Method and kit to detect and/or quantify the presence of horse DNA in isolated samples (Machine-tran

专利摘要:
Method and kit for detecting and/or quantifying the presence of horse DNA in isolated samples. The present invention relates to a method for detecting and/or quantifying horse DNA in isolated samples, especially in meat mixtures suspected of inclusion frauds of horse meat in mixtures of economically more valuable meats. The invention also relates to a kit that contains the elements for preparing an electrochemical amperometric biosensor to detect and/or quantify percentages of up to 0.5% of horse meat in mixtures of other meats, without the need to extract the DNA or from perform amplifications using the PCR technique. Said biosensor uses specific sequences of mitochondrial horse DNA, a nucleic acid duplex detector antibody, a bacterial protein capable of binding to the Fc region of immunoglobulins and a molecule for the generation and amplification of the electrochemical signal. (Machine-translation by Google Translate, not legally binding)
公开号:ES2637232A1
申请号:ES201700642
申请日:2017-06-21
公开日:2017-10-11
发明作者:José Manuel Pingarrón Carrazón；Susana Campuzano Ruiz；Francisco Javier GALLEGO RODRIGUEZ；Rosario LINACERO DE LA FUENTE；Victor RUIZ-VALDEPEÑAS MONTIEL
申请人:Universidad Complutense de Madrid；
IPC主号:

专利说明:

Method and kit to detect and / or quantify the presence of horse DNA in isolated samples Technical sector
The present invention falls within the field of methods for performing food analysis. More specifically, it is framed in the sector of methods for the detection of the possible presence of horse meat in mixtures of meat or other foods and of the kits, based on amperometric electrochemical biosensors, designed for the detection and quantification of horse DNA in meat mixtures. State of the art
At present, the detection of food fraud and, in particular, in meat products is especially relevant. Although the presence of different types of meat in other products of animal origin does not usually represent a health risk, it is consumers who must choose according to their lifestyle, customs or beliefs, what they consume. On the other hand, the inclusion of unspecified meats in food products usually has the purpose of economic fraud, offering expensive products that include variable percentages of meats of more economic origin.
In the European Union, all ingredients must be mentioned on the label of packaged food products intended for the final consumer. In particular, food products that contain meat as an ingredient must also indicate the animal species from which that meat comes. Moreover, if an ingredient is mentioned in the name of the food, its quantity expressed in percentage must also appear in the list of ingredients so as not to mislead the consumer about the identity and composition of the food.
Following the scandals in Europe caused by adulterations with horse meat, controls have been carried out and it has been found that some packaged food products contained unreported horse meat on the list of ingredients on the package or on its label. Moreover, the name of some of these food products or the list of their ingredients misleadingly mentioned that they only contained beef.

Currently, there are numerous trials and strategies to detect adulteration
5 of the meat and differentiate the animal species present in meat mixtures; These tests are based primarily on immunological, spectroscopic or molecular biology techniques. In this sense, techniques based on the identification of species-specific proteins by electrophoretic tests or by immunoassays are not usually sensitive enough to differentiate
10 nearby animal species due to the possibility of cross-reactions, neither sufficiently reliable in heat treated products due to denaturation and degradation of the proteins produced by these heat treatments. Therefore, efforts began to focus on DNA-based assays, which is more stable than proteins at high temperatures. In this regard, during
Last 15 decades, the most used technique has been the polymerase chain reaction (PCR) as well as the improvements and variations that have emerged from this technique (real-time PCR, multiple PCR, Loop mediated isothermal amplification -LAMP- ). On the other hand, it was found that better results were obtained if the PCR was applied to mitochondrial DNA fragments, which
20 is more protected and, therefore, resists better than nuclear DNA to possible thermal treatments and, in addition, is present in multiple copies. Here are some examples of this.
KR101296221B1 patent sequences of the ribosomal RNA gene are used
25 12S and sequences of the cytochrome B gene, both mitochondrial, to detect by PCR chicken, goat, deer, pig, cow, sheep and horse meat.
Patent KR101448119B1 is based on multiple PCR and the cytochrome B gene to detect horse, cattle, pig, chicken, duck, pheasant, turkey, deer, goose, sheep,
30 rabbit and dog in meat mixtures of two or more of these species.
S. Tanabe et al. describe, in Biosci. Biotechnol Biochem 71 (2007) 3131-3135, a quantitative real-time PCR method for the detection of several animal species in food: pig, chicken, veal, lamb and horse, whose primers
35 are designed from the cytochrome B. J.J. Dooley et al., In Meat Science 68 (2004) 431-438, also use cytochrome B gene sequences to
detect animal species in meats using quantitative PCR, in this case, to
Differentiate beef, chicken, lamb, pork, turkey.
In CN104293899A, pairs of primers that amplify fragments of the horse's mitochondrial AON (COX 1, COX2, COX3, N05, N06 and cytochrome B) are used to detect meat of this species even in highly processed samples, and a PCR methodology is proposed Quantitative in real time.
CN104561327A uses pairs of COX1 mitochondrial gene primers to detect horse and donkey meat by quantitative fluorescence PCR.
Other documents describe methods that do not use PCR techniques. For example: Ali et al. describe a method of detecting adulteration with pork in raw foods and in heat-treated foods, in which hybrid nanobiosondes composed of gold nano particles and small probes labeled with fluorophores are used. Again, the probe of choice belongs to the mitochondrial gene sequence of cytochrome B. (M.E. Ali, et al. J. Nanomaterials vol. 2011, Article ID 781098, 11 pages, 2011. doi: 10.1155 / 2011/781098). With this method, detection limits (LOO) of 58.6pM are reached for the synthetic target and 230¡JgL-1 for the total AON and 1% of pork can be detected in meat mixtures under conditions of temperature and pressure such that they degrade AON targets, which prevents the detection of adulterations by PCR techniques.
In the case of adulterations with horse meat, there are no known methods that do not use the PCR technique and reach detection levels equivalent to those described for this method of Ali el al. Moreover, the European Commission, in the Commission Recommendation of March 27, 2014 (2014/180 / EU), set the detection of the presence of horse meat in meat (as a ratio of mass fraction p / p) but without determining the method of detection of choice. Detailed description of the invention
Method and kit to detect and / or quantify the presence of horse AON in isolated samples
One aspect of the present invention relates to a method for detecting the presence of horse AON in isolated samples, especially in meat samples or meat mixtures. For this, the invention is based on an electrochemical biosensor with which amperometric detection of horse AON can be performed. Another aspect of the invention relates to a kit that includes the set of elements and products necessary to obtain said electrochemical biosensor to detect horse AON in isolated samples.
With the method of the invention, significant discrimination of uncontaminated beef samples can be made against beef contaminated with 0.5% (w / w), or higher percentage of horse meat, in just 60 minutes . In addition, this discrimination can even be carried out using directly mitochondrial lysates, without the need to previously extract or purify the AON and, more importantly, without the need to amplify it either by traditional PCR, or by any of its improvements or variants.
One aspect of the present invention relates to a method for the detection and / or quantification of horse AON in isolated samples that includes:
to. - bind at least one molecule of a probe with a specific AON or RNA target sequence to magnetic particles;
b. -contact the magnetic particles from step a. with the sample (s) in which it is desired to detect and / or quantify the horse AON;
C. -include the magnetic particles from step b. with a detector antibody, or a fragment of detector antibody, specific antihomoduplex AON / AON or antiheteroduplex RNA / AON;
d. -include the magnetic particles from step c. with a bacterial protein capable of binding to the Fc region of the detector antibody, or fragment of the detector antibody, conjugated with a molecule for generating and amplifying the electrochemical response;
and. -capture the magnetic particles from step d. on printed work electrodes previously placed on a magnetic support; where the probe from step a. It has a length between 15 and 144 bp. And it is comprised in SEQ ID NO: 2, the reverse sequence to SEQ ID NO: 2 or the transcription to RNA of any of the previous two.
The magnetic particles that are used in this method may or may not be superparamagnetic, that is, they may be particles with paramagnetic properties even below certain critical temperatures. They can be microparticles or nanoparticles, thus covering diameter sizes ranging from 10 IJm to 100 nm and from 100 to 10 nm, respectively.
The different capture probes that can be used are designed from the
5 D-Ioop region of horse mitochondrial DNA. The D-Ioop region is the main non-coding area of the mitochondrial DNA molecule and, due to its high mutation rate, among other features, it is a magnificent marker system for phylogenetic studies. It is considered a marker of diversity and therefore has been frequently used to conduct genetic variability studies and analysis
10 archaeological and historical comparisons of several European and American horse breeds (McGahern, AM. Et al. Anim. Genet. 37 (2006) 498-502; Cardinali 1, et al. PLoS ONE 11 (4) (2016) : e0153004. doi: 10.1371 / journal.pone. 0153004). However, when analyzing the sequences of this region published in the NCBI database (National Center for Biotechnology Information, USA), it was found
15 an exclusive region of the horse that, in addition, remained constant in the animals of this species. This region comprises 144 bp Y is characterized by SEQ ID NO: 2. To bind them to the magnetic particles, probes included in this region are used whose sequences, preferably, can vary between 15 and 144 bp And are complementary to the corresponding fragments between 15 and 144
20 bp of SEQ ID NO: 2. In addition, DNA capture probes or capture transcripts to RNA can be used, and both can be complementary to one or another strand of the DNA. Capture probes can also be used whose sequences have a 98% identity with any of the four previous sequences, the percentage of a sequence being understood as the percentage
25 coincidences of the same nucleotides between two aligned sequences, along the entire length of both sequences.
One of the differences involved in using DNA or RNA capture probes is the type of detection antibody that will be used in the detection method and quantification of horse DNA. In case the capture probe is RNA, a specific antiheteroduplex antibody is used, such as the AbS9.6 monoclonal antibody, from Kerafast, or the monoclonal antibody D5H6, from Covalab. If a DNA capture probe is used, the antibody used is antihomoduplex specific, that is, it is double-stranded anti-DNA antibodies, which can only bind to polypyribose phosphate, to deoxyguanosine deoxycytidine base pairs and deoxyadenosine-deoxythymidine and some very special conformations of the double helix. On the other hand, another option of the invention is to use
fragments of the specific antiheteroduplex or antihomoduplex antibodies as long as they maintain said specificity. In a preferred embodiment, an RNA probe characterized by SEQ ID NO: 3 or by SEQ ID NO: 4 and an RNA / AON anti-heteroduplex antibody is used.
Several systems can be used to attach the probe having a specific AON or RNA target sequence to the magnetic particles. The magnetic particles are functionalized with a first binding element that, by means of a covalent or non-covalent interaction, allows the union of the AON or RNA probe by means of a second binding element. For example, the AON or RNA molecule can be conjugated with a short peptide sequence capable of forming a covalent or non-covalent interaction with a protein molecule that has been covalently bound to the magnetic particle through a crosslink with a sulfhydryl group or a amine; Another option is to conjugate the RNA or AON molecule with an amino acid and bind it covalently to the magnetic particles activated by NHS esters (N-Hydroxysuccinimide). On the other hand, it should be noted that each magnetic particle can be joined by several molecules of the AON or RNA probe with the specific target sequence selected.
The first binding element with which the magnetic particles are modified can be biotin, biotin analog binding proteins, COOH groups, tosyl groups and / or specific AON sequences. Among the biotin analog binding proteins, streptavidin, avidin, espiavidin, neutravidin, traptavidin and / or antibiotin antibodies can be used. As for the specific sequences of AON they can be those known as "colasquot", that is, sequences of between 4 and 30 adenines (A) or between 4 and 30 thymine (T). The second binding element of the AON or RNA probe to the modified magnetic particles can be: biotin, biotin analogs, amino groups and / or bases complementary to the specific sequences of AON, to the "colasquot;" commented in the previous paragraph. Among the biotin analogs can be used: digoxigenin, biotinpropanol, biotin molecules labeled with fluorescein. In a preferred embodiment of the invention, the first binding element is streptavidin and the second binding element is biotin. Here, biotinylated capture RNA probes are referred to as bRNACp and biotinylated capture AON probes are referred to as bONACp, the number of bases that characterize each particular probe being indicated below.
Once the magnetic particles and the molecules of the specific RNA or DNA probe are attached, they are put in contact with the isolated sample in which it is desired to detect the possible presence, or not, of horse meat and, where appropriate , quantify it. For this, two procedures can be followed: since the target sequence has been selected in mitochondrial DNA (mtDNA), extraction and purification of mtDNA can be performed and used as an isolated sample; Another option is to obtain a mitochondrial lysate and, without extracting the DNA present in said lysate, use it as an isolated sample. In a preferred embodiment of the invention, it is not necessary to extract mtDNA from the sample using mitochondrial lysates for detection
10 and / or quantification of horse DNA, which greatly simplifies the assay and saves both time and cost.
An amplification of the response is then carried out which involves the combined use of a specific antibody against DNA / RNA heteroduplex or against DNA / DNA homoduplex, or a fragment of the detector antibody that maintains its specificity, (as has been discussed above) and a bacterial protein capable of binding to the Fc region of the antibody or antibody fragment conjugated with a molecule generating and amplifying the electrochemical response. The bacterial protein can be Prot A or Prot G and the molecule of
20 signal generation and amplification can be selected from HRP, poly-HRP or alkaline phosphatase. HRP is a peroxidase (horseradish peroxidase, horseradish peroxidase) that also exists in various polymeric forms (poly-HRP20, polyHRP40, poly-HRP80). In a preferred embodiment of the invention the amplification of the signal is obtained by using Prot A conjugated with poly-HRP40.
The magnetic supports on which the printed work electrodes are placed include a magnet that can be attached to the magnetic support or embedded in the magnetic support structure itself. Preferably, the magnet is neodymium that ensures the magnetic capture of the magnetic particles of
30 stable, reproducible and localized way on the working electrode.
The "work electrode"; or quot; printed work electrodequot; For electrochemical transduction it can be rigid or flexible. Preferably, the electrochemical biosensor described herein comprises a working electrode.
35 rigid made with a material selected from the group: gold, carbon, platinum, CDtrodos, printed electrodes, silver, mercury, graphite, glassy carbon, carbon nanotubes, gold nanowires, gold nanoparticles, metal oxide nanoparticles, carbon paste , diamond and compounds doped with boron. Preferably, the working electrode used in this method is a carbon printed electrode.
Some of the steps described for this method can be performed simultaneously, which shortens the detection and / or quantification time of the presence of horse DNA in isolated samples. On the one hand, incubation with the detector antibody, or with a fragment of the detector antibody, can be performed at the same time as the incubation for the generation and amplification of the electrochemical signal. On the other hand, these two incubations can also be combined with the incubation in which the magnetic particles that already carry the specific DNA or RNA probe attached to its surface are brought into contact with the isolated sample, so that they join in a single incubation steps b., c. and d. of the method of the invention. In a preferred embodiment of the invention, steps b., C are performed simultaneously. and d. or at least the steps c. and d.
In this way, the method of detection and / or quantification of horse DNA can be completed in 60 minutes from the magnetic particles modified with the specific RNA or DNA probe without the need to resort to PCR amplification of any target sequence in isolated samples, whether meat, mixtures of meat or any other type of sample, with the practical advantages that this entails in terms of number of reagents and instrumentation (for example, the thermal cycler that is used to perform the PCR is not necessary ) required to make the determination.
Another aspect of the invention relates to a kit for detecting and / or quantifying the presence of horse DNA in isolated samples that includes: - modified magnetic particles with a first binding element to which at least one molecule of a probe is attached. DNA or RNA by means of a second binding element; -a detector antibody, or a fragment of detector antibody, specific antihomoduplex DNA / DNA or antiheteroduplex RNA / DNA; -a bacterial protein capable of binding to the Fc region of the detecting antibody, or fragment of the detecting antibody, conjugated with a molecule for generating and amplifying the electrochemical signal; -and a printed work electrode on a magnetic support; in which the probe has a length between 15 and 144 bp And is comprised in SEQ ID NO: 2, the reverse sequence to SEQ ID NO: 2 or the transcription to RNA of any of the previous two.
The magnetic particles included in this kit to form the electrochemical biosensor of the invention may or may not be superparamagnetic, that is, they may be particles with paramagnetic properties even below certain critical temperatures. They can be microparticles or nanoparticles, thus encompassing diameter sizes ranging from 1 O ~ m at 100 nm and from 100 to 10 nm, respectively.
The different capture probes that the kit can include are designed from the D-Ioop region of the horse mitochondrial DNA, more specifically from a 144 bp region characterized by SEQ ID NO: 2 its inverse or RNA transcription of any of the previous two, or sequences with a 98% identity with any of the previous four. Preferably, the kit includes the probe characterized by SEQ ID NO: 4 or by SEQ ID NO: 3.
To bind them to the magnetic particles, the kit of the invention contains probes included in the region of 144 bp whose sequences, preferably, can vary between 15 and 144 bp. And are complementary to the corresponding fragments of between 15 and 144 bp of SEQ. ID NO: 2. In addition, it can include DNA capture probes or capture probes for its transcription to RNA, and both can be complementary to one or another strand of the DNA.
When the capture probe included in the kit is RNA, a specific antiheteroduplex antibody is included, such as the AbS9.6 monoclonal antibody, from Kerafast, or the monoclonal antibody D5H6, from Covalab. When a DNA capture probe is included in the kit, the antibody that incorporates the kit is antihomoduplex specific, that is, it is a double-stranded anti-DNA antibody, which can only bind to polypyribose phosphate, base pairs deoxyguanosine deoxycytidine and deoxyadenosine deoxythymidine or some very special conformations of the double helix. In another embodiment of the invention, fragments of the specific antiheteroduplex or antihomoduplex antibodies that maintain said specificity are included in the kit. In a preferred embodiment, an RNA probe characterized by SEQ ID NO: 3 or by SEQ ID NO: 4 and an RNA / DNA antiheteroduplex antibody is used.
The DNA or RNA capture probe can be attached to the magnetic particles according to different systems. Magnetic particles functionalized with a first binding element, through a covalent or non-covalent interaction, bind to the DNA or RNA probe that incorporates a second binding element. For example, the DNA or RNA molecule may be conjugated to a short peptide sequence capable of forming a covalent or non-covalent interaction with a protein molecule that is covalently bound to the magnetic particle through a crosslink with a sulfhydryl group or an amine. ; Another option is that the RNA or DNA molecule be conjugated to an amino acid and bind this amino acid covalently to the magnetic particles activated by NHS esters. On the other hand, it should be noted that in the kit that gives rise to the electromagnetic biosensor of the invention each magnetic particle can have several molecules of the DNA or RNA probe attached to the specific target sequence selected.
The first binding element of the magnetic particles can be selected from the group consisting of: biotin, biotin analog binding proteins, COOH groups, tosyl groups and / or specific DNA sequences. Biotin analogue binding proteins can be selected from: streptavidin, avidin, espiavidin, neutravidin, traptavidin and / or antibiotin antibodies. As for the specific DNA sequences, they may be those known as "colasquot", that is, sequences of between 4 and 30 adenines (A) or between 4 and 30 thymine (T). The second binding element of the DNA or RNA probe to the modified magnetic particles may be: biotin, biotin analogs, amino groups and / or bases complementary to the specific DNA sequences, to the "colasquot;" commented in the previous paragraph. Among the biotin analogs the kit may include: digoxigenin, biotinpropanol, biotin molecules labeled with fluorescein. In a preferred embodiment of the invention, the first binding element is streptavidin and the second binding element is biotin.
The bacterial protein capable of binding to the Fc region of the detector antibody
or detector antibody fragment can be selected from Prot A and Prot G. The signal generation and amplification molecule can be selected from HRP, poly-HRP (poly-HRP20, poly-HRP40, poly-HRP80) or alkaline phosphatase. In a preferred embodiment of the invention the kit includes Prot A conjugated with poly-HRP40.
The magnetic supports on which the printed electrodes of
Work preferably include a neodymium magnet.
The "work electrode"; or quot; printed work electrodequot; which includes the kit, for
5 electrochemical transduction, can be rigid or flexible. Preferably, the electrochemical biosensor of the invention comprises a rigid working electrode made of a material selected from the group: gold, carbon, platinum, CD-trodes, printed electrodes, silver, mercury, graphite, glassy carbon, carbon nanotubes, carbon nanowires gold, gold nanoparticles, metal oxide nanoparticles, paste
10 carbon, diamond and compounds doped with boron. The kit of the invention preferably includes a carbon printed electrode.
Another aspect of the invention relates to the use of a kit such as the one described in this section to detect and / or quantify the presence of horse meat in
15 meat mixtures, especially to detect and quantify possible fraud committed by adding a cheaper item (horse meat) to a more valuable one (such as beef).
Brief description of the figures
20 Figure 1. Specificity of SEO ID NO: 2 to detect horse meat. Fragments amplified by PCR on an agarose gel (1.8%) starting at 5.0; 1.0 and 0.5 ng of DNA extracted from horse meat (H .M.) And 5 ng of DNA extracted from beef (B.M.) using different commercial methods and kits
Extraction of total DNA (A) and mtDNA (B and C). The negative C (-) control consists of a conventional PCR performed in the absence of DNA.
Figure 2. Influence of the length of the biotinylated capture probe immobilized in Strep-MB on the amperometric response measured with the biosensor of the invention
30 for 0.0 (B, white columns) and 0.1 nM (S, gray columns) of the SEO ID NO target sequence: 1 and the corresponding values of the S / B ratio (red diamonds). Error bars were estimated as triple the standard deviation (n = 3).
Figure 3. Influence of the number of stages used to perform the test on
35 the amperometric responses measured with the biosensor of the invention including bRNACp-40bp and ProtA-HRP40 for 0.0 (B, white columns) and 0.1 nM (S, gray columns) of the synthetic sequence SEO ID NO: 1 and the corresponding values of the S / B ratio (red diamonds). Error bars were estimated as triple the standard deviation (n = 3).
Figure 4. Calibration graphs obtained for the target sequence characterized by SEO ID NO: 1 with the biosensor that includes bRNACp-40bp and ProtA-HRP40 (black), the biosensor that includes bRNACp-40bp and ProtA-HRP (red) and a conventional sandwich procedure (blue). Error bars were estimated as triple the standard deviation (n = 3).
Figure 5. Discrimination between beef (B.M., white striped bars) and horse (H.M., solid maroon bars) from extracted mitochondrial DNA. The amperometric responses measured with a biosensor of the invention including bRNACp-40pb and ProtA-HRP40 for different amounts of mitochondrial DNA extracted are shown. Error bars were estimated as triple the standard deviation (n = 3).
Figure 6. Discrimination of beef and horse meat using mitochondrial lysates prepared from 3.0 g of veal (B.M., white striped bars) or horse (H.M., solid maroon bars). The amperometric responses measured with a biosensor of the invention including bRNACp40pb and ProtA-HRP40 for different dilutions of mitochondrial lysates in casein blocking solution are shown. Error bars were estimated as triple the standard deviation (n = 3).
Figure 7. Amperometric response measured with a biosensor of the invention that includes bRNACp-40pb and ProtA-HRP40 from 1: 1 diluted mitochondrial lysates of beef (pink bar), turkey (blue bar), pork (orange bar), chicken (yellow bar) and horse (garnet bar) and including a negative control (white bar). Error bars were estimated as triple the standard deviation (n = 3).
Figure 8. Dependence of the amperometric signals obtained with the biosensor of the invention for mitochondrial lysates diluted 1: 1 with blocking casein solution and obtained from 3.0 g of uncontaminated beef samples (red circle) and contaminated with different percentages of horse meat (black squares) (on the abscissa axis the percentage (%) of adulteration is represented). Error bars were estimated as triple the standard deviation (n = 3).
Figure 9a Amperometric signals provided by the biosensor of the invention
5 for mitochondrial lysates obtained from 100% beef, 99.5 / 0.5% beef / horse and 99/1% beef / horse samples (abscissa represents the percentage (%) of adulteration indicating the percentage of horse meat included in the sample). Error bars were estimated as triple the standard deviation (n = 3).
Figure 9b Linear dependence between the measured cathodic current and the percentage of horse meat used in the meat mixture of the samples, in the range of 1.0 to 10.0% (% of adulteration). Error bars were estimated as triple the standard deviation (n = 3).
Embodiment of the invention Example 1. Design of RNA probes.
To select a region of AON invariable in horses and absent in the rest of
20 mammals, the sequences of the mitochondrial AON O-loop region (AONmt) published in the NCBI database were analyzed. From the sequence published in Gen Bank with the reference: OQ327940 and that includes 851 bp of mitochondrial horse AON, of haploid 1065 O-loop described by McGahem et al. (McGahern, AM. Et al. Anim. Genet. 37 (2006) 498-502), a target sequence was selected as
25 fragment of 48 bp, characterized by SEQ ID NO: 1.
From SEQ ID NO: 1, several biotinylated probes at the 5 'end, of RNA or AON, were designed to analyze the operation of the biosensor with fragments of different sizes and different sequences that totally or partially included SEQ ID NO: 1 .
Among them, we designed: -a 144 bp AON probe characterized by SEQ ID NO: 2 (bONACp-144pb) and biotinylated at the 5 'end, for whose PCR amplification primers EQ2 F (forward) were used and EQ2 R (reverse) characterized by SEQ ID NO: 5 and 6, respectively;
35 -a 40 bp RNA probe characterized by SEQ ID NO: 3 and biotinylated at the 5 'end (bRNACp-40pb);
- a 24 bp RNA probe characterized by SEa ID NO: 4 and biotinylated at the 5 'end (bRNACp-24pb).
To amplify the fragment characterized by SEa ID NO: 2, a PCR was performed in a total volume of 20 ~ I, in which a mixture was prepared with 1O ~ I of the ready-to-use mixture of Biotools "ONA AmpliTools Fast Mix 2xquot ;, 5pmol of each primer (SEa ID NO: 5 and 6), 2 ~ I of AON and nuclease-free water until volume is complete. An initial denaturation was performed at 94 ° C for 5 minutes, followed by 35 cycles that included a denaturation at 94 ° C for 15 seconds, a banding at 55 ° C for 30 seconds and a final elongation step at 72 ° C for 5 minutes. The primers and the other probes were commissioned to SigmaAldrich.
Example 2. Preparation of the modified magnetic particles with biotinylated capture probes.
Two washes were performed with B&W buffer (10 mM Tris-HCI with 1 mM EOTA and 2 M NaCl, pH 7.5) of 5 ~ I of a commercial suspension of magnetic particles functionalized with streptavidin (Strep-MB, 2.8 ~ m in diameter, 10 mg mL · 1, Oynabeads M-280 Streptavidin, 112060 -Oynal Biotech ASA-). After each wash, the Strep-MB was magnetically concentrated for 3 minutes in a magnetic concentrator, and the supernatant was discarded. They were incubated for 15-90 minutes in different tests (with stirring at 950 rpm and 37 ° C) in 25 ~ I of a solution with the biotinylated capture probe at the 5 'end (0.1 ~ M bRNACp or 0.1 ~ M cONACp) prepared in B&W buffer. Then, the modified Strep-MB (bRNACp-MB
or bONACp-MB) were washed twice with 50 ~ I of a commercial casein blocking solution (1% purified casein in PBS).
In this step, the bRNACp-MB or the bONACp-MB were stored, until the moment of use, in filtered B&W buffer pH 6.0 and at 4 ° C. Example 3. Preparation of the samples.
All samples of meat used (veal, horse, turkey, pork and chicken) were purchased at a local supermarket and stored at -80 ° C. For its determination each of these meat samples was crushed and homogenized with liquid nitrogen by using a mortar. Samples of beef with different amounts of horse meat (0.5; 1.0; 2.5; 5.0; 10; 25 and 50% (w / w)) were prepared from the crushed meats.
Example 4. Mitochondrial DNA extraction and lysate preparationmitochondrialFrom the meat samples prepared according to example 3, we proceeded toEvaluate two different procedures.
AONmt extraction. To isolate the mitochondria from the samples, 3 g of were crushedeach meat sample in 15 ml of Solution I (50 mM glucose, 10 mM EOTA and 25mM Tris-HCI, pH 8.0) And centrifuged at 1000 g for 5 minutes at 4 ° C. TOthen the supernatant was centrifuged a second time at 12000 for 10minutes, at 4 ° C. The sediment was resuspended in 800 ~ I of MLB solution (150 mMNaCI, 50 mM Na2EOTA and 10 mM Tris-HCI, pH 8.0) and then added400 ~ I of SOS solution (2% SOS in 0.4 N NaOH) and 800 ~ I of Solution 111 (29.5%(v / v) glacial acetic acid, pH 4.8), shaking manually, and kept on icefor 5 minutes. It was centrifuged at 13000 rpm for 5 minutes, at 25 ° C, and the AONmtwas extracted from the supernatant by the purification kit Speedtools plasmid ONApurification kit (Biotools).
Mitochondrial lysate. To isolate the mitochondria from the samples, 3 g of were crushedeach meat sample in 15 ml of Solution I and centrifuged at 1000 g for 5minutes at 4 ° C. The supernatant was then centrifuged a second time at12000 for 10 minutes, at 4 ° C. The mitochondrial mass obtained in200 ~ I of MLB solution and 100 ~ I of SOS solution and thus the lysates were obtainedmitochondrial
Example 5. Verification of the specificity of the mtDNA regionselectedThe AONmt of horse meat (H. M.) and beef (B. M.) was extracted asas indicated in example 4 and then an amplification was performed byPCR of the region characterized by SEO ID NO: 2 using primerscharacterized by SEO ID NO: 5 and 6 And following the procedure indicated in theexample 2.
Figure 1 shows the high specificity of the primers used and the selected fragment.
Example 6. Detection of the target sequence.
To test the biosensor, tests were performed with the modified magnetic microparticles bRNACp-MB and bDNACp-MB, obtained as explained in example 2 with a 60 minute incubation, and with samples of mtDNA extracted or mitochondrial lysates without DNA extraction , as described in example 4.
Modified magnetic microparticles with biotinylated capture probes (bRNACp-40bp-MB, bRNACp-24bp-MB, bDNACp-144bp-MB) were incubated in different assays for 15 to 90 minutes (under agitation at 950 rpm and 37 ° C) in 25 µJ of a solution containing the DNA target sequence, characterized by SEQ ID NO: 1. After two washes with 50 µJ of casein blocking solution, each mixture was incubated in different assays for 15 to 90 minutes in 25! JI of a solution with an anti-DNA / DNA or anti-RNA / DNA antibody (2! JgmL- ') and ProtA bound to poly-HRP40 (1! JgmL-'), which was previously prepared in blocking solution of casein and incubating for 1 hour. After two additional washes with casein blocking solution, the modified magnetic microparticles were resuspended with biotinylated capture probes in 45 [mu] JI of a PBS buffer (O, 05M, pH 6.0) to perform the amperometric detection.
Example 7. Amperometric detection.
The 45 µJ of suspension of the modified MB, as in example 6, resulting from the incubation test with the 30-minute target sequence and incubation with the antibody and with ProtA-poly-HRP40 also 30 minutes, were pipetted onto the Carbon printed electrodes (SPCE) where they were magnetically captured on the surface of the work electrodes placed on the magnetic support elements. The amperometric measurements were made in solutions under agitation by immersion of the SPCE, assembled in the magnetic support elements, in an electrochemical cell with 10 ml of 0.05 M PBS (pH 6.0) and 1.0 mM hydroquinone ( HQ) prepared just at the time of electrochemical measurement, and applying a detection potential of -0.20 V against a silver pseudo-reference electrode.
The amperometric measurements were made with a potentiostat 812B from CH Instruments (Austin, TX) using the CH1812B software. As electrochemical transducers, together with a specific cable connector (DRP-CAC, DropSens, Spain), SPCE (DRP-110, DropSens, Spain), composed of a 4 mm diameter carbon working electrode, a counter electrode, were used of carbon and an electrode of Ag of pseudoreference. All measurements were made at room temperature. A neodymium magnet (AIMAN GZ) embedded in a Teflon envelope was used to magnetically capture the modified MB on the surface of the SPCEs. Once the baseline was established, 50 1-11 of a solution of H20 2 (0.1 M) was added and the current was recorded until the steady state was reached. It was found that the magnitude of the measured cathodic current and the concentration of target AON were directly proportional.
In all the trials in which the different experimental variables were evaluated, the selection criterion was the highest ratio between the amperometric response measured at -0.20 V (vs. the pseudo-reference Ag electrode) for 0.0 (blank , B) and 0.1 nM of the target sequence characterized by SEO ID NO: 1 (signal, S), that is, the S / B ratio.
Example 8. Differences in the length of the biotinylated capture probes versus the target sequence. In this example, different sized RNA probes immobilized on Strep-MB were tested. As an antibody, a specific monoclonal antibody of AON / RNA heterohybrid (clone 05H6, from Covalab) was used When bRNACp40pb was used, the amperometric responses were 2.2 times higher than the measurements obtained with bRNACp-24pb. Figure 2 shows the difference in the response obtained with both probes.
Example 9. Differences in the number of steps of the hybridization procedure.
1-stage protocol: hybridization and labeling were performed in a single step by incubating for 30 minutes the bRNACp-40-MB in a solution containing a mixture of the target sequence characterized by SEO ID NO: 1, the antiONNRNA antibody (clone 05H6, from Covalab) and the ProtA-poly-HRP40 complex.
Protocol in 2 stages: a hybridization step was carried out initially by incubating the bRNACp-40-MB with the target sequence characterized by SEO ID NO: 1 for 30 minutes and, subsequently, the labeling of the binding of both elements by incubating them for 30 minutes with a mixture of the antiONNRNA antibody (clone 05H6, from Covalab) and the ProtA-poly-HRP40 complex.
3-stage protocol: hybridization of the target sequence characterized by SEQ ID NO: 1 was first performed with the bRNACp-40-MB for 30 minutes; followed by another 30 minute incubation with the antiONAlRNA antibody (clone 05H6, from Covalab) and a third incubation with the ProtA-poly-HRP40 complex, for an additional 30 minutes.
The three protocols gave positive results, although the first two were
better than the third, as can be seen in figure 3.
Example 10. Comparison with a conventional sandwich test.
Figure 4 shows the calibration chart for the target sequence characterized by SEQ ID NO: 1 using ProtA-poly-HRP40 (in black), ProtA-polyHRP (in red) and a conventional sandwich strategy (in blue). A linear dependence (r = 0.98984) was verified with the synthetic AON concentration between 0.39 and 75 pM, with slope and intercept values of 91,796 ± 728 nA nM-1 and 244 ± 20 nA, respectively. The limits of detection (LOO) and quantification (LQ) were calculated according to the criteria of 3xst! M and 10xst! M, respectively, where quot; mquot; it is the slope of the linear calibration graph "S"; It was estimated as the standard deviation of ten amperometric measurements obtained in the absence of the target sequence characterized by SEQ ID NO: 1. Values of 0.12 and 0.39 pM (3.0 and 9.75 attomol) were obtained, respectively. Table 1 summarizes the analytical characteristics of the comparison made.
The conventional sandwich sensor contained a biotinylated capture AON probe characterized by SEQ ID NO: 7 and a biotinylated detection probe characterized by SEQ ID NO: 8, which was enzymatically labeled with Strep-HRP.
Table 1. Comparison of the analytical characteristics of biosensors constructed following different strategies.
Parameter Conventional sandwich test with Strep-HRPbRNACp24bp / ProtA-poli-HRP40bRNACp40bp / ProtA-HRPbRNACp40bp / ProtA-poli-HRP40
Linear range 812-50,0001.4-2502.5-1000.39-75
/p.m
r2 0.9990.9990.9990.998
Pending / nA nM-1 119.0 ± 1.340,629 ± 2959,986 ± 8091,796 ± 728
Interception / nA 86 ± 27206 ± 2551 ± 4244 ± 20
Amplification factor vs. conventional -34184771
LOD / pM 1570.40.80.12
Trial duration, min 60606060
Example 11. Detection of horse DNA in extracted mtDNA.
The usefulness of the biosensor to discriminate between beef and horse meat was verified by analyzing different amounts of mtDNA extracted from 5 meat of both species, following the indications of example 4. The biosensor was used with bRNACp-40pb using ProtA -poli-HRP40. As can be seen in Figure 5, it was possible to clearly differentiate between both types of meat without resorting to the amplification of any DNA fragment by any type of PCR technique provided that amounts of
10 mtDNA equal to or greater than 50ng. Figure 5 also shows a decrease in the amperometric response when the amounts of DNA were very high, a fact attributable to an excess of target DNA with respect to the concentration of the capture RNA probe.
15 Example 12. Detection of horse DNA in mitochondrial lysates. The mitochondrial lysates obtained according to example 4 from 3 g of beef and horse meat were used to check if the mtDNA extraction could be dispensed with. Several dilutions of the mitochondrial lysates were prepared with casein blocking solution. Parallel measurements were made from
20 mtDNA extracted from the same amount of meat. Figure 6 shows how the biosensor of the invention was able to discriminate between beef and horse meat without the need to proceed with the extraction of mtDNA, using diluted mitochondrial lysates directly, obtaining the best results with dilution
1: 2 (mitochondrial lysate volume / total volume).
Example 13. Selectivity of the biosensor of the invention.
To analyze the selectivity of the biosensors of the invention, 1: 1 dilutions were made, in casein blocking solution, of mitochondrial lysates
5 obtained from 3 g of beef, turkey, pork, chicken and horse. Figure 7 shows how only significant amperometric response was obtained (differentiated from that obtained in the absence of a DNA target sequence, that is, in the negative control) when mitochondrial lysates prepared from horse meat were used.
Example 14. Applicability of the biosensor in the detection of fraudulent adulterations.
The most important parameter to ensure the real applicability of the biosensor in the detection of adulterations of beef with horse meat, for fraudulent purposes, is the limit of real detection of horse meat in the presence of beef that is capable of reaching the biosensor of the invention.
To analyze this parameter, different percentages of crushed beef were mixed with different percentages of crushed horse meat as described in Example 3. As shown in Figure 8, it is possible to clearly differentiate between 100% beef and samples containing different amounts of horse meat, including amounts as small as 0.5% (w / w), which was the amount established by the European Commission in its recommendation on a second coordinated control plan to establish prevalence from
25 fraudulent practices in the marketing of beef, in March 2014 (Official Journal of the European Union L 95/64, 29.3.2014).
In this example, statistically significant differences (with a 95% confidence level) were found between the amperometric signals detected by the biosensor of the invention in 100% beef and beef with 99.5% beef and 0, 5% horse (figure 9a). The existence of a linear dependence (r = 0.999) between the measured cathodic current and the percentage of horse meat used in the meat mixture in the range of 1.0 to 10.0% (Figure 9b), was verified. that the biosensor of the invention is not only useful in the detection of adulteration but
35 also in its quantification.

权利要求:
Claims (37)
[1]
1. Method for the detection and / or quantification of horse AON in isolated samples that includes:
5 a. - bind at least one molecule of a probe with a specific AON or RNA target sequence to magnetic particles;
b. -contact the magnetic particles from step a. with the sample (s) in which it is desired to detect and / or quantify the horse AON;
C. - incubate the magnetic particles from step b. with a detector antibody, or a
10 detector antibody fragment, specific antihomoduplex AON / AON or antiheteroduplex RNA / AON;
d. -include the magnetic particles from step c. with a bacterial protein capable of binding to the Fc region of the antibody, or antibody fragment, conjugated with a molecule of generation and amplification of the response
Electrochemistry;
and. -capture the magnetic particles from step d. on printed work electrodes placed on magnetic supports; where the probe from step a. It is between 15 and 144 bp in length and is comprised of SEO ID NO: 2, the reverse sequence to SEO ID NO: 2, the
20 RNA transcription of any of the previous two, or sequences with a 98% identity with any of the previous four.
[2]
2. Method according to claim 1 wherein the samples from step b. they are samples of mitochondrial AON extracted.
[3]
3. Method according to claim 1 wherein the samples from step b. they are mitochondrial lysates without extraction of the AON.
[4]
4. Method according to any of the preceding claims wherein the steps c. 30 and d. They are performed simultaneously.
[5]
5. Method according to any of claims 1-3 wherein the steps b. c and d are performed simultaneously.
Method according to any one of the preceding claims in which the magnetic particles are nanoparticles or microparticles.
[7]
7. Method according to any of the preceding claims wherein the magnetic particles have superparamagnetic properties.
[8]
8. Method according to any of the preceding claims in which the union of
5 the specific target sequence of AON or RNA to magnetic particles is performed with: - a first binding element with which the magnetic particles are modified: biotin, biotin analog binding proteins, COOH groups, tosyl groups and / or specific sequences of AON;
10 -a second element of binding of the AON or RNA probe to the modified magnetic particles: biotin, biotin analogs, amino groups and / or bases complementary to the specific AON sequences.
[9]
9. Method according to claim 8 wherein the analog binding proteins of
Biotin are selected from the group: streptavidin, avidin, espiavidin, neutravidin, traptavidin and / or antibiotin antibodies.
[10]
10. Method according to any of claims 8-9 wherein the sequences
AON-specific sequences are between 4 and 30 adenines (A) or between 4 and 30 thymine (T).
[11]
11. Method according to any of claims 8-10 wherein the biotin analogs are selected from the group: digoxigenin, biotinpropanol, fluorescein-labeled biotin molecules.
[12]
12. Method according to claim 8 wherein the first binding element is streptavidin and the second binding element is biotin.
[13]
13. Method according to any of the preceding claims in which the protein
Bacterial capable of binding to the Fc region of the detector antibody, or detector antibody fragment, is protein G or protein A.
[14]
14. Method according to any of the preceding claims in which the molecule
Generation and amplification of the electrochemical response is HRP, poly-HRP or alkaline phosphatase.
[15]
fifteen. Method according to claims 13-14 wherein the bacterial protein is Prot A and the electrochemical response generation and amplification molecule is polyHRP40.
[16]
16. Method according to any of the preceding claims wherein the probe is characterized by SEO ID NO: 3 or by SEO ID NO: 4.
[17]
17. Method according to any of the preceding claims in which the working electrode is made of a material selected from the group: gold, carbon, platinum, CO-trodes, printed electrodes, silver, mercury, graphite, glassy carbon, carbon nanotubes, nanowires of gold, gold nanoparticles, metal oxide nanoparticles, carbon paste, diamond and boron doped compounds.
[18]
18. Method according to claim 17 wherein the electrode is a carbon printed working electrode.
[19]
19. Method according to any of the preceding claims in which the magnetic support includes a neodymium magnet.
[20]
twenty. Method according to any one of the preceding claims wherein the magnetic support includes a magnet embedded in the magnetic support structure itself.
[21]
twenty-one. Kit for detecting and / or quantifying the presence of horse AON in isolated samples that includes: - modified magnetic particles with a first binding element to which at least one molecule of an AON or RNA probe is attached by means of a second connecting element; -a detector antibody, or a fragment of detector antibody, specific antihomoduplex AON / AON or antiheteroduplex RNA / AON; -a bacterial protein capable of binding to the Fc region of the detector antibody, or fragment of the detector antibody, conjugated with a molecule for generating and amplifying the electrochemical response; -and a printed work electrode on a magnetic support; in which the probe is between 15 and 144 bp in length and is comprised of SEO ID NO: 2, the reverse sequence to SEO ID NO: 2, RNA transcription of any of the previous two, or sequences with a 98 % identity with any of the previous four.
[22]
22 Kit according to claim 21 wherein the magnetic particles are nanoparticles or micro particles.
[23]
2. 3. Kit according to any of claims 21-22 in which the magnetic particles have superparamagnetic properties.
[24]
24. Kit according to any of claims 21-23 wherein the first binding element with which the magnetic particles are modified is biotin, biotin analog binding proteins, COOH groups, tosyl groups and / or specific sequences of AON.
[25]
25. Kit according to claim 24 wherein the biotin analog binding proteins are selected from the group: streptavidin, avidin, espiavidin, neutravidin, traptavidin and / or antibiotin antibodies.
[26]
26. Kit according to any one of claims 24-25 in which the specific sequences of AON are sequences of between 4 and 30 adenines (A) or between 4 and 30 thymine (T).
[27]
27. Kit according to any of claims 21-26 wherein the second element of binding of the AON or RNA probe to the modified magnetic particles is biotin, biotin analogs, amino groups and / or bases complementary to the specific AON sequences .
[28]
28. Kit according to claim 27 wherein the biotin analogs are selected from the group: digoxigenin, biotinpropanol, biotin molecules labeled with fluorescein.
[29]
29. Kit according to any of claims 21-23 wherein the first binding element is streptavidin and the second binding element is biotin.
[30]
30 Kit according to any of claims 21-29 in which the bacterial protein capable of binding to the Fc region of the detector antibody, or fragment of the detector antibody, is protein G or protein A.
[31 ]
31. Kit according to any of claims 21-30 in which the signal generation and amplification molecule is HRP, poly-HRP or alkaline phosphatase.
[32]
32 Kit according to any of claims 21-31 wherein the probe ischaracterized by SEO ID NO: 3 and SEO ID NO: 4.
[33]
33. Kit according to any of claims 21-32 wherein the electrode ofWork is done with a material selected from the group: gold, carbon, platinum,CO-trodes, printed electrodes, silver, mercury, graphite, glassy carbon, nanotubescarbon, gold nanowires, gold nanoparticles, oxide nanoparticlesmetallic, carbon paste, diamond and doped compounds with boron.
[34]
3. 4. Kit according to claim 33 wherein the electrode is a printed electrode ofcarbon.
[35]
35 Kit according to any of claims 21-34 wherein the magnetic supportIt includes a neodymium magnet.
[36]
36. Kit according to any of claims 33-35 wherein the magnetic supportIt includes a magnet embedded in the magnetic support structure itself.
[37]
37. Kit to detect and / or quantify the presence of horse AON in samplesisolated that includes:- Streptavidin modified magnetic microparticles to which aprobe characterized by SEO ID NO: 3 or 4 and modified with biotin;-a specific antibody, or detector antibody fragment, specificRNA / AON antiheteroduplex;-a Prot A bacterial protein conjugated with poly-HRP20, poly-HRP40 or polyHRP80;-And a printed work electrode on a magnetic support that includes a magnetattached to the magnetic support or embedded in its structure.
[38]
38. Use of the kit defined in claims 21-37 in the detection and / orquantification of the presence of horse meat in meat mixtures.
Fig. 1
 144bp
B. M. H. M. B. M. H. M. H. M.
•,
A B e
Fig. 2
40 8000
 r 30
<C
 r 20 al
and
CI)
-
 -4000
'one
, .. 10
oo
 bRNACp-24bp bRNACp-40bp
Fig. 3
1 2 3
Fig. 4
~ .------------------ # -------------- ~
<C
e 4000
.-... I
[SEQ ID NO: 1], nM
Fig. 5 1800 1500 1200 «900
C:
-
quot ;, 600 300
OR
5 10 50 100 250
n9 AONrnt
·
: i
6 -
to the
: i
4 ·
~
OR
500 750
Fig. 7
«800
and
'' i '~
Fig. 8
!
,
1000 «750-
I
and
'' i '~
•
500
•
•
•
250
• •
.,
OR
0 '
0.1 1 10 100
%
Fig. 9a
300 -T
T
l.
lt;
...
C ~ 150 -T
quot;
or I I I
or 0.5 1.0
%
Fig. 9b
700 600 500
lt;
and
'jquot; ~
300 200
OR
O 2 4 6 8 10%

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ES201700642A|ES2637232B2|2017-06-21|2017-06-21|Method and kit to detect and / or quantify the presence of horse DNA in isolated samples|ES201700642A| ES2637232B2|2017-06-21|2017-06-21|Method and kit to detect and / or quantify the presence of horse DNA in isolated samples|