivities?of?Some?Derivatives?of?5-Methylphenazinium?Methyl?Sulfate(PhenazineMethosulfate)，J.Appl.Biochem.，1981，3，535-538.

(6)C.J.F.V.Noorden，J.Tas，The?Role?of?Exogenous?Electron?Carriers?inNAD(P)-dependent?Dehydrogenase?Cytochemistry?Studied?in?vitro?and?with?a?Model?Systemof?Polyacrylamide?Films，J.Histochem.Cytochem.，1982，30，12-15.

(7)H.Tsuge，Y.Kuroda，A.Iwamoto，K.Ohashi，Partial?Purification?and?Property?ofPyridoxine(Pyridoxamine)-5’-phosphate?Oxidase?Isozymes?from?Wheat?Seedlings，Arch.Biochem.Biophys.，1982，217，479-483.

(8)M.Rabinovitch，J-P.Dedet，A.Ryter，R.Robineaux，G.Topper，E.Brunet，?Destruction?of?Leishmania?Mexicana?Amazonensis?Amastigotes?Within?Macrophages?inCulture?by?Phenazine?Methosulfate?and?Other?Electron?Carriers，J.Exp.Med.，155，1982.415-416.

(9) Tabe Kazuhiro, Kawasaki Takao, the former Tianchang Zi, Tsuji Akio,?

inside Masahiko, one - メ Suites キ shifter Oh Nana ji nn メ Suites thermal Hikaru フ Oh, a coat and shareholders' イ ソ Hikaru Amino one Hikaru を use iru also neuronal Nico チ nn ア mini cloth ア terrestrial ni nn ji ヌ CLEA Io チ cloth Full Chemical Development ray analysis and Biological Full ingredient Full analysis of heavy Applied Analytical Chemistry ,1987,36,82-84.

(10) Sudo Yukio, Danze Chun sheng, former Tianchang Zi, Tsuji Akio, one - メ Suites キ shifter Oh Nana ji nn メ Suites thermal Hikaru フ Oh, a coat and shareholders' イ ソ Hikaru Amino one Hikaru affiliates also neuronal Nico チ nn ア mini cloth ア terrestrial ni nn ji ヌ ku Io チ cloth Full Chemical Development light を use iru 17α-Hiroshi cloth ro キ Shipley ro ge su Te ro nn Full chemical Development of light enzymes, analytical chemistry ,1988,37,185-187. (11) K.Yano? et? al, Formazan? Metal? complexes, US? Patent? 6,2003,645,595.

Existing polymerase chain reaction (PCR) product qualitative checking method needs gel electrophoresis or real-time fluorescence PCR method.Detect small amount of sample only through gel electrophoresis method, advantage is to measure accurately.But for detecting a large amount of samples, gel electrophoresis is cumbersome, takes time and effort the consumption funds.Carry out detection by quantitative with the real-time fluorescence quantitative PCR method, the instrument that require to use is more valuable, complicated operation and receive the restriction of conditions such as place, and application popularization property is poor.

Summary of the invention

In order to solve the problem that exists in the prior art, the invention provides qualitative and half-quantitative detection test kit (colored PCR test kit) of a kind of PCR product and uses thereof.

Qualitative and the semi-quantitative detection method of PCR product provided by the invention, mensuration to as if the by product tetra-sodium (PPi) of polymerase chain reaction (PCR).Utilize reagent and tetra-sodium (PPi) reaction in the test kit, end product be first and, English formazan by name is to contain H ₂The general name of the compound of N-N=CH-N=NH structure takes on a red color.

Through the positive findings of colour developing phenomenon (redness) judgement polymerase chain reaction (PCR), realize the qualitative detection of PCR reaction; Or the content of the by product tetra-sodium (PPi) that generates in absorbance half-quantitative detection polymerase chain reaction (PCR) process through mensuration 490nm place; The growing amount of indirect half-quantitative detection PCR product is because the nucleic acid moiety proportions constant of tetra-sodium that in the PCR reaction process, produces (PPi) and PCR product.Chemical reaction structural formula and the chemical equation that the present invention relates to are as depicted in figs. 1 and 2.

In order to realize the foregoing invention purpose, the invention provides following technical scheme:

A kind of qualitative and half-quantitative detection test kit of PCR product that is used for; Comprise component A, B component, component C and component D; Wherein component A contains inosine list phosphoric acid and XOD; B component contains chlorination iodonitrotetrazolium and HGPRT, and component C contains tetra-sodium, and component D is a water.

In the above-mentioned test kit, each component is preferably by following proportional arrangement: component A (2ml): comprise inosine list phosphatase 11 4mg; XOD 0.8U adds to 2ml with aqua sterilisa; B component (8ml): comprise chlorination iodonitrotetrazolium 10mg; HGPRT 8U uses aqua sterilisa

Add to 8ml;

Component C (100ml): comprise tetra-sodium 10mg, aqua sterilisa adds to 100ml;

Component D (100ml): aqua sterilisa.

The present invention also further provides the mentioned reagent box to be used for the purposes of qualitative detection PCR product, comprises the steps:

Get component A and mix with the PCR reaction product, place 60 ℃ following 10 minutes, add B component and component D then, wherein contrast adds component D, all the other add B component, place 60 ℃ following 20 minutes, observe the color of end product, there is the PCR product in explanation if take on a red color promptly.

The present invention also further provides the mentioned reagent box to be used for the purposes of half-quantitative detection PCR product, comprises the steps:

1) typical curve of drafting tetra-sodium: dilute times to component C respectively with component D, form the tetra-sodium diluent of finite concentration gradient;

Getting 20 μ l component A mixes with 100 μ l tetra-sodium diluents; Place 60 ℃ following 10 minutes, add 80 μ l B component and component D then, wherein contrast adds component D; All the other add B component; Place 60 ℃ following 20 minutes, get 150 μ l reaction solutions are surveyed the 490nm place on ultraviolet spectrophotometer absorbance, respectively with Δ OD=OD ₄₉₀-OD _BlankMake ordinate zou, tetra-sodium concentration (denary logarithm value) is X-coordinate, the production standard curve;

2) working sample: get 20 μ l component A and mix with 100 μ l PCR products; Place 60 ℃ following 10 minutes, add 80 μ l B component and component D then, wherein contrast adds component D; All the other add B component; Place 60 ℃ following 20 minutes, get 150 μ l reaction solutions are surveyed the 490nm place on ultraviolet spectrophotometer absorbance, ask Δ OD=OD respectively ₄₉₀-OD _BlankValue is calculated tetra-sodium concentration from the typical curve equation, and then is calculated the PCR production concentration.

The present invention has following technique effect:

Qualitative and the half-quantitative detection test kit of PCR product of the present invention is used for qualitative detection PCR reaction product or half-quantitative detection PCR product growing amount, and is simple to operate.Qualitative detection PCR reaction product need not carried out gel electrophoresis, and the condition that half-quantitative detection PCR product growing amount experimental implementation requires is low, has a spectrophotometer just can accomplish the mensuration of absorbancy, is not tested the restriction in place etc., application popularization property height.This test kit is more suitable for the qualitative screening of high-throughput, the many more meliority of using this test kit that can demonstrate more of working sample.Using this test kit qualitative detection does not need the production standard curve, and half-quantitative detection needs the production standard curve.

The susceptibility (2.5ng) that colored PCR test kit method detects the PCR product is suitable with the susceptibility (4ng) that gel electrophoresis detects the PCR product.

Description of drawings

Fig. 1: the chemical reaction structural formula that the present invention relates to

Fig. 2: the chemical equation that the present invention relates to

Fig. 3: tetra-sodium typical curve

Fig. 4: agarose gel electrophoresis figure

Fig. 5: colored PCR detects design sketch

Fig. 6: the active figure as a result of colored PCR test kit detection by quantitative T4DNA ligase enzyme (3 batches)

Fig. 7: the active figure as a result of colored PCR test kit detection by quantitative dna polymerase i

Fig. 8: colored PCR test kit is used for the figure as a result of sense terminals ribodesose transferase active capture range

Embodiment

Following experimental implementation chemical reagent is mainly purchased the company in Sigma; Pyrophosphate salt purchase in and light (Wako) company; Thymus nucleic acid (DNA) polysaccharase that uses, dna ligase etc.; All purchase in Takara company if no special instructions, all experiments are all by the routine operation of biological chemistry and molecular biology experiment.Below only exemplary explanation test kit of the present invention and purposes, but be not construed as limiting the invention.

Detection kit provided by the invention comprises (100 secondary response amount):

Component A (2ml):

Comprise inosine list phosphoric acid (Inosine 5 '-monophosphate, IMP; Molecular weight is 348.20) (Sigma, Code No.:I 2879) 14mg; XOD (Xanthine oxydase, XOD; Specification is 0.4U/ml) (Sigma, Code No.:X 2252) 0.8U (1U is defined as under 25 ℃, the condition of pH7.5, and PM changes into uric acid to the xanthine of 1.0 μ mol).Add to 2ml. with aqua sterilisa

B component (8ml):

Comprise the chlorination iodonitrotetrazolium ((Iodonitrotetrazolium chloride, INT); Molecular weight is 505.70) (Sigma, Code No.:I 8377) 10mg; HGPRT (hypoxanthine-guanine phosphoribosyltransferase, HGPRT; Concentration is 5U/ml) (Sigma; Code No.:3389) (1U is defined as under 37 ℃, the condition of pH7.5 8U; PM catalysis guanylic acid (guanine) and Phosphoribosyl tetra-sodium (phosphoribosyl pyrophosphate; PRPP) generate 1nmol guanosine monophosphate (guanosine 5 '-monophosphate, GMP)).Add to 8ml. with aqua sterilisa

Component C (100ml):

Comprise tetra-sodium (PPi, molecular weight are 330.34) (Wako, Code No.:349-01503) 10mg.Aqua sterilisa adds to 100ml.

Component D (100ml):

Aqua sterilisa.

The transportation of test kit, preservation and precaution:

This test kit transports under-20 ℃ of conditions, under 4 ℃ of conditions, preserves, and component A must keep in Dark Place; Each component stability is more than 6 months in the test kit; Reagent allows to uncap at ambient temperature.

Answer the rapid test absorbance after utilizing the reagent react end in the test kit.

The use of test kit provided by the invention:

1, draws the typical curve of tetra-sodium (PPi): dilute 300 times, 150 times, 60 times, 30 times, 15 times, 10 times to component C respectively with component D; With component D constant volume to 100 μ l; The final concentration of tetra-sodium is respectively 10 μ mol/L, 20 μ mol/L, 50 μ mol/L, 100 μ mol/L, 200 μ mol/L, 300 μ mol/L, and the typical curve that supplies to draw tetra-sodium (PPi) is used.

Getting 20 μ l component A mixes with 100 μ l tetra-sodium diluents; Place 60 ℃ following 10 minutes; Add 80 μ l B component or component D (contrast adds component D, and all the other add B component) then, place 60 ℃ following 20 minutes; Get 150 μ l reaction solutions and on ultraviolet spectrophotometer, survey the absorbance at 490nm place, ask Δ OD=OD respectively ₄₉₀-OD _BlankMake ordinate zou, lg (PPi μ mol/L) is an X-coordinate, the production standard curve.Typical curve should be as shown in Figure 3, the standard variance (R of typical curve ²) should be less than 0.980.

2, working sample: get 20 μ l component A and mix with 100 μ l PCR products; Place 60 ℃ following 10 minutes; Add 80 μ l B component or component D (contrast adds component D, and all the other add B component) then, place 60 ℃ following 20 minutes; Get 150 μ l reaction solutions and on ultraviolet spectrophotometer, survey the absorbance at 490nm place, ask Δ OD=OD respectively ₄₉₀-OD _BlankValue is calculated lg (PPi μ mol/L) value from the typical curve equation, calculates PPi concentration, and then calculates the PCR production concentration.

Embodiment 1: colored PCR test kit qualitative detection effect experiment, confirm that the Schwellenwert of detection polymerase chain reaction product is that template is an example with lambda DNA, and carry out the amplification of 500bp purpose fragment PCR, but be not limited to this.

The PCR system is: 10 * PCR damping fluid, 10 μ l, 15mM MgCl ₂4 μ l, 2.5mM dNTPmix 8 μ l, 20 μ M lambda1primer, each 1 μ l of lambda 2primer, template DNA 20～200pg, Taq archaeal dna polymerase (5U/ μ l) 0.5 μ l, moisturizing to 100 μ l.

Reaction conditions is: 94 ℃ of preparatory sex change 30 seconds; 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds, 72 ℃ of lengthening reactions 30 seconds; 25 circulations.

1, agarose gel electrophoresis detects:

PCR product amount is 100ng/uL, dilutes 100 times, and concentration is 1ng/uL, agarose gel electrophoresis (1.0%), and applied sample amount is respectively: 100uL, 50uL, 20uL, 15uL, 10uL, 8uL, 6uL, 4uL, 2uL, 1uL, 0.5uL, 0.1uL.

Electrophoresis result is as shown in Figure 4, can be found out by Fig. 4, and the DNA band can be seen slightly in the duct that is labeled as 4ng, explains that the product of 4ng PCR can be detected by agarose gel electrophoresis.Agarose gel electrophoresis is difficult to detect the product that is lower than 4ng PCR.

2, colored PCR test kit detects:

PCR product amount is 100ng/ μ L, dilution different concns (1000,750,500,250,125; 25,12.5,2.5,1.25,0.25ng/100 μ L); Each concentration product sample 100 μ l adds component A 20 μ l respectively, concussion mixing, 60 ℃, 10 minutes; Add B component 80 μ l, the concussion mixing, 60 ℃, 20 minutes, the result was as shown in Figure 5.

Can find out by Fig. 5, be labeled as 2.5 the apparent slightly redness of EP pipe, explain that the PCR product of 2.5ng can be detected.The minimum quantity that the minimum quantity of colored PCR test kit detection PCR product and agarose gel electrophoresis detect the PCR product is suitable.

Embodiment 2, the experiment of colored PCR test kit detection by quantitative effect

1, is example with active (three batches: K2900, K3000, the K3100) detection by quantitative of T4DNA ligase enzyme (Ligase), confirms colored PCR test kit detection by quantitative effect.

The pBgl II Linker 15uL that in the EP pipe, adds 5nM, 10 times damping fluid 3uL, T ₄Dna ligase (different concns) 1uL, water adds to 30uL, and reaction is 70 minutes under 16 ℃ of conditions, and 90 ℃ were heated 5 minutes down, add 70uL water.Add 20uL component A again, 60 ℃ were reacted 10 minutes down, added the 80uL B component, and 60 ℃ were reacted 20 minutes down, measure OD ₄₉₀, calculate Δ OD=OD ₄₉₀-OD _Blank, result such as following table and shown in Figure 6.

Three batches of detected results, K2900 is that 353U/ μ l, K3000 are that 345U/ μ l, K3100 are 358U/ μ l, and three crowdes of results are very approaching, and it is active to explain that colored PCR test kit can be used in quantitatively determined T4DNA ligase enzyme.

2, detecting with dna polymerase i (Polymerase I) active level is example, confirms colored PCR test kit detection by quantitative effect.

Get 100 μ l dna polymerase i (Polymerase I) determination of activity reaction solutions respectively, add dna polymerase i (different extension rate) 1 μ l respectively, 1 part of contrast is done in 37 ℃ of heating 15 minutes simultaneously, and promptly 100 μ l do not add dna polymerase i and react.Add component A concussion mixing, 60 ℃ were heated 10 minutes, added 80 μ l B component, the concussion mixing, and 60 ℃ were heated assaying reaction liquid OD 20 minutes ₄₉₀Value, measure control samples simultaneously, calculate Δ OD=OD ₄₉₀-OD _Blank, result such as following table and shown in Figure 7.

polI	U	OD490	OD490-B
				B	?	0.099	0
×1000	0.0035	0.095	-0.004
				×500	0.007	0.113	0.014
×200	0.0175	0.109	0.01
				×100	0.035	0.102	0.003
×50	0.07	0.115	0.016
				×10	0.35	0.333	0.234
×3	0.7	0.575	0.476
				1μl	3.5	1.456	1.357
2μl	7	1.43	1.331
				3μl	10.5	1.515	1.416

Can find out by table and Fig. 7, in the scope of extension rate 5～100, the very good (R of the linear relationship of colored PCR test kit ²Be 0.999), explain that colored PCR test kit can be applied to DNA Polymerase I active level and detect, measure DNA Polymerase I activity thereby replaced common radioactive rays method, for experimental implementation person has brought convenience.

3: colored PCR test kit is applied to the mensuration of the refining active capture range of terminal ribodesose transferring enzyme (Terminal DeoxynucleotidylTransferase (TdT)), is that radioactive rays are surveyed active method and done one relatively with traditional method simultaneously:

Get 100 μ l reaction solution terminal ribodesose transferase active assaying reaction liquid respectively; Add terminal ribodesose transferring enzyme (different extension rate) 1 μ l respectively; 1 part of contrast is done in 37 ℃ of heating 15 minutes simultaneously, and promptly the sample that do not add in the active collection tube of 100 μ l reacts.Add component A concussion mixing, 60 ℃ were heated 10 minutes, added 80 μ l B component, the concussion mixing, and 60 ℃ were heated assaying reaction liquid OD 20 minutes ₄₉₀Value, measure control samples simultaneously, calculate Δ OD=OD ₄₉₀-OD _Blank, active (fraction assay), result such as the following table and shown in Figure 8 of detecting of the refining Sephadex 6-100 part of TDT.

Can find out by table and Fig. 8; Radioactive rays detect (RI; Black line peak part) the collection tube scope of terminal ribodesose transferase active being arranged is 57～68 pipes; It also is 57～68 pipes that colored PCR test kit detection (blue line peak part) has the collection tube scope of terminal ribodesose transferase active, and colored PCR test kit detection method is consistent with radioactive rays detection of active method (RI method), can replace the radioactive rays detection method.

Claims (2)

1. one kind is used for the qualitative and half-quantitative detection test kit of PCR product; It is characterized in that comprising component A, B component, component C and component D; Wherein component A contains inosine list phosphoric acid and XOD; B component contains chlorination iodonitrotetrazolium and HGPRT, and component C contains tetra-sodium, and component D is a water;

Each component is pressed following proportional arrangement:

Component A: comprise inosine list phosphatase 11 4mg; XOD 0.8U adds to 2ml with aqua sterilisa;

B component: comprise chlorination iodonitrotetrazolium 10mg; HGPRT 8U adds to 8ml with aqua sterilisa;

Component C: comprise tetra-sodium 10mg, aqua sterilisa adds to 100ml;

Component D: aqua sterilisa;

Wherein, the qualitative and half-quantitative detection of PCR product to as if the by product tetra-sodium of PCR reaction, utilize the by product tetra-sodium reaction of component A in the test kit, B component and PCR reaction, end product be redness first and; It is 490nm that product is measured wavelength;

Described test kit transports under-20 ℃ of conditions, under 4 ℃ of conditions, preserves, and component A must keep in Dark Place; Each component stability is more than 6 months in the test kit; Reagent allows to uncap at ambient temperature.

2. test kit is used for the purposes of half-quantitative detection PCR product according to claim 1, comprises the steps:

1) typical curve of drafting tetra-sodium: dilute times to component C respectively with component D, form the tetra-sodium diluent of finite concentration gradient;

Getting 20 μ l component A mixes with 100 μ l tetra-sodium diluents; Place 60 ℃ following 10 minutes, add 80 μ l B component and component D then, wherein contrast adds component D; All the other add B component; Place 60 ℃ following 20 minutes, get 150 μ l reaction solutions are surveyed the 490nm place on ultraviolet spectrophotometer absorbance, respectively with Δ OD=OD ₄₉₀-OD _BlankMake ordinate zou, tetra-sodium concentration is X-coordinate, the production standard curve;

2) working sample: get 20 μ l component A and mix with 100 μ l PCR products; Place 60 ℃ following 10 minutes, add 80 μ l B component and component D then, wherein contrast adds component D; All the other add B component; Place 60 ℃ following 20 minutes, get 150 μ l reaction solutions are surveyed the 490nm place on ultraviolet spectrophotometer absorbance, ask Δ OD=OD respectively ₄₉₀-OD _BlankValue is calculated tetra-sodium concentration from the typical curve equation, and then is calculated the PCR production concentration.

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