CN1281052A - Endoreference of equivalent competition polymerase chain reaction and its preparing process and application - Google Patents

Abstract

The endoreference able to be equivalently amplified relatively to target gane in competition PCR is a mutation fragment with a length equal to that of target fragment and the same content of various bases. Its positions from 21 to 70 are mutation part. The mutation fragment obtained by PCR through mutation primer and downstream primer of target gene is just the endoreference which can be used in equivalent competition PCR and enzyme-linked hybridization of PCR resultants to quantitatively detect virus gene. Its advantages include high accuracy of quantitative analysis, and simple preparing process.

Description

Equivalent competition polymerase chain reaction,PCR internal reference, preparation method and application thereof

The present invention relates to virogene detection technique field, be that an a kind of part of virogene is suddenlyd change specifically, the mutator gene fragment that obtains is isometric with the purpose fragment of intending detecting, serve as the internal reference of competition polymerase chain reaction,PCR (PCR) with this, the equivalence of internal reference and purpose fragment is increased in the PCR process, can be used for the PCR detection by quantitative of various virogenes.

The PCR method is the highest a kind of method of susceptibility in the gene diagnosis technology, is used for qualitative detection at the beginning, expands to detection by quantitative afterwards.Showing through years of researches, adopt round pcr to carry out detection by quantitative, must be the competitive PCR method, that is to say internal reference must be set, otherwise its quantitative result not only can't stdn, and out of true very.So internal reference has become the key component of PCR quantitative technique.Two problems the most basic should noting of preparation competitive PCR internal reference (sudden change template) are: guarantee to greatest extent that 1. the sudden change template increases with the equivalence of wild template in the PCR process; 2. easy and can accurately carry out identification and analysis to sudden change fragment in the product and wild fragment.There is the most frequently used method of reported literature to have three kinds at present: to add regular way, truncation method and enzyme-added site method (Diviacco S, Norio P, Zentilin L, the et al:Gene 1992 of cutting; 122:3013, Clementi M, Manzo S, Bagnareli P, etal:PCR Methods Appl 1993; 2:191, Wu J, Sullivan DE and Gerber MA:J VirolMethods 1994; 49:331).Lengthening and the prepared sudden change template of truncation method, though when the PCR product analysis, have directly perceived, easy advantage, but because the sudden change template is lengthening or shortening on the basis of wild template sequence, with the obvious difference of wild template on length, can't guarantee the unanimity of both amplification efficiencies, influence competitive PCR quantitative analysis tolerance range; Enzyme-added cut the prepared internal reference of site method and the purpose fragment isometric, can guarantee both equivalence amplifications, but this method has two defectives: the first, selecting or increasing proper restriction site has higher difficulty, is subjected to certain limitation during practical application; The second, as not guaranteeing complete degestion, then can reduce the tolerance range of product analysis to the internal reference amplified production.1993, reports such as Jalava, the complementary sequence of the segmental upstream and downstream primer of the purpose that is used to increase is added in one section segmental both sides of allogeneic dna sequence DNA respectively, after with this primer being made PCR again, also obtain sudden change fragment (the Jalava T isometric with wild template, Lehtovaara P, Kallio A:BioTechniques1993; 1:134).This method still has deficiency: the preparation method is loaded down with trivial details; The G+C content of selected allos fragment sequence and target gene fragment relatively have certain difference inevitably, thereby also can cause the difference of two kinds of fragment amplification efficiencies.Someone adopt chemical synthesis prepare about 100bp, with isometric sudden change internal reference (Gerlich WH, Heermann KH, the Thomssen R:Viral HepRev 1995 of wild fragment; 1:53), and introduce product hybridization quantitative analysis, than preceding method major progress is arranged all, but still there is big difficulty technically in the long oligonucleotide of synthetic 100nt at present, only can be for it for the very few laboratories of number, also because output is very low cost is too high.In addition, must synthetic two complementary oligonucleotide, and just can be used as internal reference after becoming double-stranded DNA through hybridization, not only expense is higher, and the preparation process complexity.

For this reason, the purpose of this invention is to provide a kind of equivalent competition PCR internal reference, it is a kind of sudden change fragment.Another object of the present invention provides the preparation method of this internal reference, is by the downstream primer with mutant primer and goal gene, carries out PCR and makes.Another purpose of the present invention provides the application of this internal reference, is used for the detection by quantitative of virogene.

One, the invention provides a kind of in competitive PCR can with the internal reference of goal gene equivalence amplification, it is that a kind of and purpose fragment length equate and A, C, T, sudden change fragment that G content is identical, in the 21-70 position is the sudden change part, except that the base sequence of sudden change part was different with the purpose fragment, all the other positions were all identical with the purpose fragment.

Two, the invention provides the preparation method of equivalent competition PCR internal reference, it comprises the following steps:

1, the design of mutant primer

The conservative region sequence of the pathogenic agent that the selection amount of drafting detects is as goal gene, and according to the sequence of goal gene and the Position Design mutant primer in genome thereof.Mutant primer is made up of three parts, i.e. PCR upstream primer sequence, mutant nucleotide sequence and catenation sequence.The long 40-80 of a mutant primer base.Mutant nucleotide sequence is at the 21-70 bit base, mutant form is the base sequence that changes the sudden change part randomly, but the absolute number of base A, C, T, G is constant, mutant nucleotide sequence length is 10-50 base, the Tm value of mutant nucleotide sequence, form the Energy value of hairpin structure and primer dimer, all identical with former target gene sequences.

2, the segmental preparation that suddenlys change

By downstream primer with mutant primer and goal gene, be template with the goal gene, carrying out PCR products therefrom sudden change fragment promptly is internal reference.

This sudden change fragment just is 21-70 bit base sequence with the difference of the purpose fragment of intending detecting (or claiming wild fragment), other position especially PBR at two ends is identical, thereby can guarantee that two kinds of segmental amplification efficiencies are in full accord when making competitive PCR.

3, segmental clone of sudden change and evaluation

A, clone clone's purpose has two: the first, and the sudden change fragment is inserted certain carrier, obtains containing the segmental plasmid of sudden change, thus can be in intestinal bacteria massive duplication, become the raw material sources of internal reference; The second, containing the segmental plasmid of sudden change is ring-type, and size is similar to viral genome to be detected, thereby more meet the requirement of internal reference, cloning process is to adopt PCR product TA cloning, promptly uses PCR clone test kit, and the sudden change fragment that newly increases directly is cloned in the carrier.The positive colony of selecting is done further to identify.

B, evaluation purpose are to confirm that whether the sudden change fragment has successfully inserted carrier, will confirm also simultaneously whether the base sequence of mutant nucleotide sequence is consistent with design requirements.Method is as follows:

(1) the PCR method has been synthesized a primer in addition, and its sequence is identical with the 1-20 bit base of mutant primer, and with downstream primer, to positive colony plasmid DNA performing PCR, the positive person that increases remakes further evaluation with it.

(2) dna sequence analysis is got pcr amplification male plasmid DNA, and cloned sequence is done the sequence analysis.The clone that reservation queue is correct.

Three, equivalent competition PCR internal reference of the present invention is applied to the detection by quantitative virogene

Utilize the prepared internal reference of the present invention, set up competitive PCR and product enzyme thereof connection hybridization technique, with the detection by quantitative virogene.Detection comprises two parts: the first, and competitive PCR contains goal gene to be measured and internal reference at same reaction tubes, carries out polymerase chain reaction,PCR; The second, the hybridization of enzyme connection as solid phase carrier, is carried out enzyme connection hybridization with a kind of micro reaction plate.This detection can reach three purposes: 1. distinguish wild fragment and internal reference fragment after increasing; 2. quantitative to above-mentioned two kinds of fragments respectively; 3. further improve the tolerance range and the sensitivity of gene test.

Advantage of the present invention: add regular way, truncation method and the enzyme-added site legal system of cutting is equipped with competitive PCR internal reference technology and compares with commonly used, the present invention has following advantage: first, preparation method's advantage: on the purpose fragment, select the ideal amplification region, and design suitable mutant primer, mutant primer is done one with another root pairing primer and goal gene takes turns PCR and can obtain the fragment of suddenling change, compare the simplest, convenient, the easy row of this method with present all report methods.The second, equivalence amplification fully: suddenly change segmental length and A, T, C, G form all identical with wild fragment, therefore can guarantee the equivalence amplification in the PCR process of two kinds of templates, have improved the tolerance range of quantitative analysis.The 3rd, for detecting, the hybridization of competitive PCR product provides top condition: the mutant nucleotide sequence after the segmental 21-70 position sudden change that suddenlys change, it is the special hybridization site of sudden change fragment, and all hybridization parameters of this hybridization site are all identical with the segmental corresponding section of purpose of intending amplification, therefore can set up the enzyme connection hybridization quantitative analysis method of competitive PCR product on this basis.Compare with length dna internal references such as artificial chemosynthesis, identical characteristics are arranged aspect application principle, promptly can guarantee and wild fragment equivalence amplification, can in the hybridization testing process, distinguish again with wild fragment, but on the preparation method then than simpler, easily the row and economical, any one has the laboratory of the most basic molecular biology experiment condition and all can implement.

Below be that goal gene is that example is further elaborated in conjunction with the accompanying drawings and embodiments with the HBV gene, but do not place restrictions on scope of the present invention.

Description of drawings

Fig. 1. the structural representation of mutant primer

Up: HBV gene 2278-2333 bit sequence, wherein 2299-2318 position (black line

In the frame) for intending the sudden change zone

Descending: mutant primer sequence, length are 56nt, are mutant nucleotide sequence in the red line frame

Fig. 2. sudden change produced in fragments process synoptic diagram

1.PCR add mutant primer, downstream primer, wild template etc. in the reaction tubes;

2. mutant primer and downstream primer annealing, extension after the double-stranded template sex change is unwind;

3. the 1st PCR circulation back forms 2 new fragments, and one is the sudden change fragment, another

Individual is wild fragment;

4. enter the 2nd PCR circulation;

5. the 2nd PCR circulation back forms 3 sudden change fragments and 1 wild fragment;

6. through after 30～40 circulations, product is exponential amplification, and the sudden change fragment accounts for most extremely

Number, minute quantity is wild fragment (can ignore).

Fig. 3. sudden change fragment MS299 and TA cloning vector pCR2.1 reorganization synoptic diagram

Embodiment 1. usefulness mutant primers preparation sudden change fragment (internal reference)

One, the design of mutant primer is with synthetic

The genomic S gene line of HBV conserved regions is so select fragment to be amplified in this district.Adopt robot calculator Nucleotide Sequence Analysis Software HYBsimulator Version 4.0 (available from U.S. ACGT Corp.) to design.Require fragment to be amplified (wild fragment) to meet the following conditions: 1. length is 100-500bp; 2. 1-20 bit base and 21-40 bit base sequence strictly meet primer and probe design requirement (comprise Tm value, G+C content, repeat the base number, form Energy value of hairpin structure and primer dimer or the like) respectively; 3. the 41-56 bit base more strictly meets the design of primers condition.After the plan amplified fragments is selected, list the number of 4 kinds of bases in 21-40 position (A, T, C, G) respectively, the oligonucleotide sequence that synthesizes a plurality of 20nt by the absolute number random groups of every kind of base, these sequence input robot calculator, analyze with Nucleotide Sequence Analysis Software, select one to several sequences (being referred to as mutant nucleotide sequence), its Tm value, form the Energy value of hairpin structure and primer dimer, all equate or very approaching with wild fragment 21-40 bit base sequence.Get the wild fragment sequence in 1-56 position, wherein 21-40 bit base sequence is replaced into mutant nucleotide sequence (hybridization site when this section mutant nucleotide sequence is exactly the hybridization of PCR product) constitutes mutant primer.With this primer and another downstream primer HBV genome that goes to increase, just can obtain the sudden change fragment that the present invention needs, as internal reference.At HBV gene S district design mutant primer (Pm) sequence (56nt) comprised three parts (Fig. 1): first part (20nt) is identical with competitive PCR upstream primer sequence, is positioned at 5 ' hold; Second section is mutant nucleotide sequence (20nt), in the middle of being positioned at; Third part is positioned at 3 ' end, with the 41-56 bit base complementation of fragment minus strand to be amplified for connecting primer (16nt).Rely on this section to play the primer of ligation, just might make the annealing of mutant primer and HBV gene, finish the process of PCR, obtain the fragment of suddenling change, see Fig. 2.

Two, preparation sudden change fragment

(1) main raw

1. hepatitis B virus (adr type) plasmid DNA is cut rear clone to the pBR322 carrier by the full gene of adr type HBV through the BamHI enzyme.Plasmid is so kind as to give by doctor Jiang that acts like a bully of molecular biology teaching and research room of The 2nd Army Medical College.

Mutant primer (Pm, it is synthetic and with PAGE method purifying 56nt) to give birth to worker engineering corporation by Shanghai, sequence is seen Fig. 1.

3. conventional pcr amplification primer upstream primer (Pu, 20nt) sequence: 5 '-GTTGC CCGTTTGTCC TCTAC-3 '; Downstream primer (Pd, 22nt) sequence is as follows: 5 '-GCCCC CAATACCACA TCATC-3 '.

4.PCR damping fluid, Taq enzyme and substrate etc. are conventional reagent, give birth to worker engineering corporation available from Shanghai Huamei Bio-Engrg Co., and Shanghai.

(2) pcr amplification sudden change fragment is got a 0.2ml thin-walled PCR reaction tubes, presses following prescription application of sample:

Composition application of sample amount (μ l)

10 * damping fluid 5

15mmol／L?MgCl ₂?????????????5

DNTP mixed solution (each 10mmol/L) 2

Pm(25μmol／L)?????????????????1

Pd(25μmol／L)???????????????1

HBV DNA plasmid (μ g/L) 1

Taq archaeal dna polymerase (1u/ μ l) 1

Bi-distilled water 34

The pcr amplification parameter: 94 ℃ 5 minutes; 94 ℃ 1 minute, 55 ℃ 30 second, 72 ℃ of 30 second, totally 35 circulations; 72 ℃ were extended 5 minutes again.

(3) the PCR product analysis adopts agarose gel electrophoresis method: the PCR product is splined on 1.5% sepharose, ethidium bromide staining behind the electrophoresis, observe under the UV-light, amplification gained fragment length is about 300bp, and there are not other assorted bands, consistent with the result who infers, promptly tentatively confirm as the sudden change fragment, can clone.

Three, the clone and the evaluation of PCR product (sudden change fragment)

(1) clone

1. 1. TA cloning vector pCR2.1 carrier of main raw, 25ng/ μ l; T ₄Dna ligase (4.0Weiss unit/μ l) and damping fluid (available from COLON TECH company), 2. bacillus coli DH 5 alpha strain (preserve in this laboratory).

2. operating process is as follows:

(1) in the 0.5mlEppendorf pipe, add successively:

Fresh PCR sudden change fragment product 3 μ l

10 * connection damping fluid, 1 μ l

PCR2.1 carrier 1 μ l

Distilled water 4 μ l

T ₄Dna ligase 1 μ

Total amount 10 μ l, mixing is put reaction tubes and is spent the night in 14 ℃;

(2) prepare DH5 α calcification bacterium routinely;

(3) connector is added in the 100 μ l calcification bacterium, placed 1 hour for 4 ℃, 42 ℃ of water-baths 90 seconds;

(4) add LB substratum 400 μ l, 37 ℃ of slow joltings of constant temperature shaking table 1 hour;

(5) centrifugal, 4000rpm, 3 minutes.Draw supernatant, retain the about 50 μ l of liquid, add 20 μ lIPTG (100mmol/L), 5 μ l X-Gal (40mg/ml) inhale gently and beat at the pipe end, and bacterium is fully suspended;

(6) bacterium liquid is coated the solid LB-agar culture plate that contains penbritin (50 μ g/ml), put 37 ℃ of overnight incubation;

(7) picking white colony is inoculated in the 5ml LB substratum that contains 100 μ g/ml penbritins, in 37 ℃ of thermostat containers, and jolting (250 rev/mins);

(8) bacterial growth to the logarithm later stage is collected bacterium, extracts and plasmid DNA purification with separator column;

(9) add Xba I double digestion with EcoR I single endonuclease digestion and BamH I respectively, carry out agarose gel electrophoresis again, with the preliminary screening positive colony.

The internal reference fragment is inserted the synoptic diagram of carrier and is seen Fig. 3

(2) the evaluation positive colony is also further done the sequence analysis with the PCR preliminary evaluation.

1．PCR

The reaction system prescription:

Composition application of sample amount (μ l)

10 * damping fluid 5

15mmol／L?MgCl ₂?????????5

DNTP mixed solution (each 10mmol/L) 2

Pu(25μmol／L)????????????1

Pd(25μmol／L)????????????1

Positive colony plasmid (μ g/l) 1

Taq archaeal dna polymerase (1u/ μ l) 1

Bi-distilled water 34

The pcr amplification parameter: 94 ℃ 5 minutes; 94 ℃ 1 minute, 56 ℃ 30 second, 72 ℃ of 30 second, totally 35 circulations; 72 ℃ were extended 5 minutes again.

Product is splined on 2% sepharose, electrophoresis and with confirming behind the ethidium bromide staining that amplified fragments is about 300bp, waits until further evaluation.

2. sequential analysis automatic sequencing method records amplified production and is positioned at HBV S gene regions 2278-2576 position, sheet segment length 299bp.The correct clone of preface is read in retention, wherein a strain called after PCR2.1-MS299.

Four, the amplification of recombinant plasmid PCR2.1-MS299, purifying and quantitatively get recombinant plasmid PCR2.1-MS299 and transform DH5 α intestinal bacteria once more; with this plasmid of a large amount of amplifications; with using phenol-chloroform extracting once (ordinary method) again behind plasmid DNA separator column (available from Shanghai China Shun bio-engineering corporation, the by specification operation) purifying.The purifying product are used agarose gel electrophoresis and ultraviolet spectrometry standard measure respectively.

Embodiment 2. competitive PCRs amplification experiment

One, design of primers and synthetic

The upstream primer sequence is Pu, and the sequence of downstream primer is Pd (with embodiment 1), but at 5 ' end biotin modification, called after Pb.Primer is synthetic to provide service with modification by the living worker in Shanghai bio-engineering corporation.

Two, preparation HBV DNA standard substance

(1) HBV DNA plasmid amplification and purifying increase routinely in this laboratory, adopt post to separate and add phenol chloroform extraction method purifying.

(2) agarose gel electrophoresis and quantitative criterion product ratio method and ultraviolet spectrophotometer method are identified and quantitatively adopted to plasmid.

Three, competitive PCR

Get 10 0.2ml thin-walled PCR reaction tubess, at first by following prescription application of sample:

Composition application of sample amount (μ l)

10 * damping fluid 5

15mmol/L MgCl ₂5DNTP mixed solution (each 10mmol/L) 2

Pu(25μmol／L)?????????1

Pb(25μmol／L)?????????1

Taq archaeal dna polymerase (1u/ μ l) 1

Bi-distilled water 33 adds pADR-1 DNA and each 1 μ l of PCR2.1-MS299 then, application of sample amount such as following table (table 1): the application of sample amount of pADR-1DNA and PCR2.1-MS299 in the table 1.10 side reaction pipe

The pipe number	1	????2	????3	????4	????5	????6	????7	????8	????9	?10
											PADR-1DNA (fM/ pipe) PCR2.1-MS299 (fM/ pipe)	10 -310 -3	10 -310 -4	10 -310 -5	10 -410 -3	10 -410 -4	10 -410 -5	10 -510 -3	10 -510 -4	10 -510 -5	0 0

The pcr amplification condition: 94 ℃ 5 minutes; 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 45 seconds, totally 35 circulations; 72 ℃ were extended 5 minutes again.Increase products therefrom through agarose gel electrophoresis alleged occurrence purpose band, and the product hybridization of going is again identified and is quantitative.

Embodiment 3. enzymes connection hybrid method detects HBV gene competitive PCR amplified production

One, main raw

(1) micro-bar is arranged the production of DNA hybridization plate Coyning Costar company.

(2) streptavidin-alkaline phosphatase enzyme complex (Say-Ap) and substrate right-nitrophenyl Di-Sodium Phosphate (pNPP) is respectively Gibco company and Merck company product.

(3) probe design is with synthetic

1. wild fragment capture probe (CPw) 5 '-end is with amido modified.This probe and wild fragment (detecting the purpose fragment of HBV gene) 21-40 bit base complementation, sequence is as follows: 5 '-amine-TGAGACTAAC CTCCG ATTGA-3 '.

2. suddenly change fragment capture probe (CPm) 5 '-end with amido modified, (21-40 bit base) 20 base complementrities of this probe and sudden change fragment (internal reference) normal chain mutant nucleotide sequence, sequence is as follows: 5 '-amine-ACGTA CTGAA TAGCT GCTAC-3 '.

(4) damping fluid and prescription

1. binding buffer liquid (Bb) 50mmol/L Na ₂HPO ₄, 1mmol/L EDTA, 0.02%NaN ₃, pH 8.5

2.20 * SSC 3mol/L NaCl, the 0.3mol/L sodium citrate.

3. contain 3%BSA in the confining liquid binding buffer liquid, 0.1mg/ml salmon sperm dna, 0.02%NaN ₃

4.2 * hybridization buffer 10 * SSC, 0.2%N-lauroylsarcosine, 0.1mg/ml salmon sperm dna, 0.05%NaN ₃, 2.0%BSA, 0.04%SDS.

5. maleate damping fluid 0.1mol/L maleic acid, 0.15mol/L NaCl, pH7.5.

6.Sav-Ap damping fluid: contain in the maleate damping fluid: 3%BSA, 0.05% tween 20,0.02%NaN ₃

7. washings I 2 * SSC contains 0.1%SDS.

8. contain 0.05% tween 20 in the washings II maleate damping fluid.

9.AP substrate (pNPP) damping fluid 30mmol/L trolamine, lmmol/L MgCl ₂

Two, the hybridization of micro-reaction plate enzyme connection quantitatively

(1) bag is diluted capture probe CPw and CPm respectively with binding buffer liquid (Bb), and concentration is 500nmol/L, gets two parts of DNA trace bar row Sptting plates, add CPw and CPm after diluting respectively, every hole 100 μ l, the rearmounted 37 ℃ of water baths of sealing of hole, 1hr, or 4 ℃ spent the night.

(2) sealing discards hole endoperidium thing, and every hole adds 200 μ l confining liquids, puts 37 ℃ of water baths, and 30min abandons confining liquid, pats dry.

(3) hybridization is done 10 times of dilutions with the PCR product with distilled water, puts ice bath immediately behind 100 ℃ of 5min that unwind; Get in the 50 μ l adding hybridization hole after adding 2 times of hybridization solutions of equal-volume, put Sptting plate in 56 ℃ of water baths, 30min; It is inferior to give a baby a bath on the third day after its birth with the washing lotion I, and each 56 ℃, 5min abandons liquid and pats dry.

(4) the every hole of signal detection adds the Sav-AP mixture of 50 μ l dilution in 1: 1000; Place 20min under the room temperature, dry, wash four times with the washing lotion II, each 37 ℃, 3min; Patting dry the back develops the color with the pNPP substrate; Read 405nm OD value.

Three, the result of competitive PCR and product enzyme connection hybridization detection by quantitative

Table 2 shows the result that different concns internal reference template and the PCR product of wild template when nine kinds of various combinations detect with the hybridization of enzyme connection.The result shows, when the amount and the plasmid HBV DNA of internal reference measure when identical, the ratio of ODm and ODw (the OD value that on behalf of capture probe CPm and CPw bag, ODm and ODw surveyed by the hole respectively) is in close proximity to 1.0, conforms to expected value.

Table 2. internal reference and wild template different concns combination back PCR product enzyme connection hybridization detected result

The pipe number	1	????2	????3	????4	????5	????6	????7	????8	?9	?10
											PADR-1DNA (fm/ pipe) PCR2.1-MS299 (fm/ pipe)	10 -310 -3	10 -310 -4	?10 -3?10 -5	?10 -4?10 -3	?10 -4?10 -4	?10 -4?10 -5	?10 -5?10 -3	?10 -5?10 -4	10 -510 -5	????0 ????0
ODm value ODw value ODm/ODw ratio	1．98 ?2．00 ?0．99	1．99 0．57 3．49	?2．33 ?0．21 11．10	?0．46 ?2．11 ?0．22	?1．66 ?1．60 ?1．04	?1．72 ?0．29 ?5．93	?0．54 ?2．14 ?0．25	?0．66 ?1．69 ?0．39	?1．67 ?1．59 ?1．05	?0．03 ?0．04 ????-

Four, the initial method for determination of amount of HBV gene (equation) is pressed (Clementi Met al., PCR Methods Appl 1993 such as Clementi; 2:191) the competitive PCR product amount calculation formula of Jie Shaoing: M/W=M '/W ' (1).M and W represent the original bulk of sudden change template (being internal reference) and wild template respectively in the formula, and M ' and W ' then are respectively sudden change fragment and target gene fragment amount in the PCR product.Formula (1) can be rewritten as: M/W=ODm/ODw (2).In the equation (2), W is a unknown number, and M is a known number, the ODm value, and promptly by the OD value after the hole hybridization, ODw is the OD value after CPm probe bag is hybridized by the hole to the PCR product at CPm probe bag.Since behind the integrated enzyme reaction gained OD value not with product application of sample amount proportionlity linearly, only internal reference and plasmid HBV DNA original bulk relatively near the time, above-mentioned formula is just set up, therefore can pass through the production standard curve, or per minute analyses a sample to be measured and uses 3 above internal reference concentration pipes, thereby tries to achieve HBV dna content in the sample to be measured more accurately.

Embodiment 4. competitive PCRs and product enzyme connection hybridization detection by quantitative serum HBV DNA thereof

One, main raw

(1) serum specimen: collected all serum specimens of the positive (i.e. so-called " great three positive ") of 30 parts of HBsAg, HBeAg, anti-HBcAg altogether, all from chronic hepatitis B patient.The qualitative technology for detection of the equal PCR of above-mentioned all samples confirms hepatitis B virogene (HBV DNA) positive.

(2) material is used in the hybridization of PCR and enzyme connection: see embodiment 2,3.

Two, the preparation of serum specimen processing and amplified reaction pipe

Adopt the direct boiling method of serum, be about to 100 μ l and place in the 0.5mlEppendorf pipe, 100 ℃ were boiled 5 minutes, drew 5 μ l supernatants as pcr template.Every part of sample all is provided with 3 amplification pipes, and contained internal reference is respectively 10 in 3 pipes ^-5, 10 ^-3, 10 ^-1The fmol/ pipe, other PCR reagent are application of sample routinely, and reaction volume is 50 μ l.

Three, amplification condition

The pcr amplification condition: 94 ℃ 5 minutes; 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 45 seconds, totally 32 circulations; 72 ℃ were extended 5 minutes again.Amplified production is gone product hybridization quantitatively again through agarose gel electrophoresis alleged occurrence purpose band.

Four, micro-reaction plate enzyme connection hybridization

Method is seen embodiment 3.

Five, detection by quantitative result

30 parts of samples see all that through the agarose gel electrophoresis analysis molecular weight gets positive band about 300bp.Get 2 μ l and do enzyme connection hybridization detection, and according to HBV dna content before the amplification of aforementioned formula calculating serum specimen.As a result, 30 routine hepatitis B patient serum HBV dna contents distribute and just do not wait, and the soprano reaches 3 * 10 ⁸Copy/ml, the lowest 4 * 10 ³Copy/ml.

The above results shows that the prepared internal reference of the present invention can be used for competitive PCR and enzyme connection hybridization detection by quantitative HBV gene, and clinical value is arranged.

Claims (8)

1, a kind of equivalent competition polymerase chain reaction,PCR internal reference, it is characterized in that it being a kind of and each base contents identical sudden change fragment equal with the purpose fragment length, in the 21-70 position is mutant nucleotide sequence, except that the base sequence of sudden change part was different with the purpose fragment, all the other positions were all identical with the purpose fragment.

2, the preparation method of the described equivalent competition polymerase chain reaction,PCR of claim 1 internal reference is characterized in that comprising the following steps:

(1) the conservative region sequence of selection goal gene, the design mutant primer;

(2) by the downstream primer with mutant primer and goal gene, be template with the goal gene, carry out PCR, products therefrom promptly is an internal reference.

3, as the preparation method of internal reference as described in the claim 2, it is characterized in that according to goal gene conserved regions fragments sequence and the Position Design mutant primer in genome thereof.

4,, it is characterized in that described mutant primer is made up of three parts: PCR upstream primer sequence, mutant nucleotide sequence and catenation sequence as the preparation method of internal reference as described in claim 2 or 3.

5, as the preparation method of internal reference as described in claim 2 or 3, it is characterized in that the long 40-80 of a described mutant primer base, mutant nucleotide sequence is at the 21-70 bit base, change the base sequence of sudden change part randomly, but the absolute number of each base is constant, the Tm value of mutant nucleotide sequence, the Energy value of formation hairpin structure and primer dimer is all identical with former target gene sequences.

6, as the preparation method of internal reference as described in the claim 4, it is characterized in that described mutant nucleotide sequence length is 10-50 base.

7, the application of the described equivalent competition multienzyme of claim 1 chain internal reference is characterized in that described internal reference is used for equivalent competition PCR and PCR product enzyme connection hybridization detection by quantitative virogene.

8,, be to be hybridization site with the mutant nucleotide sequence when it is characterized in that described PCR product enzyme connection hybridization detection by quantitative as the application of internal reference as described in the claim 7.

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