CN112921075B - Hand-taking-free reagent for blood sample fluorescence PCR and application thereof - Google Patents

Abstract

A non-extraction reagent for fluorescence PCR of a blood sample specifically comprises: the hand-free reagent and a blood sample form a PCR reaction system, wherein an alcohol substance in the blood diluent is PEG200, and an alkaline substance is NaOH; and the concentration of each substance in the blood diluent is respectively as follows: 0.01 to 0.1 weight percent of PEG200, 10 to 100mM of NaOH, 5 to 50mM of Tris-HCl and 0.1 to 1 weight percent of Tween nonionic surfactant. The extraction-free reagent can obtain unexpected system stability, reduce the interference capability of inhibitors in blood and improve the accuracy of test results, wherein the extraction-free reagent can fully denature various inhibitors in the blood, and the tolerance capability of a subsequent PCR system to the inhibitors is effectively improved.

Description

Hand-taking-free reagent for blood sample fluorescence PCR and application thereof

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to an extraction-free reagent suitable for Taqman SNP typing blood direct fluorescence PCR amplification and an application method thereof.

Background

In recent years, the fluorescent PCR technology has been widely used in clinical and inspection and quarantine fields due to its characteristics of rapidness, accuracy and simplicity. The Taqman SNP typing technology is one of important applications of a fluorescence PCR technology, is widely applied to aspects of pathogen drug-resistant mutation detection, genetic disease detection, prenatal and postnatal care, tumor-targeted drug target detection and the like, becomes an important technical means for molecular diagnosis and clinical examination, and is widely popularized in clinic.

Human genome DNA is an important analysis sample for clinical Taqman SNP typing detection, and blood is an important biological source commonly used in clinic. However, since blood contains a large amount of DNA polymerase inhibitors, such as hemoglobin, protein, fat, etc., which seriously interfere with the accuracy of PCR detection, in order to perform taqman SNP typing detection, the prior art usually adopts a method of extracting and purifying nucleic acid in blood, and then performing PCR detection, such as phenol-chloroform purification, column purification, magnetic bead separation, etc., which are commonly used methods for extracting blood genome DNA. Although these methods can obtain high-purity DNA, they have disadvantages such as large DNA loss, complicated operation, and easy cross-contamination. Even though the improvement and the simplification of the operation steps are carried out, the extraction reagent used in the operation can affect the effect of the fluorescence PCR, the operation can be completed only by multiple operations such as centrifugation and transfer, the operation process is relatively complex, and the risk of cross contamination exists.

Researchers in the prior art have developed hands-free reagents that can be used for direct PCR detection. For example, CN103305499B discloses a direct amplification reagent 1a prepared from 20mmol of Tris-HCl, 1.6mM MgCl₂，200uM each dNTP，50mM KCl，25mM NH₄Cl, 1200ug/ml BSA, 0.2% (volume percent) octylphenyl-polyethylene glycol, 0.2% (volume percent) N-octanoyl-N-methylglucamine, 5% (mass percent) PEG6000, 0.3U/. mu.l GoldTaq DNA polymerase and water. The direct amplification reagent and the primer are added into a blood card sample for testing, so that the aim of the extraction-free fluorescent PCR test is fulfilled, wherein the effect of a stable system can be obtained when the concentration of PEG6000 reaches the range of 4.5-5.5%. The prior art adopts STR parting fluorescence PCR to obtain a detection result. Compared with STR typing fluorescent markers, SNP typing fluorescent markers have the technical effects of high specificity and more reliable results, and accordingly have higher requirements on elimination of interference of inhibiting substances in a sample, stability of a reaction system and the like.

In addition, the prior art CN109402239A discloses a hand-free direct amplification reagent for real-time fluorescence quantitative PCR, which is used for detecting nucleic acid in non-blood samples such as respiratory tract secretion and the like, the difference of the types of inhibiting substances in different types of samples in the field is very large, the reagent used in the prior art is used for Taqman SNP typing detection of DNA in blood, and the result has the problem of low accuracy, probably because the interfering substances in the blood are more and higher in content, the inhibiting substances are not easy to effectively denature.

In order to solve the problems, the invention expects to establish a non-extraction reagent which can be suitable for the direct fluorescence PCR amplification of blood of Taqman SNP typing so as to obtain the technical effects of simple operation process and detection result.

Disclosure of Invention

Based on the technical problems in the prior art, the invention provides a non-extraction reagent for fluorescence PCR of a blood sample, which specifically comprises the following components: blood diluent and PCR reaction solution, the hand-free reagent and the blood sample form a PCR reaction system, wherein,

the blood diluent comprises: alcohol substances, alkaline substances, Tris-HCl and Tween nonionic surfactants;

the PCR reaction solution comprises: PCR buffer solution, hot start enzyme, UNG enzyme, Taqman SNP typing primer and probe,

the alcohol substance in the blood diluent is PEG200, and the alkaline substance is NaOH; and the concentration of each substance in the blood diluent is respectively as follows: 0.01 to 0.1 weight percent of PEG200, 10 to 100mM of NaOH, 5 to 50mM of Tris-HCl and 0.1 to 1 weight percent of Tween nonionic surfactant.

Preferably, the blood diluent contains 0.01 wt% to 0.02 wt% of PEG200 and 45mM to 55mM of NaOH.

Preferably, in the blood dilution: the Tween nonionic surfactant is Tween 20.

Preferably, the pH of the PCR reaction solution is 9 to 10.

Preferably, the concentrations of the substances in the PCR reaction solution are respectively as follows: PCR buffer solution, wherein the content of hot start enzyme is (1-5) U/25 muL, the content of UNG enzyme is (0.05-0.5) U/25 muL, and the content of each primer and probe is 0.10-0.50M respectively; wherein, the concentration of each substance in the PCR buffer solution in the PCR reaction solutionThe degrees are respectively: 5 mM-50 mM Tris-HCl, 5 mM-30 mM KCl, 10 mM-50 mM (NH)₄)₂SO₄、2mM～5mM MgCl₂0.1-1M betaine, 0.1-1 wt% glycerol, 0.1-0.5 mg/mL BSA, 0.1-0.5 mM dNTPs, 1-5 wt% formamide, 0.5-3 wt% DMSO.

Preferably, in the PCR buffer, the hot start enzyme is Apexdirect DNA polymerase, and the concentration of the Apexdirect DNA polymerase in the PCR reaction solution is 0.25U/25. mu.L.

The invention also provides an application method of the extraction-free reagent, which specifically comprises the following steps:

(1) preparing a blood diluent;

(2) mixing a blood sample with the blood diluent to form a diluted blood sample;

(3) preparing a PCR reaction solution;

(4) and (3) mixing the diluted blood sample formed in the step (2) with the PCR reaction solution to form a PCR reaction system, amplifying by adopting a fluorescence PCR instrument, and collecting fluorescence.

Preferably, in step (2), the blood sample is diluted to 15-25 times, preferably 20 times, its original concentration by the mixing process.

Preferably, the blood sample in step (2) contains an anticoagulant, and the anticoagulant is one or more of EDTA, sodium citrate and heparin.

Preferably, the fluorescent PCR instrument amplification procedure in step (4) is as follows:

the first step is as follows: circulating for 1 time at 37 ℃ for 5 min;

the second step is that: circulating for 1 time at 95 ℃ for 5 min;

the third step: 95 ℃ for 15 s; the fluorescence was collected by cycling 40 times at 60 ℃ for 1 min.

By adopting the technical means, the invention can obtain the following technical effects:

(1) according to the invention, a great number of innovative attempts are made to find that when the alcohol substance used in the hands-free reagent system is PEG200, the alkaline substance is NaOH, and the concentrations of the two substances in the blood diluent are respectively as follows: PEG200 content of 0.01 wt% -0.1 wt%, NaOH content of 10 mM-100 mM. On one hand, the inventor unexpectedly finds that when the system contains NaOH with a specific content in an alkaline environment, the system is combined with PEG200 with an extremely low content, unexpected system stability and anti-interference capability can be obtained, and the accuracy of a test result is improved, wherein the extraction-free reagent can fully denature various inhibitors in blood, and the tolerance capability of a subsequent PCR system to the inhibitors is effectively improved.

(2) The direct fluorescence PCR detection process and the result of the extraction-free reagent on the blood sample are ensured to be quicker and more accurate by optimizing the content of each component in the reagent.

(3) The invention adopts a two-step method to form a PCR reaction system, and ensures that various DNA polymerase inhibitors in a blood sample are fully denatured in a specific diluent system in the first step before adding the PCR reaction solution. In the second step, the pH value of the PCR reaction solution is adjusted to be within the range of 9-10, so that the pH value of the system is maintained in a specific alkaline environment as much as possible during the PCR reaction process, the activity of various reaction enzymes in the system is not damaged, the nucleic acid in the system is fully released to the maximum extent, and the sensitivity and the accuracy of detection are further improved.

Drawings

FIG. 1 is a graph showing the amplification effect of different PEG types in ALDH2 gene 1/1 homozygous wild type whole blood samples;

FIG. 2 is a graph showing the amplification effect of different PEG types in ALDH2 gene 1/2 heterozygous mutant whole blood samples;

FIG. 3 is a graph showing the amplification effect of different PEG types in ALDH2 gene 2/. times.2 homozygous mutant whole blood samples;

FIG. 4 is a graph showing the amplification effect of different PEG200 concentrations in ALDH2 gene 1/. sup.1 homozygous wild type whole blood samples;

FIG. 5 is a graph showing the amplification effect of different PEG200 concentrations in ALDH2 gene x 1/x 2 heterozygous mutant whole blood samples;

FIG. 6 is a graph showing the amplification effect of different PEG200 concentrations in ALDH2 gene 2/. times.2 homozygous mutant whole blood samples;

FIG. 7 is a graph showing the effect of amplification of different NaOH concentrations in a sample of ALDH2 gene x 1/' 1 homozygous wild type whole blood;

FIG. 8 is a graph showing the amplification effect of different NaOH concentration dilutions of ALDH2 gene x 1/x 2 heterozygous mutant whole blood samples;

FIG. 9 is a graph showing the amplification effect of different NaOH concentration dilutions on ALDH2 gene x 2/' 2 homozygous mutant whole blood samples;

FIG. 10 is a graph of the amplification effect of different concentrations of Tween20 in ALDH2 gene 1/' 1 homozygous wild type whole blood samples;

FIG. 11 is a graph of the amplification effect of different concentrations of Tween20 in ALDH2 gene x 1/x 2 heterozygous mutant whole blood samples;

FIG. 12 is a graph of the amplification effect of different concentrations of Tween20 in ALDH2 gene 2/' 2 homozygous mutant whole blood samples;

FIG. 13 is a graph showing the amplification effect of different dilution ratios of ALDH2 gene x 1/' 1 homozygous wild type whole blood samples;

FIG. 14 is a graph showing the amplification effect of ALDH2 gene 1/2 heterozygous mutant whole blood samples at different dilution ratios;

FIG. 15 is a graph showing the amplification effect of different dilution ratios of ALDH2 gene x 2/' 2 homozygous mutant whole blood samples;

FIG. 16 is a graph showing the amplification effect of 1 example of ALDH2 gene 1/1 homozygous wild type different anticoagulants;

FIG. 17 is a graph showing the amplification effect of 1 example of ALDH2 gene 1/2 hybrid mutant different anticoagulants;

FIG. 18 is a graph showing the amplification effect of 1 example of different anticoagulants of ALDH2 gene x 2/' 2 homozygous mutant type;

FIG. 19 is a graph showing the amplification effect of 1 example of the ALDH2 gene 1/1 homozygous wild type different enzymes;

FIG. 20 is a graph showing the amplification effect of 1 example of different ALDH2 genes 1/2 hybrid mutant types;

FIG. 21 is a graph showing the amplification effect of 1 example of different enzymes of the ALDH2 gene x 2/' 2 homozygous mutant;

FIG. 22 is a graph showing the amplification effect of 1 example of ALDH2 gene 1/. sup.1 homozygous wild type on different enzyme amounts;

FIG. 23 is a graph showing the amplification effect of different enzyme amounts of 1 example of the ALDH2 gene 1/2 hybrid mutant;

FIG. 24 is a graph showing the amplification effect of different enzyme amounts of 1 example of the homozygous mutant type 2/. times.2 of the ALDH2 gene;

FIG. 25 is a graph showing the effect of amplification of 1 example of the ALDH2 gene 1/1 homozygous wild type at different pH values;

FIG. 26 is a graph showing the amplification effect of 1 example of ALDH2 gene 1/2 hybrid mutant at different pH values;

FIG. 27 is a graph showing the amplification effect of 1 example of the ALDH2 gene 2/. multidot.2 homozygous mutant at different pH values;

FIG. 28 is a graph showing the results of testing ALDH2 gene x 1/' 1 homozygous wild type whole blood samples;

FIG. 29 is a graph showing the results of testing ALDH2 gene x 1/x 2 heterozygous mutant whole blood samples;

FIG. 30 is a graph showing the results of testing ALDH2 gene x 2/' 2 homozygous mutant whole blood samples;

FIG. 31 is a comparison of the results of testing the genomic DNA of ALDH2 gene x 1/' 1 homozygous wild type whole blood samples;

FIG. 32 is a graph comparing the results of testing the genomic DNA of ALDH2 gene x 1/x 2 heterozygous mutant whole blood samples;

FIG. 33 is a comparison of the results of testing the genomic DNA of ALDH2 gene x 1/x 2 heterozygous mutant whole blood samples.

Detailed Description

The technical contents of the present invention will be described in detail with reference to the accompanying drawings and specific embodiments.

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention.

One, hand free reagent composition and screening

Aiming at the technical defects in the prior art, the invention designs a detection reagent and a detection method which are suitable for a blood sample of Taqman SNP typing on the premise of realizing extraction-free and direct fluorescence PCR detection by optimizing the composition of a non-extraction reagent. Through extensive trial and innovative development, the inventors finally determined a hands-free reagent of a two-solution system, i.e., a blood diluent system for dissolving a blood sample, and a PCR reaction solution system for providing a PCR reaction environment. The composition and effect verification of the extraction-free reagent of the present invention will be described in detail below.

1. The hand-free reagent taking system consists of

(1) Blood diluent

The blood diluent comprises: alcohol substance, alkaline substance, Tris-HCl, Tween nonionic surfactant.

Wherein, the alcohol substance in the system is PEG200, the alkaline substance is NaOH, and the concentration of each substance in the blood diluent is respectively as follows: the content of PEG200 is 0.01 wt% -0.1 wt%, preferably 0.01 wt% -0.02 wt%; the NaOH content is 10 mM-100 mM, preferably 45 mM-55 mM; the Tris-HCl content is 5 mM-50 mM, the Tween type nonionic surfactant content is 0.1 wt% -1 wt%, and the Tween type nonionic surfactant is preferably Tween 20.

(2) PCR reaction solution

The PCR reaction solution comprises: PCR buffer solution, hot start enzyme, UNG enzyme, Taq man SNP typing primer and probe.

Wherein, the concentrations of all substances in the PCR reaction solution are respectively as follows: PCR buffer solution with hot start enzyme content of 1-5U/25 μ L and 2.5U/25 μ L; the UNG enzyme content is (0.05-0.5) U/25 muL, preferably 0.1U/25 muL; the content of each primer and probe is 0.10M-0.50M. Wherein the concentrations of all substances in the PCR buffer solution in the PCR reaction solution are respectively as follows: 5mM to 50mM Tris-HCl, preferably 25 mM; 5mM to 30mM KCl, preferably 20 mM; 10mM to 50mM (NH)₄)₂SO₄Preferably 20 mM; 2 mM-5 mM MgCl₂Preferably 3 mM; 0.1M to 1M betaine, preferably 0.5M; 0.1 wt% to 1 wt% glycerol, preferably 0.5 wt%; 0.1mg/mL to 0.5mg/mL BSA, preferably 0.2 mg/mL; 0.1 mM-0.5 mM dNTPs; 1 to 5 wt% formamide, preferably 2.5 wt%; 0.5 wt% to 3 wt% DMSO, preferably 1.5 wt%.

Further, the concentration ratio of each substance of dNTPs in the PCR reaction solution is dATP: dCTP: dGTP: dUTP ═ 1: 1: 1: 1.5, dATP: dCTP: dGTP: the preferred concentration level of dUTP is 0.3 mM: 0.3 mM: 0.3 mM: 0.45 mM.

The hot start Taq DNA polymerase capable of tolerating whole blood to a certain extent is an important component in a fluorescence PCR reaction system, and Apexdirect DNA polymerase (product number AG12205) of Aicori bioengineering Limited in Hunan is preferably selected through an experimental test of Taq enzyme which is called by main manufacturers on the market and can directly perform blood PCR. Preferably, 2.5U of whole blood-tolerant hot start Taq DNA polymerase (i.e., 2.5U/25. mu.L) is added to 25. mu.L of the PCR reaction solution.

In the invention, NaOH is added into the blood diluent, and the pH value of the PCR reaction solution is increased to be within the range of 9-10, so that inhibitors such as heme and the like in a whole blood sample can be furthest promoted to be denatured, and the tolerance of the PCR reaction can be increased. Experimental tests show that the amplification effect is best when the pH value of the PCR reaction solution is 9.6.

Furthermore, the Taqman SNP typing primer and the probe adopted in the invention can select an upstream/downstream primer, a wild type probe and a mutant type probe of different SNPs according to the detection requirement. Wherein, the proportion and the concentration of the upstream primer, the downstream primer, the wild type probe and the mutant type probe are optimized according to the SNP sequence to be detected. As a preferred mode, in the Taqman SNP typing system, the upstream primer: a downstream primer: wild-type probe: the concentration ratio of the mutant probe is 1: 1: 2: 4, the concentration of each primer and probe in the PCR reaction solution is preferably 0.1. mu.M to 2. mu.M, and the concentration of the upstream primer: a downstream primer: wild-type probe: the concentration content of the mutant probe is preferably 0.3 mu M: 0.3. mu.M: 0.6. mu.M: 1.2. mu.M. In order to enhance the recognition specificity for certain SNP sites, upstream and/or downstream ARMS primers can also be added, and the amount of ARMS primers added needs to be optimized according to the sequence of the SNP sites.

Based on the experimental result verification and the comprehensive judgment of the structure of the compound, the Taqman SNP typing PCR reaction solution has the following functions: Tris-HCl in a Taq man SNP typing PCR reaction solution provides a stable reaction environment; k provided by KCl⁺The ions may facilitate annealing; (NH)₄)₂SO₄NH of (2)₄ ⁺NH can be generated under high temperature condition₃And H⁺，NH₃The condition of generating hydrogen bonds is available, unstable hydrogen bonds between primers and probes which are not specifically annealed and a template can be destroyed, and the specificity is improved; MgCl₂Mg of (2)²⁺Auxiliary ions necessary for the function of DNA polymerase can increase the activity of Taq enzyme and improve the amplification efficiency; dNTPs areRaw materials for PCR reaction; DMSO, betaine, glycerol, BSA and formamide play a role in enhancing the PCR effect by reducing the annealing temperature on one hand, protect NDA polymerase on the other hand, and improve the tolerance of PCR to inhibitors in blood; dNTPs containing dUTP and UNG enzyme can prevent nonspecific PCR amplification and ensure the amplification accuracy.

According to the technical scheme, various DNA polymerase inhibitors in the blood sample are denatured on the basis of adding the blood diluent and the PCR reaction solution with specific components in sequence. The Taq DNA polymerase capable of tolerating the hot start of whole blood to a certain extent is selected in a Taq man SNP typing fluorescence PCR amplification system, the added specific additive can effectively inhibit inhibitors capable of inhibiting the DNA polymerase in blood such as heme and protein, and the concentration and the proportion of the primer probe are adjusted, so that the steps of extracting and purifying blood nucleic acid are omitted, and the whole blood is used as a template to carry out specific and efficient Taq man SNP typing detection.

(3) Application of extraction-free reagent

The extraction-free direct fluorescence PCR reaction detection method for blood samples by adopting the extraction-free reagent comprises the following steps:

preparing a blood diluent;

mixing the blood sample with the blood diluent to form a diluted blood sample;

preparing PCR reaction liquid;

mixing the diluted blood sample formed in the step (2) with the PCR reaction solution to form a PCR reaction system, amplifying by adopting a fluorescence PCR instrument, and collecting fluorescence.

Wherein the blood sample is diluted to 15-25 times its original concentration, preferably to 20 times its original concentration, by the mixing process. The blood sample is firstly diluted in the blood diluent and fully releases nucleic acid substances, so that the full denaturation of enzyme inhibitors in the blood is ensured, the diluted solution is mixed with PCR reaction liquid and then the fluorescence PCR reaction process can be directly carried out, the tolerance of a PCR system to the inhibitors is enhanced, and the detection accuracy is improved.

2. Screening of extraction-free reagent compositions

In this section, the screening test process of the hands-free reagent of the present invention is described in detail by taking human aldehyde dehydrogenase (ALDH) as an example. ALDH is a quadruplex protease that catalyzes the oxidation of acetaldehyde and other aliphatic aldehydes. The major mutant of human ALDH2 gene is Glu504Lys (rs671), a single base mutation located in exon 12. The normal allele was designated as ALDH2 x 1 (x 1510G) and the mutant allele was designated as ALDH2 x 2 (x 1510A). The primers and probes used in this section are specifically as follows:

ALDH2 upstream primer: 5'-TTGGAGCCCAGTCAC-3' (SEQ ID NO: 1);

ALDH2 downstream primer 5'-CCTCAAGCCCCAAC-3' (SEQ ID NO: 2);

ALDH2 × 1 wild-type probe: 5 '-FAM-TACACTGAAGTGAA-NFQ-MGB-3' (SEQ ID NO: 3);

ALDH2 × 2 mutant probe: 5 '-VIC-CATACACTAAAGTGAA-NFQ-MGB-3' (SEQ ID NO: 4);

an internal standard forward primer: 5'-CGCAATACCTCCGGATT-3' (SEQ ID NO: 5);

internal standard reverse primer: 5'-TCCGCAGAGGCACTGAG-3' (SEQ ID NO: 6);

internal standard probe: ROX-5'-GGTCGCTGCATGGCTG-3' -BHQ2(SEQ ID NO: 7).

The specific application method of the extraction-free reagent is as follows:

(1) preparing blood diluent comprising polyethylene glycol, NaOH, Tris-HCl and Tween 20;

(2) mixing a blood sample with the blood diluent to form a diluted blood sample;

(3) preparing a PCR reaction solution containing: 25mM Tris-HCl, 20mM KCl, 20mM (NH4)₂SO₄、3mM MgCl₂0.5M betaine, 0.5 wt% glycerol, 0.2mg/mL BSA, 2.5U/25 μ L Apexdirect DNA polymerase from Esci bioengineering, Inc. of Hunan, 0.1U/25 μ L UNG enzyme, 2.5 wt% formamide, 1.5 wt% DMSO; 0.3mM dATP, 0.3mM dCTP, 0.3mM dGTP, 0.6mM dUTP; 0.3. mu.M ALDH2 upstream primer, 0.3. mu.M ALDH2 downstream primer, 0.6. mu.M ALDH 2. mu.1 wild-type probe, 1.2. mu.M ALDH 2. mu.2 mutant probe, 0.15. mu.M internal standard forward primerPrimers, 0.15 mu M internal standard reverse primers and 0.2 mu M internal standard probes; the pH of the PCR reaction solution was 9.6.

(4) mu.L of the diluted blood sample was mixed with 20. mu.L of the LPCR reaction solution and amplified using an ABI7500 fluorescent PCR instrument using the following PCR amplification procedure:

the first step is as follows: circulating for 1 time at 37 ℃ for 5 min;

the second step is that: circulating for 1 time at 95 ℃ for 5 min;

the third step: 95 ℃ for 15 s; the fluorescence was collected by cycling 40 times at 60 ℃ for 1 min.

It should be noted that, in this section, the components and the screening process of the non-extraction reagent of the present invention are exemplarily shown only by using fluorescent PCR reaction detection of human ALDH2 gene G1510A SNP, and the application range of the non-extraction reagent of the present invention is not limited to the above-mentioned human ALDH2 gene G1510A SNP detection.

Next, the screening and determination process of the main components of the extraction-free reagent for fluorescence PCR of blood samples according to the present invention will be described in detail.

(1) Screening of polyethylene glycol substances and concentration

Separately, 1 part of each EDTA anticoagulated whole blood was taken from ALDH2 gene G1510 SNP site 1/'1 homozygous wild type (' 1510G/G), '1/' 2 heterozygous mutant ('1510G/a) and' 2/'2 homozygous mutant (' 1510A/a). Preparing a PCR reaction system according to the steps recorded in the specific application method of the extraction-free reagent, wherein the concentration of each substance in the blood diluent is as follows: 0.015 wt% polyethylene glycol, 50mM NaOH, 20mM Tris-HCl, 0.3 wt% Tween20, test ApexeDirect DNA polymerase from Aicori bioengineering, Inc. of Hunan. And amplifying the PCR reaction system, and collecting fluorescence values, thereby realizing the polymorphism detection of the G1510 SNP locus of the ALDH2 gene. And testing and evaluating the effects of the hands-free taking and amplification reaction of the hands-free reagent when the polyethylene glycol in the blood diluent is PEG200, PEG400, PEG600, PEG800, PEG1000, PEG2000, PEG4000, PEG6000 and PEG 8000.

Specific results are shown in fig. 1-3, and it can be seen from the trend of the fluorescence PCR amplification curve shown in the figure that different molecular weight polyethylene glycol has significantly different effect on amplification, as shown in fig. 1, PEG6000 preferred by CN103305499B in the prior art has poor amplification effect for the characteristic whole blood system of the present invention, while as shown in fig. 3, the amplification effect of the reaction system using PEG200 is significantly better than that of any other reaction system for the × 2/× 2 homozygous mutant. Therefore, the comparison of the amplification effects can judge that the extraction-free reagent adopting PEG200 in the invention can obviously improve the amplification effect of the system on the basis of realizing extraction-free and direct fluorescence detection for the direct fluorescence PCR amplification of blood of Taqman SNP typing. In contrast to the teachings given in the prior art with respect to polyethylene glycol selection per se, the inventors have found that the use of a low molecular weight PEG200 instead provides better stability for the particular reaction system of the present invention, leading to unexpected technical effects.

Next, the choice of PEG200 concentration in the system was tested. And (3) respectively taking 1 part of EDTA anticoagulant from sites of G1510 SNP of the ALDH2 gene, namely 1/1 homozygous wild type (1510G/G), 1/2 heterozygous mutant type (1510G/A) and 2/2 homozygous mutant type (1510A/A). Preparing a PCR reaction system according to the steps recorded in the specific application method of the extraction-free reagent, wherein the substances and the concentrations in the blood diluent are as follows: PEG200, 50mM NaOH, 20mM Tris-HCl, 0.3 wt% Tween 20. And amplifying the PCR reaction system, and collecting fluorescence values, thereby realizing the polymorphism detection of the G1510 SNP locus of the ALDH2 gene. The effects of the hands-free and amplification reactions of the hands-free reagents were tested and evaluated at concentrations of 0.01 wt%, 0.015 wt%, 0.02 wt%, 0.05 wt%, and 0.1 wt% of PEG200 in the blood dilutions.

The specific results are shown in fig. 4-6, and it can be seen from the trend of the fluorescence PCR amplification curve shown in the figure that the PEG200 concentration is in the range of 0.01 wt% to 0.1 wt%, although effective amplification of the reaction system of the present invention can be achieved, it can be seen from the figure that there is a significant difference in amplification effect at different concentrations, and it can be seen from fig. 4 and 6 that the amplification effect is significantly better than other concentrations when the PEG200 concentration is 0.015 wt%. Compared with the content of polyethylene glycol with the concentration of about 4 wt% in the prior art, the content is different from the understanding of the conventional technical personnel in the field, as shown in fig. 4-6, the inventor unexpectedly finds that when the concentration of PEG200 in the system is reduced, the amplification effect of the reaction system can be greatly improved, and an unexpected technical effect is obtained.

(2) Screening of NaOH content

Separately, 1 part of each of the EDTA-anticoagulated whole blood was taken at sites x 1/' 1 homozygous wild (x 1510G/G), x 1/' 2 heterozygous mutation (x 1510G/a), and x 2/' 2 homozygous mutation (x 1510A/a) of the G1510 SNP of the ALDH2 gene. Preparing a PCR reaction system according to the steps recorded in the specific application method of the extraction-free reagent, wherein the concentration of each substance in the blood diluent is as follows: 0.015 wt% PEG200, NaOH, 20mM Tris-HCl, 0.3 wt% Tween20, tested using ApexeDirect DNA polymerase from Aicori bioengineering, Inc. of Hunan. And amplifying the PCR reaction system, and collecting fluorescence values, thereby realizing the polymorphism detection of the G1510 SNP locus of the ALDH2 gene. The effects of the hands-free extraction and amplification reactions of the hands-free reagent were measured and evaluated at NaOH concentrations of 1mM, 10mM, 50mM, 100mM, and 150mM, respectively, in the blood dilutions.

As can be seen from FIGS. 7-9, NaOH, at a concentration of 1mM, in the blood dilution, was not efficiently amplified; NaOH with the concentration of 150mM, the amplification result is poor; 10mM to 100mM can effectively amplify the DNA. Among them, 50mM NaOH is preferable as the optimum concentration of NaOH in the diluent because the fluorescence signal is high and the Ct value is relatively high. NaOH can achieve the purpose of cell lysis, and the pH environment is increased to denature inhibitors such as heme and the like, so that the tolerance of a subsequent PCR system to the inhibitors is enhanced. Compared with the prior art, the method has the advantages that the blood diluent system at least comprising PEG200 and NaOH with specific concentration is adopted, the direct fluorescence PCR amplification of blood for Taqman SNP typing is effectively realized, and the technical problems that the conventional alkaline system in the prior art cannot effectively amplify whole blood samples and the detection accuracy is relatively poor are solved.

(3) Screening of Tween20 content

Separately, 1 part of each of the EDTA-anticoagulated whole blood was taken at sites x 1/' 1 homozygous wild (x 1510G/G), x 1/' 2 heterozygous mutation (x 1510G/a), and x 2/' 2 homozygous mutation (x 1510A/a) of the G1510 SNP of the ALDH2 gene. Preparing a PCR reaction system according to the steps recorded in the specific application method of the extraction-free reagent, wherein the concentration of each substance in the blood diluent is as follows: 0.015 wt% PEG200, 50mM NaOH, 20mM Tris-HCl, Tween20, test ApexeDirect DNA polymerase from Aikery bioengineering, Inc. of Hunan. And amplifying the PCR reaction system, and collecting fluorescence values, thereby realizing the polymorphism detection of the G1510 SNP locus of the ALDH2 gene. The effects of the hands-free and amplification reactions of the hands-free reagent were measured and evaluated at concentrations of Tween20 of 0.1 wt%, 0.3 wt%, 0.5 wt%, 0.7 wt%, and 1 wt%, respectively, in the blood diluent.

Tween20 can increase cell permeability, destroy membrane structure and depolymerize protein-DNA complex, denature protein and fat in blood, and enhance nucleic acid release ability. As can be seen from the detection results shown in FIGS. 10-12, the amplification effect is not changed regularly with the change of the concentration of Tween20, and good amplification effect cannot be obtained at too low or too high concentration, but through a great deal of verification, Tween20 with the concentration of 0.3 wt% can exert the best effect in the special hands-free reagent system of the invention.

(4) Screening of dilution ratio of Whole blood

Separately, 1 part of each of the EDTA-anticoagulated whole blood was taken at sites x 1/' 1 homozygous wild (x 1510G/G), x 1/' 2 heterozygous mutation (x 1510G/a), and x 2/' 2 homozygous mutation (x 1510A/a) of the G1510 SNP of the ALDH2 gene. Preparing a PCR reaction system according to the steps recorded in the specific application method of the extraction-free reagent, wherein the concentration of each substance in the blood diluent is as follows: 0.015 wt% PEG200, 50mM NaOH, 20mM Tris-HCl, 0.3 wt% Tween20, test Apexdirect DNA polymerase from Ex Bio-engineering, Inc. of Hunan. And amplifying the PCR reaction system, and collecting fluorescence values, thereby realizing the polymorphism detection of the G1510 SNP locus of the ALDH2 gene. And testing and evaluating the effect of the hands-free extraction and amplification reaction of the hands-free reagent when the diluted blood sample is diluted by 5 times, 10 times, 20 times, 50 times and 100 times by using the PCR reaction solution.

The specific results are shown in FIGS. 13-15, where the amplification effect did not reach good effect when the dilution factor was too high or too low, and when the dilution factor was 20 times, the amplified fluorescence curve was the best in line form, the Ct value was the smallest, and the fluorescence height was higher.

(5) Screening for anti-coagulant agents in blood samples

10 portions of EDTA, sodium citrate and heparin anticoagulated whole blood of each genotype of ALDH2 gene G1510 SNP site 1/. multidot.1 homozygous wild (1510G/G),. multidot.1/. multidot.2 heterozygous mutation (1510G/A) and. multidot.2/. multidot.2 homozygous mutation (1510A/A) were taken, respectively (as shown in Table 1). Preparing a PCR reaction system according to the steps recorded in the specific application method of the extraction-free reagent, wherein the concentration of each substance in the blood diluent is as follows: 0.015 wt% PEG200, 50mM NaOH, 20mM Tris-HCl, 0.3 wt% Tween20, test Apexdirect DNA polymerase from Ex Bio-engineering, Inc. of Hunan. And amplifying the PCR reaction system, and collecting fluorescence values, thereby realizing the polymorphism detection of the G1510 SNP locus of the ALDH2 gene.

TABLE 1

The specific results are shown in fig. 16-18, and it can be seen from the amplification line pattern of 3 different genotype samples that the amplification effects of EDTA, sodium citrate, heparin anticoagulated whole blood are relatively similar, and the requirements of SNP typing can be satisfied. The method of the patent can be at least suitable for whole blood anticoagulated by EDTA, sodium citrate and heparin.

(6) Screening for Hot Start enzymes

Separately, 1 part of each of the EDTA-anticoagulated whole blood was taken at sites x 1/' 1 homozygous wild (x 1510G/G), x 1/' 2 heterozygous mutation (x 1510G/a), and x 2/' 2 homozygous mutation (x 1510A/a) of the G1510 SNP of the ALDH2 gene. Preparing a PCR reaction system according to the steps recorded in the specific application method of the extraction-free reagent, wherein the concentration of each substance in the blood diluent is as follows: 0.015 wt% PEG200, 50mM NaOH, 20mM Tris-HCl, 0.3 wt% Tween 20. And amplifying the PCR reaction system, and collecting fluorescence values, thereby realizing the polymorphism detection of the G1510 SNP locus of the ALDH2 gene.

The hot-start Taq enzymes were respectively enzyme 1 (AK Taq DNA Polymerase (cat # MD099) from Fipeng Bio Inc.), enzyme 2 (Apexdirect DNA Polymerase (cat # AG12205) from Ex Bio Inc. of Hunan, and enzyme 3 (MightyAmp from Bao Bio Inc. ((Dalian))^TMDNA Polymerase Ver.2 (catNumber R071)), enzyme 4 (heihh Unicon0 hotspot Direct Taq DNA Polymerase (cat number 10171) from shisai biotech (shanghai) ltd) were tested and evaluated for the hands-free extraction of the hands-free reagents and the effect of the amplification reaction.

As shown in FIGS. 19 to 21, it can be seen from the amplification line graphs of 3 samples with different genotypes that Apex direct DNA polymerase (corresponding to enzyme 2 in the graph) of Aikory bioengineering, Inc. in Hunan is the highest in fluorescence height and the smallest in Ct value, and the system of the present invention can obtain the best amplification detection effect.

The amount of hot start enzyme was further screened.

Separately, 1 part of each of the EDTA-anticoagulated whole blood was taken at sites x 1/' 1 homozygous wild (x 1510G/G), x 1/' 2 heterozygous mutation (x 1510G/a), and x 2/' 2 homozygous mutation (x 1510A/a) of the G1510 SNP of the ALDH2 gene. Preparing a PCR reaction system according to the steps recorded in the specific application method of the extraction-free reagent, wherein the concentration of each substance in the blood diluent is as follows: 0.015 wt% PEG200, 50mM NaOH, 20mM Tris-HCl, 0.3 wt% Tween20, test Apexdirect DNA polymerase from Ex Bio-engineering, Inc. of Hunan. And amplifying the PCR reaction system, and collecting fluorescence values, thereby realizing the polymorphism detection of the G1510 SNP locus of the ALDH2 gene. The effects of the hands-free extraction and amplification reaction of the hands-free reagent when the contents of the hot start enzyme in the 25 mu LPCR reaction system are respectively 1U, 2.5U and 5U are tested and evaluated.

As shown in FIGS. 22 to 24, the amount of 1U/25. mu.L enzyme was significantly insufficient, the fluorescence was low, and the Ct value was large. The reaction system results of hot start enzyme contents of 2.5U/25 μ L and 5U/25 μ L are similar, and the hot start enzyme concentration of 2.5U/25 μ L is preferable for cost.

(7) Screening of pH value of PCR reaction solution

Separately, 1 part of each of the EDTA-anticoagulated whole blood was taken at sites x 1/' 1 homozygous wild (x 1510G/G), x 1/' 2 heterozygous mutation (x 1510G/a), and x 2/' 2 homozygous mutation (x 1510A/a) of the G1510 SNP of the ALDH2 gene. Preparing a PCR reaction system according to the steps recorded in the specific application method of the extraction-free reagent, wherein the concentration of each substance in the blood diluent is as follows: 0.015 wt% PEG200, 50mM NaOH, 20mM Tris-HCl, 0.3 wt% Tween20, test Apexdirect DNA polymerase from Ex Bio-engineering, Inc. of Hunan. And amplifying the PCR reaction system, and collecting fluorescence values, thereby realizing the polymorphism detection of the G1510 SNP locus of the ALDH2 gene. The effects of the hands-free extraction and amplification reactions of the hands-free reagent when the pH values of the PCR reaction solution were 9, 9.2, 9.4, 9.6, 9.8, and 10, respectively, were tested and evaluated.

As shown in FIGS. 25 to 27, the amplification effect was significantly more excellent at a pH of 9.6 in comparison with other pH values, indicating that the effect of the extraction-free reagent protected by the present invention was the best at a pH of 9.6 in the PCR reaction solution.

Second, it is free to take reagent and exempt from to take the effect to prove

Verification example 1

In this embodiment, a "2. screening consisting of the non-extraction-free reagent" section PCR reaction system is still used, that is, the fluorescent PCR reaction detection of the human ALDH2 gene G1510A SNP site polymorphism is taken as an example to exemplarily show the process of the verification effect of the non-extraction-free reagent of the present invention.

The specific application method of the extraction-free reagent is as follows:

(1) preparing blood diluent, which comprises 0.015 wt% of PEG200, 50mM of NaOH, 20mM of Tris-HCl and 0.3 wt% of Tween 20;

(2) mixing a blood sample with the blood diluent to form a diluted blood sample diluted by 20 times;

(3) preparing a PCR reaction solution containing: 25mM Tris-HCl, 20mM KCl, 20mM (NH4)₂SO₄、3mM MgCl₂0.5M betaine, 0.5 wt% glycerol, 0.2mg/mL BSA, 2.5U/25 μ L Apexdirect DNA polymerase from Esci bioengineering, Inc. of Hunan, 0.1U/25 μ L UNG enzyme, 2.5 wt% formamide, 1.5 wt% DMSO; 0.3mM dATP, 0.3mM dCTP, 0.3mM dGTP, 0.6mM dUTP; 0.3. mu.M ALDH2 upstream primer, 0.3. mu.M ALDH2 downstream primer, 0.6. mu.M ALDH 2. mu.1 wild type probe, 1.2. mu.M ALDH 2. mu.2 mutant probe, 0.15. mu.M internal standard forward primer, 0.15. mu.M internal standard reverse primer, 0.2. mu.M internal standard probe.

(4) mu.L of the PCR reaction was mixed with 5. mu.L of the diluted blood sample and amplified using an ABI7500 fluorescent PCR instrument using the following PCR amplification procedure:

the first step is as follows: circulating for 1 time at 37 ℃ for 5 min;

the second step is that: circulating for 1 time at 95 ℃ for 5 min;

the third step: 95 ℃ for 15 s; the fluorescence was collected by cycling 40 times at 60 ℃ for 1 min.

10 portions of each of EDTA anticoagulated whole blood of ALDH2 gene G1510 SNP sites 1/'1 homozygous wild type (' 1510G/G), '1/' 2 heterozygous mutant type ('1510G/a) and' 2/'2 homozygous mutant type (' 1510A/a) were subjected to Sanger sequencing and direct fluorescence PCR amplification using the extraction-free reagent of the present invention, respectively, to test the 30 samples, and the results of the tests were compared. The results of the experiment are shown in table 2 below. Among them, Sanger sequencing method has accurate results, and is generally used as a "gold standard" for SNP detection.

TABLE 2

As can be seen from the test results in the above table, the direct fluorescence PCR method using the hands-free reagent described in the present invention showed 100% agreement with the Sanger sequencing method.

Verification example 2

In this example 2, the results of direct fluorescent PCR amplification of whole blood using the extraction-free reagent used in example 1 and the results of fluorescent PCR amplification of genomic DNA after whole blood extraction using a conventional blood genomic DNA extraction kit were compared.

Wherein, the conventional blood genome DNA extraction process comprises the following steps: 10 portions of each of the ALDH2 genes x 1/' 1 homozygous wild type (× 1510G/G), 1/' 2 heterozygous mutant (× 1510G/a), and 2/' 2 homozygous mutant (× 1510A/a) EDTA anticoagulated whole blood used in verification example 1 were collected and extracted using a common domestic blood genome DNA extraction kit. The extracted genomic DNA was detected using the same PCR reaction solution as in example 1 and the fluorescent PCR amplification procedure as in example 1, and the detection results are shown in Table 3 below.

TABLE 3

As can be seen from the results, the direct fluorescence PCR method test results of whole blood using the non-extraction reagent described in the present invention are 100% identical to the fluorescence PCR test results of genome DNA extracted by the conventional extraction kit. Specific results can be shown in fig. 31-33, and as can be seen from the amplification line graphs of 3 samples with different genotypes, the difference between the signal height and Ct value of amplification using whole blood and the amplification using extracted genomic DNA in the validation example 2 of the present invention is small, and the requirements of clinical examination are completely met.

Verification example 3

And (3) verifying the function of the extraction-free reagent system in other SNP site typing systems.

Adopting the hand-free reagent taking system and the direct fluorescence PCR amplification program in the verification example 1 to respectively carry out the treatment on SLCO1B1 gene 388A > G and 521T > C SNP sites, ApoE gene 526C > T and 388T > C SNP sites related to statin metabolism; MTHFR gene 677C > T and 1298A > C SNP sites, MTRR gene 66A > G SNP sites related to folate metabolism; CYP2C9 gene x 3 and VKORC1 gene 1639G > A SNP site related to warfarin metabolism, and parallel comparison tests are carried out. Adopting the extraction-free reagent used in the verification example 1 to respectively treat 388A > G and 521T > C SNP loci and 526C > T and 388T > C SNP loci of human SLCO1B1 genes in whole blood; MTHFR gene 677C > T and 1298A > C SNP sites, MTRR gene 66A > G SNP sites related to folate metabolism; direct fluorescent PCR amplification detection is carried out on SNP sites of CYP2C9 gene x 3 and VKORC1 gene 1639G > A related to warfarin metabolism, and compared with the result of fluorescent PCR amplification of genomic DNA extracted from whole blood by using a conventional blood genomic DNA extraction kit. The primers and probes used in each system are shown in Table 4, and the results are shown in Table 5.

TABLE 4

TABLE 5

The result is verified, and the direct fluorescence PCR method test result of different types of human genes in the whole blood is 100% consistent with the fluorescence PCR detection result of genome DNA extracted by the conventional extraction kit. Therefore, the hand-free reagent disclosed by the invention has universality for detecting multiple human gene polymorphisms in the existing whole blood, and is suitable for wide popularization.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> Wuhan Yongzhiyou medical science and technology GmbH

<120> taking-free reagent for fluorescence PCR of blood sample and application thereof

<160> 55

<170> PatentIn version 3.3

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Claims (9)

1. A non-extraction reagent for fluorescence PCR of a blood sample specifically comprises: blood diluent and PCR reaction solution, the hand-free reagent and the blood sample form a PCR reaction system, wherein,

the blood diluent comprises: alcohol substances, alkaline substances, Tris-HCl and Tween nonionic surfactants;

the PCR reaction solution comprises: PCR buffer solution, hot start enzyme, UNG enzyme, Taqman SNP typing primer and probe,

the method is characterized in that:

the alcohol substance in the blood diluent is PEG200, and the alkaline substance is NaOH; and the concentration of each substance in the blood diluent is respectively as follows: 0.01 to 0.02 weight percent of PEG200, 45 to 55mM of NaOH, 5 to 50mM of Tris-HCl and 0.1 to 1 weight percent of Tween nonionic surfactant.

2. The extraction-free reagent according to claim 1, wherein:

in the blood diluent: the Tween nonionic surfactant is Tween 20.

3. The extraction-free reagent according to claim 1, wherein:

the pH value of the PCR reaction solution is 9-10.

4. A strip-free reagent according to any one of claims 1 to 3, wherein:

the concentrations of the substances in the PCR reaction solution are respectively as follows: PCR buffer solution, wherein the content of hot start enzyme is (1-5) U/25 muL, the content of UNG enzyme is (0.05-0.5) U/25 muL, and the content of each primer and probe is 0.1-0.50M respectively;

wherein the concentrations of all substances in the PCR buffer solution in the PCR reaction solution are respectively as follows: 5 mM-50 mM Tris-HCl, 5 mM-30 mM KCl, 10 mM-50 mM (NH)₄)₂SO₄、2mM～5mM MgCl₂0.1-1M betaine, 0.1-1 wt% glycerol, 0.1-0.5 mg/mL BSA, 0.1-0.5 mM dNTPs, 1-5 wt% formamide, 0.5-3 wt% DMSO.

5. A strip-free reagent according to claim 4, wherein:

in the PCR buffer, the hot start enzyme was Apexdirect DNA polymerase, and the concentration thereof in the PCR reaction solution was 0.25U/25. mu.L.

6. A method of use of the extraction-free reagent of any of claims 1-5 for non-disease diagnostic and therapeutic purposes, comprising the steps of:

(1) preparing a blood diluent;

(2) fully mixing the blood sample with the blood diluent to form a diluted blood sample;

(3) preparing a PCR reaction solution;

(4) and (3) mixing the diluted blood sample formed in the step (2) with the PCR reaction solution to form a PCR reaction system, amplifying by adopting a fluorescence PCR instrument, and collecting fluorescence.

7. The application method according to claim 6, wherein the application program,

in step (2), the blood sample is diluted to 15-25 times of its original concentration by a mixing process.

8. The application method according to any one of claims 6 to 7, characterized in that,

the blood sample in the step (2) contains an anticoagulant, and the anticoagulant is one or more of EDTA, sodium citrate and heparin anticoagulant.

9. The application method according to any one of claims 6 to 8, characterized in that,

the fluorescent PCR instrument amplification procedure in the step (4) is as follows:

the first step is as follows: circulating for 1 time at 37 ℃ for 5 min;

the second step is that: circulating for 1 time at 95 ℃ for 5 min;

the third step: 95 ℃ for 15 s; the fluorescence was collected by cycling 40 times at 60 ℃ for 1 min.

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