• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    Method of molecular determination of the sex of birds by isothermal amplification mediated by loops. The invention provides sequences of primers, reagents and protocols for sex determination in the aves class employing the loop-mediated isothermal amplification (lamp) technique. Unlike other molecular techniques based on pcr, this invention does not require thermocyclers or specialized laboratories and thus allows the determination of the sex of birds in any place where incubation is allowed at a single temperature. Its application will be extended to all sectors related in some way to ornithology (poultry, ecology and conservation, mainly). (Machine-translation by Google Translate, not legally binding)
    公开号:ES2647134A1
    申请号:ES201630653
    申请日:2016-05-19
    公开日:2017-12-19
    发明作者:Alejandro CENTENO-CUADROS；Martina CARRETE；Miguel DELIBES DE CASTRO；José Luis TELLA ESCOBEDO
    申请人:Consejo Superior de Investigaciones Cientificas CSIC；Universidad Pablo de Olavide；
    IPC主号:
    专利说明:

    Method of molecular determination of bird sex by loop-mediated isothermal amplificationFIELD OF THE INVENTION
     5
    The invention falls within the field of biotechnology, more specifically to the amplification (making of multiple copies) of polynucleotides. Primer sequences, reagent compositions and sex determination protocols applicable to the Bird class are provided.
      10
    STATE OF THE TECHNIQUE

    The determination of sex in birds (DSA hereinafter) is a key step for the management of wild populations and captive breeding programs, as well as in studies of ethology and ecology. A correct DSA is also basic to develop 15 evolutionary theories on differentiated parental investment or on how females can determine the sex of their offspring, theories that, despite decades of research, their mechanisms and consequences are not yet well known. . This step is especially critical in those bird species that have sexual monomorphism (species in which there is no phenotypic or behavioral differentiation between males and females), in early stages of the life cycle (embryos, chickens and volatones) and / or when Biological material sampling has been performed without the possibility of capturing individuals (as in non-invasive sampling). These cases tend to be problematic because they show high error rates when methodologies are applied in which it is not necessary to capture individuals, 25 such as those based on ethological studies (Gray & Hamer, 2001; Anim Behav 62 (1): 117– 121) or differences in songs (Volodin et al. 2009; Bioacoustics 18 (3): 277-290). These errors in the determination of sex that show non-invasive techniques as well as those based on palpation or visualization of the bird's cloacal area can be partially solved by taking a blood sample 30 for hormonal analysis (Bercovitz et al. 1978; J Zool Anim Med 9: 114-124) or taking morphometric measurements (Reynolds et al. 2007; J Zool 274 (1): 2-8). In more extreme cases, more invasive techniques such as laparoscopy (Richner, 1989; JF Ornithol 60: 137-142), laparatomy (Maron & Myers, 1984; JF Ornithol 55: 336–342) or genital area examination have been applied. (Miller & Wagner, 1955; Auk 35
    72: 279-285). Although these traditional methods have proven effective, their application has not been exempt from logistics beyond the reach of a large part of the interested public and endangers the physical integrity of the animal examined.
    The DSA has been specially studied in poultry farming by moving millions of euros annually in the market generated by the tens of thousands of 5 sex determinations requested daily by poultry farmers and private breeders (mainly) and therefore depend on the truthfulness and speed to which said determination is made. They have even been able to select genetic lines that allow to determine the sex by feather coloration in chickens of a new turkey breed (WO 9719588 A1), something completely impracticable in the case of the determination of sex in wild species. In the context of livestock species, techniques have been developed for the determination of sex in chickens before hatching thanks to ultrasound scanning (US 6512839 B1) or hormonal analysis (JP 06153742 A).
    The application of molecular techniques based on deoxyribonucleic acid (DNA) 15 has led to a revolution in DSA. Isolation and characterization of the karyotype of the species was the first DNA-based DSA technique. By means of the karyotype, the males can be differentiated from the females after identifying the chromosomes of each species (two copies of the Z chromosome in the case of the males and a single copy of the Z chromosome and another one of the W when the karyotype was one female) (Harris & Walters, 1982; Genetics 60: 19–20). Other techniques of molecular determination of sex have been based on the differential measurement between sexes of variations of DNA molecules after being irradiated with ultraviolet (DE 102007013107 A1). However, it has been the sequencing and gene characterization of the sex chromosomes that has allowed a greater advance in the DSA. For example, the design and hybridization of probe sequences with DNA and subsequent fluorescence detection have been useful for differentiation between male and female birds (US 5508165 A). However, the vast majority of DSA molecular techniques are based on the polymerase chain reaction (PCR for its acronym in English). As a result of this technique, a number of copies of 30 orders of magnitude greater than the initial copy quantity can be obtained after applying thermal cycling (DNA denaturation, primer alignment and DNA copy synthesis). DSA is possible today thanks to the relative conservation along the evolutionary scale in the Birds class of the DNA-binding chromohelicase protein (CHD) located on the sex chromosomes Z (CHDZ) and W (CHDW) (WO 35
    1996039505 A1), which has allowed to find oligonucleotides characteristic of sex (US 8232382 B2) and, to a greater extent, due to its easy diagnosis by electrophoresis in agarose gels. The introns of the CHD gene vary in size depending on the sex chromosome in which they are located, so that by PCR and subsequent separation of fragments using agarose gels it can be determined if the individual is the carrier of two copies of different sizes (ZW , females) or of the same size (ZZ, males) (eg Griffiths et al. 1998; Mol Ecol 7 (8): 1071-1075) (CN 203200273 U). More recently, loop-mediated isothermal amplification (Loop-Mediated Isothermal Amplification, LAMP) (Notomi et al. 2000; Nucleic Acids Res 28 (12): E63) has been added to the set of molecular techniques for the synthesis of 10 DNA at the DSA. LAMP amplifies any region of the flanked genome by the combination of two pairs of primers that align with six specific regions. It has been applied in bird species with commercial interest (Kim et al. 2015; Turkish J Vet Anim Sci 39 (5): 583-588) although there are still no molecular markers that allow the determination of sex in other bird species of commercial interest (eg breeding 15 of exotic species) or that are useful in disciplines dedicated to the study of birds (eg ecology, conservation biology). The works published to date, in addition, lack an appropriate marker that serves as a positive control of DNA and determine the assignment of false negatives or use an mitochondrial marker inappropriate for this purpose due to a clear amplification bias in their favor. to the reduced number of copies of the nuclear genes used located in the sex chromosomes) (Chan et al. 2012; Theriogenology 78 (6): 1329–38).

    DESCRIPTION OF THE INVENTION
     25
    Every day millions of sex determinations of birds are made on which decisions with important repercussions on poultry farming and biodiversity conservation and management depend. A large majority of these reactions are based on the polymerase chain reaction (PCR) for which appropriate temperature cycling is required for denaturation, primer alignment and synthesis of new DNA copies. As a result, a number of copies of the DNA fragment under study of numerous orders of magnitude greater than the initial copy quantity is obtained by applying thermal cycling (denaturation of DNA, alignment of primers and synthesis of DNA copies). The results of these amplifications are usually interpreted by applying techniques 35
    electrophoretic in agarose gels and subsequent DNA staining by intercalating agents.
    The determination of sex in birds is possible today thanks to the amplification by PCR and subsequent separation of fragments of the DNA-binding chromohelicase protein (CHD) located on the sex chromosomes Z (CHDZ) and W (CHDW) 5 using gels agarose The introns of the CHD gene vary in size depending on the sex chromosome in which they are located, so that it can be determined if the individual is the carrier of two copies of different sizes (ZW, females) or of the same size (ZZ, males) after applying electrophoretic techniques to the amplification product. This technique was undoubtedly a revolution in the determination of 10 sex in birds, especially in monomorphic species (ie those in which males and females cannot be distinguished by any morphological or behavioral character) although it is limited to specialized laboratories equipped for both Molecular tests such as separation and visualization of diagnostic fragments in agarose gels. Consequently, molecular determination of sex in birds is only possible today away from the sampling site and makes it difficult to obtain the results until a few days after the shipment or transport of the sample.
    The molecular determination of sex based on DNA described below presents three fundamental characteristics that differentiate it, in turn, from the 20 methods based on PCR: simplicity, portability and speed. The simplicity of the invention allows the determination of sex in birds outside specialized laboratories and minimizes the possibility of error and contamination, a characteristic that makes it especially attractive both for scientists in their field work and for bird breeders. A direct consequence of the simplicity and characteristic of the technique is portability, allowing to determine sex and make experimental approaches in scenarios unthinkable to date (e.g. in the study area with the help of a car battery). The invention is further characterized by the rapidity with which sex is determined (less than 90 minutes at the sampling site itself) compared to the several hours of specialized laboratory work that amount to 30 days or weeks if time is considered of sending the sample from the sampling site to the laboratory.
    The invention is based on the loop-mediated isothermal amplification (Loop-Mediated Isothermal Amplification, LAMP) of a DNA-binding chromohelicase protein fragment located on the female sex chromosome in birds.
    (W chromosome) (CHDW) and a fragment of an ultraconserved element (ECU) in the genome of the Aves class in both males and females. This ECU is used as a positive control to identify false negatives in the CHDW marker and, therefore, minimizes the possibility of erroneously assigning sex to the individuals examined. The combination of the LAMP results for both markers will be diagnostic of the sex of the analyzed sample, with CHDW + / ECU + the result observed in females and CHDW- / ECU + that of males. The diagnosis of each LAMP reaction is made after the change or not of color (positive and negative reactions, respectively) of the added staining agent that reacts with the pyrophosphate ions released in an amount proportional to the amount of DNA synthesized. 10 Since LAMP is an isothermal reaction, only one thermoblock is needed for the incubation reaction and due to the colorimetric properties of the added staining agent no device is required to see the reaction results. The designed primers are versatile and extensible to the orders of tested birds. fifteen
    Thus, the invention relates to a method for the molecular determination of sex in birds, which comprises in a first stage the extraction and preparation of DNA by any protocol of extraction of the genetic material from the samples, followed by a stage of amplification by molecular methods of a fragment of the nucleotide sequence of the DNA-binding chromohelicase protein (CHD) gene 20 between positions 2246 and 2445 of the reference sequence of the same gene sequenced in Gallus gallus located on chromosome W (CHDW) of species of the class Birds and determinant of sex itself. In another step a reference amplification reaction of the ultraconserved element (ECU) located on chromosome 6 of the birds and present in both sexes is carried out through 25 specific primers according to sequences SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8. The determination of sex in birds is performed by comparing the results of the CHDW and UCE amplifications described above, so that a positive result of the amplifications of the specific region of CHDW and UCE is diagnosis, characteristic and determining of the females of the birds; a negative result of the amplification of the specific region of CHDW and positive of the amplification of ECU is diagnostic, characteristic and determining of the males of the birds; and a negative result of the amplification of the specific region of CHDW and negative of the amplification of UCE requires a replication of DNA extraction and / or amplification of CHDW and UCE for the determination of sex. 35
    CHDW and UCE amplifications are preferably performed using the LAMP (Loop-Mediated Isothermal Amplification) technique.
    In a preferred embodiment of the invention, amplification of the nucleotide sequence fragment of the DNA-binding chromohelicase protein (CHD) gene is performed by using a group of specific primers according to 5 sequences SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4 to determine the sex in birds.
    In another preferred embodiment, specific primers according to sequences SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 12 are used to determine the sex in birds of the order Falconiformes. 10
    The invention also relates to a kit for the determination of sex in birds, comprising at least the means and reagents necessary for the extraction, preparation, amplification of DNA by means of the LAMP technique (Loop-Mediated Isothermal Amplification) and the determination of sex by electrophoretic or fluorescence techniques, and a set of primers selected from the 15 groups of specific primers SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4 for sex determination in birds and / or SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 12 for the determination of sex in birds of the order Falconiformes.
     twenty
    DETAILED DESCRIPTION OF THE INVENTION
    The invention relates to a method of molecular determination of sex based on loop-mediated isothermal amplification (Loop-Mediated Isothermal Amplification, LAMP) which amplifies any region of the genome flanked by the combination of two pairs of primers (i.e. nucleic acid chain that serves as a starting point for DNA replication) that aligns with six specific regions. This method uses Bst polymerase, obtained from Bacillus stearothermophilus characterized by a 5’-3 ’polymerase activity and displacement activity that allows the synthesis of the new DNA chain through a double-chain autocylic displacement system. This displacement activity together with the characteristic loop structure generated as a result of the primer design synthesizes large amounts of DNA product in a single reaction and at a single temperature.
    The invention comprises the following phases:
    1. DNA extraction and preparation 35
    2. Amplification of the female specific marker (CHDW) and positive DNA control (ECU)
    3. Diagnosis of the amplification result by LAMP
    4. Determination of sex
    In an embodiment of the first phase of the invention, any protocol 5 for extracting the genetic material from the samples is required, although protocols based on DNA purification and isolation (such as those based on phenol chloroform or protocols using membrane columns) of silica) ensure a lower interaction of inhibitors with the enzyme (Bst polymerase) and higher performance of the amplification reaction. 10
    The second phase of the invention comprises the amplification by LAMP of a fragment of the DNA-binding chromohelicase protein gene located on the female specific sex chromosome (W chromosome) (CHDW) and a fragment of an ultraconserved element (ECU) located in the genome of males and females of the Birds class. The combination of the LAMP results for both markers is the diagnostic criterion of the sex of the analyzed sample.
    The female-specific marker is defined by a nucleotide sequence located between positions 2246 and 2445 of the first fragment of the gene encoding the DNA-binding chromohelicase protein (CHD1) sequenced in Gallus gallus (GenBank Acc.No. AF181826) in the W-specific sex chromosome of females of the class 20 Birds. This region has an average of 92% homology among the CHDW sequences of the total bird species available on GenBank ( http://www.ncbi.nlm.nih.gov/nuccore) and is synthesized using the specific primers designed for this purpose.
    In a preferred embodiment of the invention, primers SEQ ID 25 NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4 have been designed from the CHDW fragment sequences available in GenBank. This set of primers has been selected against other designed primers (eg the set of primers SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16 or the set of primers formed by SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19 and SEQ ID NO 20) as it does not work in any of the species of the various taxonomic orders evaluated.
    While the synthesis of this specific fragment of the CHDW region is amplified using primers SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4, it is possible to design other sets of specific primers for a given order such as primers SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 35
    and SEQ ID NO 12 that specifically amplify this same region of the CHDW fragment in species of the order Falconiformes.
    Primers to amplify the UCE marker (SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8) are designed using the sequence of the ultra-preserved element 4126 located on chromosome 6 presenting 99.9% homology when considering 5 thirty species of different orders within the superorder Neoaves (McCormack et al. 2013; PLoS One 8 (1): e54848). Said marker serves to identify false negatives in the CHDW marker and, therefore, minimizes the possibility of erroneously assigning sex to the individuals examined. Primers specific for UCE are designed using Primer Explorer v.4 software ( http://primerexplorer.jp/e/) 10 and the recommendations specified in the manual. The invention requires two LAMP reactions per individual (one for each marker described above) and the determination of sex will be done jointly considering the result of both reactions.
    The third phase of the invention (diagnosis of the result of the amplification reaction of each marker) can be carried out by two techniques that are not necessarily exclusive. On the one hand, the amplified fragments can be visualized by electrophoretic techniques. The positive amplification reactions will show the characteristic ladder pattern of the LAMP reactions (result of the concatenation of the amplification product) while the negative reactions will not show any amplified fragment except the possible primer dimers. On the other hand, the amplification results of the LAMP reactions can be visualized by adding a fluorescent agent to the test microtube where the amplification reaction was performed. Said fluorescent agent has the property of interacting with the magnesium pyrophosphate ions produced in an amount proportional to the amount of DNA synthesized. This interaction causes a color change of the fluorescent agent that indicates a positive LAMP reaction. In the event that an amplification product has not been synthesized with the specific primers, a color change in the test microtube will not occur and the LAMP reaction will be determined as negative.
    The fourth phase of the invention (sex determination) comprises the joint interpretation of the results of the specific LAMP reactions for each marker (CHDW and ECU) described in the previous phases. Individuals in which there has been an amplification reaction in the specific LAMP reactions for the CHDW (CHDW +) and UCE (UCE +) markers will be assigned as females 35
    while individuals where there is no amplification in the CHDW marker (CHDW-) but in UCE (UCE +) will be assigned as males. For those cases where the same sample is CHDW- / UCE- the analysis should be repeated to avoid erroneous assignments of males due to poor DNA quality or problems associated with DNA synthesis in LAMP (false negatives). 5

    DESCRIPTION OF THE FIGURES

    Figure 1. Gel electrophoresis with a 2.5% agarose concentration of the loop-mediated isothermal amplification (LAMP) of the fragment of the CHD gene of the 10 W chromosome of a female and a male Atlantic gannet (Sula bassana) (Sba ), peregrine falcon (Falco peregrinus) (Fpe), turquoise parakeet (Neophema pulchella) (Npu), flycatcher (Ficedula hypoleuca) (Fhy), eagle owl (Bubo bubo) (Bbu), royal swift (Apus melba) (Ame) and digger owl (Athene cunicularia) (Acu). The odd lines (1 to 13) show the characteristic ladder pattern of the amplification by 15 LAMP. Since the CHDW specific primers are designed to amplify the CHD gene fragment of the W chromosome present only in females, a positive LAMP reaction with the CHDW primers is diagnostic, characteristic and determining for females of the Aves class. The non-amplification of the samples located in the even lines (2 to 14) with the CHDW primers (once the false negatives have been ruled out, see figure 2) is diagnostic, characteristic and determining of the birds of the class Aves. The last line on the agarose gel (S) is the size marker from 100 to 1000 base pairs.

    Figure 2. Gel electrophoresis with a 2.5% agarose concentration of the loop-mediated isothermal amplification (LAMP) of the ultraconserved element UCE4126 located on chromosome 6 of the birds of a female and an Atlantic gannet male (Sula bassana) (Sba), peregrine falcon (Falco peregrinus) (Fpe), turquoise parakeet (Neophema pulchella) (Npu), flycatchers (Ficedula hypoleuca) (Fhy), eagle owl (Bubo bubo) (Bbu), royal swift (Apus melba ) (Ame) and owl 30 excavator (Athene cunicularia) (Acu) tested in Figure 1. These elements show 99.9% homology in all species of the class Birds, regardless of sex, so that all individuals Tested with the UCE primers should amplify with the LAMP technique. The UCE marker therefore serves as a positive amplification control and diagnosis of DNA quality. In 35
    Combination with Figure 1, therefore, it can be confirmed that the samples located on the odd lines (1 to 13) are female while those on the even lines (2 to 14) are male. The last line on the agarose gel (S) is the size marker from 100 to 1000 base pairs.
     5
    Figure 3. Irradiation with ultraviolet light of the fourteen microtubes with the LAMP reaction of CHDW of females (odd samples: 1 to 13) and males (even samples: 2 to 14) of the seven species of different orders of the class Birds tested (see legend of figure 1). The emitted fluorescence is the result of the interaction of pyrophosphate residues produced by the DNA synthesis reaction with the fluorescent agent Sybr Green. In this way, you can visually determine if the LAMP reaction has been positive (i.e. it emits fluorescence: samples on odd lines 1 to 13) or negative (i.e. it does not emit fluorescence: samples on even lines 2 to 14).

    Figure 4. Irradiation with ultraviolet light of the CHDW LAMP reaction performed for 15 24 individuals of flycatchers (Ficedula hypoleuca). Samples 1, 2, 3, 4, 6, 7, 13, 14 and 16 correspond to female flycatchers where amplification reaction occurs and interaction occurs between pyrophosphate residues and the fluorescent agent.
     twenty
    EXAMPLES

    EXAMPLE 1: Method of molecular determination of bird sex by loop-mediated isothermal amplification and diagnosis of results by electrophoretic and fluorescent techniques 25

    The DNA of the individual for whom it is desired to determine sex is extracted from a cell lysis with proteinase K of 10-100 µL of blood. The resulting lysate is subjected to a DNA extraction and purification protocol based on chloroform and isoamyl alcohol, the resulting DNA being resuspended in 100 µL of TLE (1 M Tris-HCl, 0.5 M EDTA) at a final concentration of 25-50 ng / L.

    Next, reagents are prepared for two reactions per sample (one to amplify CHDW and another to amplify UCE). For each reaction, 0.4 mM dNTP, 1-2 M betaine, 8 units of Bst, 1x buffer (20 mM Tris-HCl, 10 mM 35 are added)
    (NH4) 2SO4, 50 mM KCl, 2 mM MgSO4, 0.1% Tween® 20, pH 8.8), 1-2 M of the internal primers (FIP-BIP; CHDW: SEQ ID NO 3 and SEQ ID NO 4; UCE: SEQ ID NO 7 and SEQ ID NO 8) and a concentration between 5 and 10 times lower of the external primers (F3-B3; CHDW: SEQ ID NO 1 and SEQ ID NO 2; UCE: SEQ ID NO 5 and SEQ ID NO 6), 2 L of the DNA and H2O extract to complete 25 L in microtubes. These are incubated at the temperatures specified in table 1 in a thermal cycler for 80 minutes. The amplification results are visualized in an ultraviolet light transilluminator after separating by means of electrophoretic techniques 5  of product in an agarose gel stained with Sybr Safe (ThermoFisher Scientific). The females show the characteristic ladder pattern of the LAMP reactions in the reactions made with the specific set of primers to amplify CHDW and UCE while the males will only show a similar pattern in the reactions that amplify UCE (Figures 1 and 2) . To check the amplification results by fluorescence, 5 L of Sybr Green I Nucleic Acid Stain (Life Technologies) is added to each microtube and irradiated with a portable ultraviolet light lamp. The microtubes 15 that show fluorescence will be positive, while the absence thereof will denote lack of amplification (Figure 3).

    TABLE 1
    Relationship of incubation temperatures for amplification by LAMP of the 20 CHDW and UCE markers for seven species belonging to six different orders of the Bird class.

     Order  Family Species Common name CHDW (ºC) UCE (ºC)
     Suliformes  Sulidae Sula bassana Alcatraz Atlantic 51 59-61
     Falconiform  Falconidae Falco peregrinus Peregrine Falcon 53 61
     Psittaciformes  Psittacidae Neophema pulchella Turquoise parakeet 59 61
     Passeriformes  Muscicapidae Ficedula hypoleuca Flycatcher 59 57
     Strigiformes  Strigidae Bubo bubo Eagle owl 55 59
     Apodiform  Apodidae Apus melba Royal Swift 55 59
     Strigiformes  Strigidae Athene cunicularia Excavator owl 59-61 57-63

     25
    EXAMPLE 2: Amplification of the CHDW fragment by loop-mediated isothermal amplification applied to DNA extracts obtained with NaOH and diagnosis of results using fluorescent agents.

    1. Extraction and preparation of DNA. 5
    100mM NaOH is added to a sample of between 10-100 µL of blood from 24 individuals of flycatchers (Ficedula hypoleuca) and boiled at 100 ° C for 10 minutes. The extract obtained is allowed to cool to room temperature and a 1: 100 dilution is made.
    2. Amplification of the specific marker of females (CHDW). 10
    On the one hand, i) lyophilized primers (or evaporated using a vacuum pump) are prepared in the same microtube at 1.6 mM concentrations (FIP, BIP; SEQ ID NO 3 and SEQ ID NO 4) and 0.2 mM (F3 , B3; SEQ ID NO 1 and SEQ ID NO 2), and, on the other hand, ii) 10x buffer composed of 750 mM KCl, 35 mM MgCl2 and Tris (pH = 8.3) 100 mM. The mixture of reagents that will be prepared for each reaction will be done in microtubes and 15 for a total of 24 samples so that each reaction should have a final composition of 1x buffer, 0.4 mM dNTP, 1-2 M betaine, sucrose 2 -10% and 8 units of Bst. The lyophilized primers are rehydrated with a volume of H2O equal to the starting one (prior to lyophilization) and added to the reagent mixture. Finally, 23 µL is dispensed to each microtube where 20 LAMP amplification will be performed and 2 µL of the 1: 100 dilution of the DNA extract is added. The incubation of the reactions is carried out at 55-70 ° C for 60 to 90 minutes in a thermoblock.
    3. Diagnosis of the amplification result by LAMP.
    Once the incubation is complete, 5 L of Sybr Green I Nucleic Acid Stain (Life Technologies) is added and irradiated with a portable ultraviolet light lamp. The 25 microtubes that irradiate fluorescence will indicate a positive amplification reaction and will be female (because there is interaction between pyrophosphate residues and the fluorescent agent), while the absence of it will denote lack of amplification and, therefore, will be assigned as males (figure 4).
    30
    权利要求:
    Claims (4)
    [1]
    1. Method for the molecular determination of sex in birds, comprising:
    i. DNA extraction and preparation using any protocol for extracting genetic material from samples.
    ii. amplification by means of the LAMP (Loop-Mediated Isothermal 5 Amplification) technique of a fragment of the nucleotide sequence of the DNA-binding chromohelicase protein (CHD) gene between positions 2246 and 2445 of the reference sequence of the same sequenced gene in Gallus gallus located on the W chromosome (CHDW) of species of the Bird class and sex-determining itself; and 10
    iii. a reference amplification reaction using the LAMP (Loop-Mediated Isothermal Amplification) technique of the ultraconserved element (ECU) located on chromosome 6 of the birds and present in both sexes through specific primers according to sequences SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8; where 15
    to. a positive result of the amplifications of the specific region of CHDW and of the ECU is diagnostic, characteristic and determining of the females of the birds;
    b. a negative result of the amplification of the specific region of CHDW and positive of the amplification of ECU is diagnostic, characteristic and determining of the birds' males;
    C. a negative result of the amplification of the specific region of CHDW and negative of the amplification of UCE requires a replication of DNA extraction and / or amplification of CHDW and UCE for the determination of sex
    25

    [2]
    2. Method according to claim 1, characterized in that the amplification of the nucleotide sequence fragment of the DNA-binding chromohelicase protein (CHD) gene is performed by using the specific primers SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4 for 30 sex determination in birds.
    [3]
    3. Method according to claim 1, characterized in that the amplification of the nucleotide sequence fragment of the DNA-binding chromohelicase protein (CHD) gene is performed by using the primers
    specific SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 12 for the determination of sex in birds of the order Falconiformes.

    [4]
    4. Kit for the determination of sex in birds by a method as defined in claims 1 to 3, comprising at least:
    i. media and reagents necessary for the extraction, preparation, amplification of DNA by means of the LAMP technique (Loop-Mediated Isothermal Amplification) and the determination of sex by electrophoretic or fluorescence techniques; Y
    ii. a set of primers selected from the groups of specific primers 10:
    to. SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4 for sex determination in birds; I
    b. SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 12 for the determination of sex in birds of the order Falconiformes. fifteen
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    ES2647134B1|2018-10-15|
    WO2017198889A1|2017-11-23|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    TWI326712B|2007-09-26|2010-07-01|Univ Kaohsiung Medical|Method for eagles gender identification and sequence for eagles gender identification|
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    PCT/ES2017/070331| WO2017198889A1|2016-05-19|2017-05-18|Method for the molecular determination of the sex of birds by means of loop-mediated isothermal amplification|
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