CN103698506A - Method for detecting content of pollutants in sample by utilizing direct immuno-PCR (polymerase chain reaction) assay - Google Patents

Abstract

The invention discloses a method for detecting the content of pollutants in a sample by utilizing a direct immuno-PCR (polymerase chain reaction) assay. The method comprises the following steps of preparing a labeled DNA (deoxyribonucleic acid)-standard product conjugate by utilizing a standard product of a detected object; fixing an antibody which can be in specific binding with the detected object on a supporting object, adding a detected sample and the labeled DNA-standard product conjugate to have the competitive combination reaction, and washing after the reaction is completed; adding the reaction product into a PCR reaction system after the washing, executing the real-time fluorescent quantitative PCR by adopting the labeled DNA as a template to obtain a CT value; calculating the content of the detected object in the detected sample by utilizing the CT value according to a standard curve. By adopting the method, the content detection of a detected object in an ambient sample can be completed only through three steps of specific antibody coating, competitive combination and PCR augmentation; the standard product of the detected object is preliminarily coupled with the DNA directly, and the antigen and the antibody are in specific binding, so that the detection sensitivity and the detection accuracy can be greatly improved.

Description

Utilize direct immunization PCR to detect the method for pollutant load in sample

Technical field

The invention belongs to environmental contaminants detection technique field, be specifically related to a kind of method of utilizing direct immunization PCR to detect pollutant load in sample.

Background technology

Environmental pollutants is along with mankind's activity, to occur, understand environment for human survival and the healthy class chemical substance exerting an influence.Such as, salbutamol (SAL) is the member of beta-stimulants family, is usually used in clinically treating the respiratory diseases such as bronchial astehma.Because it can also promote growth of animal, reduce animal tallow and accumulate, increase lean meat output, thereby be used as feed addictive by domestic and international many plants.Yet, if too much take in SAL, can have a negative impact to health, comprise and occur palpitaition, headache, dizzy, nauseating, vomiting, severe patient even causes the damage of liver kidney.Therefore a plurality of countries that, comprise China classify such medicine as forbidden drugs and feed addictive.For another example, contain the thousands of kinds of materials that health existed to potential threat in tobacco, initiatively smoking and passive smoking all can cause pneumonia, lung cancer, asthma, angiocardiopathy, hypertension and reproductive development etc.Although all these environmental pollutants molecular weight are little, can cause huge potential threat to human health.Raising along with people's living standard, increasing people starts to focus on the health of living environment and health, therefore, in order to react more accurately human body, whether be exposed in various surrounding materials pollutions, be necessary to set up the content that some quick, easy, highly sensitive methods are measured the polluter in biological sample (as urine sample, blood sample etc.) and environmental sample (as water body, soil etc.).

The method of existing testing environment polluter content has By Capillary Zone Electrophoresis, vapor-phase chromatography, liquid phase chromatography, high performance liquid chromatography and tablets by HPLC-MS.But these instrument analytical method complex pretreatment, consuming time many and cost is high.By contrast, euzymelinked immunosorbent assay (ELISA) to have that specificity is good, highly sensitive, easy and simple to handle, testing cost is low relative with the Sample pretreatment advantage such as simple.But, along with people are to the improving constantly of health requirements, be necessary to set up some more highly sensitive detection methods.

Immuno-PCR (Immuno-PCR, IPCR) is proposed by people such as Sano, is to utilize the specificity of antigen-antibody reaction and the high sensitivity of pcr amplification reaction and a kind of trace antigen detection technique of setting up, and its main program comprises:

(1) antigen+biotinylated antibody → antigen-biotinylated antibody compound;

(2) add affinity element → antigen-biotinylated antibody-affinity element compound;

(3) add biotinylation DNA → antigen-biotinylated antibody-affinity element-biotinylation DNA compound;

(4) pcr amplification biotinylation DNA part.

The specific antibody of being combined with antigen is combined with marker DNA by connecting molecule, then carries out pcr amplification, quantitative detectable antigens, makes susceptibility higher than ELISA and RIA thus.But immune PCR technique is applied to the detection of lot of trace albumen and toxin mostly at present, the research detecting for small-molecule substance (molecular weight is less than 1000 dalton) is relatively less.

Document (H.-Y.Chen and H.-S.Zhuang.A Real-Time Immuno-Polymerase Chain Reaction Assay for Detecting Polychlorinated Biphenyls in the Environment.Analytical and bioanalytical chemistry, 2009, 394, 1205-1211.) polychlorinated biphenyl in employing immuno-PCR testing environment sample is disclosed, its key step comprises: antigen coated-specific antibody and little molecule (being polychlorinated biphenyl) are competed-added biotin labeling two and resist-add Avidin-add biotinylated DNA-pcr amplification.

The method belongs to indirect Immunal PCR, identical with other Immuno-PCRs in prior art, all by biotin and Avidin system, specific antibody to be connected with DNA, but an Avidin molecule can be in conjunction with four biotin molecules, be difficult to get rid of the possibility that can have error in testing result, and whole process steps is loaded down with trivial details, is unfavorable for practical application.

Summary of the invention

The invention provides a kind of method of utilizing direct immunization PCR to detect pollutant load in sample, the method step is simple, and testing result sensitivity and accuracy all higher.

Utilize direct immunization PCR to detect the method for pollutant load in sample, comprising:

(1) get determinand standard items, prepare marker DNA-standard items conjugate;

(2) can be fixed on the antibody of determinand specific binding on holder, and add testing sample and marker DNA-standard items conjugate, being at war with property association reaction, has reacted rear washing;

(3) after having washed, add PCR reaction system, take marker DNA as template, carry out real-time fluorescence quantitative PCR, obtain CT value;

(4), according to typical curve, utilize CT value to calculate the content of determinand in testing sample.

The present invention is directed to pollutant in testing sample and set up a set of direct immunization PCR detection system, only need " be coated with-competition of specific antibody combination-pcr amplification " three steps can complete the detection to determinand content in testing sample, and determinand standard items are direct and DNA coupling in advance, in reaction system, do not introduce other and connect molecule, antigen-antibody specific bond, has improved detection sensitivity and accuracy greatly.

Described testing sample can be biological sample, as urine sample, blood sample etc., can be environmental sample, as water sample, and soil sample etc.Described pollutant refers to that molecular weight is less than 1000 daltonian compounds, this compounds due to can not direct coated to ELASA plate, therefore cannot carry out content detection with existing elisa technique.

In step (1), the preparation method of described marker DNA-standard items conjugate is:

(1.1) in determinand standard items, introduce the group easily reacting with carboxyl or amino, obtain standard items derivant;

Introduce the group easily reacting with carboxyl or amino, be convenient to amino or carboxyl coupling on standard items derivant and carrier protein, and increased the probability that determinand standard items are exposed to carrier protein outside surface.The introducing mode of group is generally: the small-molecule substance that selection can be reacted with determinand standard items is as linking arm, on this small-molecule substance with easy this group maintenance free state with carboxyl or amino group react and after reacting.

As preferably, described group is carboxyl, amino or sulfydryl.

(1.2), by described standard items derivant and carrier protein couplet, obtain coupling protein;

As preferably, adopt NHS active ester method to carry out coupling, described carrier protein can be selected BSA, OVA or KLH.

(1.3) in marker DNA sequence, introduce 5 ' end carboxyl or 3 ' end amino, obtain marker DNA derivant;

After standard items derivant and carrier protein couplet, the amino of carrier protein or carboxyl are occupied, and for ease of linkage flag DNA sequence dna on coupling protein, need in marker DNA sequence, introduce 5 ' end carboxyl or 3 ' end amino.

Marker DNA molecule in immuno-PCR can be selected any DNA, but will guarantee DNA purity, and should have good homogenieity, and does not select as far as possible and be subject to the DNA that may exist in sample product, generally can select plasmid DNA or PCR product.The base sequence of marker DNA of the present invention is (marker DNA derivant of the present invention is the marker DNA with 5 ' end carboxyl) as shown in SEQ ID No.1, to take HSA gene as template, the PCR product that carries out pcr amplification acquisition with the downstream primer shown in the upstream primer shown in SEQ ID No.2 (5 ' end of upstream primer is with carboxyl) and SEQ ID No.3.

(1.4), by coupling protein and the coupling of marker DNA derivant, obtain described marker DNA-standard items conjugate.

As preferably, adopt NHS active ester method to carry out coupling.

For example, while utilizing the inventive method to measure the content of salbutamol in testing sample (SAL), the construction method of marker DNA-SAL conjugate comprises:

(a) on SAL, introduce carboxyl, obtain SAL derivant; Wherein, can select p-aminobenzoic acid or bromoacetate to introduce carboxyl as linking arm;

(b) carboxyl on SAL derivant is reacted with the amino on BSA albumen, obtain BSA-SAL conjugate;

(c) preparation is held the marker DNA derivant of carboxyl with 5 ', and by BSA-SAL conjugate and the coupling of marker DNA derivant, obtains marker DNA-SAL conjugate.

Obtain after marker DNA-standard items conjugate, after marker DNA-standard items conjugate and the testing sample that doubly dilutes through 10-1 are mixed with volume ratio 1:1, join in the holder that is fixed with specific antibody being at war with property association reaction.Described holder can be selected immuno-PCR pipe or microwell plate.Competitive reaction is washed after completing, and removes unreacted testing sample and marker DNA-standard items conjugate, is convenient to exactly the content of marker DNA-standard items conjugate of combination be detected.

After having washed, add PCR reaction system, take marker DNA as template, with the downstream primer shown in the upstream primer shown in SEQ ID No.4 and SEQ ID No.5, carry out real-time fluorescence quantitative PCR, obtain CT value; And according to typical curve, utilize CT value to calculate the content of determinand in testing sample.

Real-time fluorescence quantitative PCR response procedures is: 94 ℃ of denaturation 30s; 94 ℃ of sex change 20s, 52 ℃ of extension 30s, 72 ℃ of extensions 15s, altogether 40 circulations; 72 ℃ are continued to extend 10min.

The preparation method of described typical curve is:

(4.1) prepare the determinand standard items that one group of concentration in gradient distributes;

(4.2) described determinand standard items are mixed with volume ratio 1:1 with marker DNA-standard items conjugate, join be fixed with can with the holder of the antibody of determinand specific binding in, being at war with property association reaction, has reacted rear washing;

(4.3) after having washed, add PCR reaction system, take marker DNA as template, carry out real-time fluorescence quantitative PCR, record CT value;

(4.4) draw and to obtain the typical curve that the logarithm value of CT value and determinand standard items concentration is proportionate.

Compared with prior art, beneficial effect of the present invention is:

The present invention has set up the method for utilizing a certain pollutant load in direct immunization PCR method testing environment sample, only need " be coated with-competition of specific antibody combination-pcr amplification " three steps can complete the detection to determinand content in environmental sample, and determinand standard items are direct and DNA coupling in advance, in reaction system, do not introduce other and connect molecule, antigen-antibody specific bond, has improved detection sensitivity and accuracy greatly.

Accompanying drawing explanation

Fig. 1 is the preparation flow figure of salbutamol derivant;

Fig. 2 is the preparation flow figure of BSA albumen-salbutamol conjugate;

Fig. 3 is the pcr amplification curve after variable concentrations salbutamol standard items competition combination, and wherein, △ Rn represents fluorescence intensity, and cycle represents PCR period;

Fig. 4 is the typical curve of salbutamol content in testing sample in direct immunization PCR, and wherein, CT represents the period that the fluorescence signal in each PCR immunity pipe experiences while arriving setting threshold; Log(C) represent the logarithm value of testing concentration in testing sample.

Embodiment

Embodiment 1 direct immunization PCR detects the content of SAL

The preparation of 1SAL derivant

Take p-aminobenzoic acid as linking arm, introduce carboxyl on SAL, derivatization process as shown in Figure 1, comprises the following steps:

(1) 1mL, 2.5mmol p-aminobenzoic acid are dissolved in 5mL, 5%NaOH solution, 4 ℃ are stirred precooling, obtain p-aminobenzoic acid solution;

(2) by the NaNO of 3mL, 3mmol ₂after mixing with 2.5mL, 12%HCl solution, be dropwise added drop-wise in p-aminobenzoic acid solution and form diazotising solution, 0-4 ℃ is reacted to KI-starch paper change basket, adds a small amount of urea stopped reaction;

(3) SAL of 2.5mmol is dissolved in 3mL acetonitrile, then adds 1.5mL, 10%NaOH solution, 4 ℃ are stirred precooling, obtain SAL solution;

(4) the diazotising solution obtaining is dropwise added in SAL solution, after stirring 5min, add again two-phase catalyst tetrabutyl phosphonium bromide ingot, reaction 30min, reactant liquor layering, lower layer of water becomes the turbid solution of brown color mutually, obtains brown color precipitation after suction filtration, with ethanol/water (1:1, v/v) recrystallization, obtains the SAL derivant with carboxyl.

The preparation of 2BSA-SAL conjugate

Adopt active ester method that the carboxyl on SAL derivant is reacted with the amino on BSA albumen, obtain BSA-SAL conjugate, coupling process as shown in Figure 2, comprises the following steps:

(1) according to EDC(1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride): NHS(N-N-Hydroxysuccinimide): the ratio of SAL derivant=1:1.2:1, take respectively EDC3.8mg, NHS4.2mg, SAL derivant 5.0mg, with the DMF(dimethyl formamide of trying one's best few) dissolve, activate 1.5h at 37 ℃ to obtain A liquid;

(2) take 3mg BSA albumen and dissolve with 8mL PBS, under room temperature, stir, obtain B liquid;

(3) strengthen the stirring rate (as far as possible not producing the lower maximal rate that can reach of situation of foam) of B liquid, A liquid is joined in B liquid lentamente along wall, after A liquid adds completely, reduce rotating speed, put under 4 ℃ of conditions and react and spend the night;

(4) utilize the PBS damping fluid (pH7.4) of 0.01M to dialyse, after dialysis completely, obtain BSA-SAL solution.

The preparation of the 3 marker DNA derivants with 5 ' end carboxyl

Take HSA gene as template, take a1/a2 as primer, with PCR instrument, increase, wherein,

Upstream primer a1:5 '-COOH-AAACAGCTATGACCATGA-3 ';

Downstream primer a2:5 '-AGTCACGACGTTGTAAAA-3 '.

Amplification system is:

PCR reaction system is:

Amplification program is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 1min, 52 ℃ of extension 30s, 72 ℃ of extension 15s, circulate 30 times; 72 ℃ are continued to extend 10min, finally 4 ℃ of maintenances.

After determining that by DNA gel electrophoresis band is correct, adopt glue to reclaim kit and carry out purifying recovery, obtain the marker DNA with 5 ' end carboxyl, the about 200bp of length, the base sequence of marker DNA is as shown in SEQ ID No.1.

The preparation of 4 marker DNAs-SAL conjugate

Adopt active ester method by BSA-SAL and the coupling of marker DNA derivant, comprise the following steps:

(1) get in 20 μ L marker DNA derivant to 180 μ L borate buffer solutions, add each 4 μ L of 10mg/mL NHS and s μ Lfo-EDC, 4 ℃ of activation 10min, obtain activating solution;

(2) the BSA-SAL solution of getting 20 μ L16.5mg/mL adds in activating solution, gentle agitation 2h at 4 ℃;

(3) super filter tube that is 100KD with molecular cut off carries out ultrafiltration to reactant, remove unnecessary coupling agent and the reactant in coupling not, trapped substance after ultrafiltration dissolves with the 100mMPBS damping fluid that contains 5mM EDTA, obtains marker DNA-SAL conjugate that coupling is good.

The foundation of 5 direct competitive Immuno-PCRs

(1) with carbonate buffer solution, SAL monoclonal antibody is diluted to 1 μ g/mL, gets 30 μ L/ holes and add in immuno-PCR pipe, in 37 ℃ of coated 2h;

(2) with the PBST damping fluid of pH7.4 (containing the 10mM PBS of 0.05%Tween-20) washing 3 times, each 3min, then with pH7.4, seal 1h containing the 10mM PBS sealing damping fluid of 0.4% gelatin, 1mg/mL salmon sperm dna in 37 ℃;

(3) in immuno-PCR pipe coated and that sealed, add 30 μ L through 10 ^-4doubly synthetic marker DNA-SAL conjugate and 30 μ L variable concentrations (10fg/mL～10ng/mL) SAL standard items in the step 4 of dilution, after 37 ℃ of reaction 1h, wash 5 times with PBST damping fluid (pH7.4), each 3min;

(4) in the immuno-PCR pipe of wash clean, add quantitative PCR reaction system, with quantitative real time PCR Instrument, measure;

Quantitative PCR reaction system is:

Wherein, the base sequence of upstream primer b1 is: 5 '-TGACCATGATTACGAATT-3 ';

The base sequence of downstream primer b2 is: 5 '-AGTCACGACGTTGTAAAA-3 '.

Amplification program is: first 94 ℃ of denaturation 30s, then 94 ℃ of sex change 20s, 52 ℃ of extension 30s, 72 ℃ of extensions 15s, altogether 40 circulations; 72 ℃ are continued to extend 10min, finally 4 ℃ of maintenances.

The result that real-time fluorescence quantitative PCR is analyzed is as Fig. 3 and Fig. 4.

The immuno-PCR typical curve that is drawn the present embodiment by Fig. 3 and Fig. 4 is: y=12.82+1.05x (r=0.997, n=3), and wherein y is PCR cycle index, x is the logarithm value of SAL concentration (fg/mL) in testing sample.The present embodiment reaches 46fg/mL to the detection sensitivity of SAL.

Comparative example 1

According to document (H.Wang, Y.Zhang, H.Li, B.Du, H.Ma, D.Wu and Q.Wei.A silver-palladium alloy nanoparticle-based electrochemical biosensor for simultaneous detection of ractopamine, clenbuterol and salbutamo.Biosens Bioelectron, 2013,49:14-19.) disclosed method, the content that detects SAL, its sensitivity is 1.44pg/mL.

Claims (8)

1. utilize direct immunization PCR to detect the method for pollutant load in sample, comprising:

(1) get determinand standard items, prepare marker DNA-standard items conjugate;

(2) can be fixed on the antibody of determinand specific binding on holder, and add testing sample and marker DNA-standard items conjugate, being at war with property association reaction, has reacted rear washing;

(3) after having washed, add PCR reaction system, take marker DNA as template, carry out real-time fluorescence quantitative PCR, obtain CT value;

(4), according to typical curve, utilize CT value to calculate the content of determinand in testing sample.

2. the method for claim 1, is characterized in that, the preparation method of described marker DNA-standard items conjugate is:

(1.1) in determinand standard items, introduce the group easily reacting with carboxyl or amino, obtain standard items derivant;

(1.2), by described standard items derivant and carrier protein couplet, obtain coupling protein;

(1.3) in marker DNA sequence, introduce 5 ' end carboxyl or 3 ' end amino, obtain marker DNA derivant;

(1.4), by coupling protein and the coupling of marker DNA derivant, obtain described marker DNA-standard items conjugate.

3. method as claimed in claim 2, is characterized in that, the base sequence of marker DNA is as shown in SEQ ID No.1.

4. method as claimed in claim 3, is characterized in that, the primer that real-time fluorescence quantitative PCR adopts is:

Upstream primer: 5 '-TGACCATGATTACGAATT-3 ';

Downstream primer: 5 '-AGTCACGACGTTGTAAAA-3 '.

5. method as claimed in claim 3, is characterized in that, real-time fluorescence quantitative PCR response procedures is: 94 ℃ of denaturation 30s; 94 ℃ of sex change 20s, 52 ℃ of extension 30s, 72 ℃ of extensions 15s, altogether 40 circulations; 72 ℃ are continued to extend 10min.

6. method as claimed in claim 2, is characterized in that, step (1.2) and step (1.4) all adopt NHS active ester method.

7. the method for claim 1, is characterized in that, by marker DNA-standard items conjugate with through 10 ^-1after doubly the testing sample of dilution mixes with volume ratio 1:1, join in holder being at war with property association reaction.

8. the method for claim 1, is characterized in that, the preparation method of described typical curve is:

(4.1) prepare the determinand standard items that one group of concentration in gradient distributes;

(4.2) described determinand standard items are mixed with volume ratio 1:1 with marker DNA-standard items conjugate, join be fixed with can with the holder of the antibody of determinand specific binding in, being at war with property association reaction, has reacted rear washing;

(4.3) after having washed, add PCR reaction system, take marker DNA as template, carry out real-time fluorescence quantitative PCR, record CT value;

(4.4) draw and to obtain the typical curve that the logarithm value of CT value and determinand standard items concentration is proportionate.

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