WO2024005656A1 - Kit de sexage précoce non invasif par pcr en temps réel pour paiche (arapaima gigas) - Google Patents

Kit de sexage précoce non invasif par pcr en temps réel pour paiche (arapaima gigas) Download PDF

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Publication number
WO2024005656A1
WO2024005656A1 PCT/PE2022/050016 PE2022050016W WO2024005656A1 WO 2024005656 A1 WO2024005656 A1 WO 2024005656A1 PE 2022050016 W PE2022050016 W PE 2022050016W WO 2024005656 A1 WO2024005656 A1 WO 2024005656A1
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msr
real
paiche
gigas
time pcr
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PCT/PE2022/050016
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English (en)
Spanish (es)
Inventor
Eliana Victora ZELADA MÁZMELA
Juan Ignacio FERNANDINO
Tomás Horacio DELGADIN
Edgar Alfonso LOPEZ LANDAVERY
Alan MARIN GUERRERO
Carmen Gabriela YZASIGA BARRERA
Lorenzo Eduardo REYES FLORES
Luis Enrique SANTOS ROJAS
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Universidad Nacional Del Santa
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Universidad Nacional Del Santa
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Priority to CONC2024/0018107A priority Critical patent/CO2024018107A2/es
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms

Definitions

  • the present invention is framed in the technical field of biotechnology and the pharmaceutical industry for continental aquaculture for the identification of sex in paiche (Arapaima gigas), mainly in the field of reproduction and cultivation of this species.
  • document CN104808005 is known, which describes a method to identify male giant grouper (Epinephelus lanceolatus).
  • the method includes the steps of selection of the giant grouper in the breeding season; obtain serum from it: select blood from the caudal vein of the giant grouper and perform centrifugation to obtain the serum; measure the content of vitellogenin in serum by ELISA (enzyme-linked immunosorbent assay); measure the content of 1 1 -KT (11 - ketotestosterone) in serum with ELISA; and identification of male giant grouper in combination with vitellogenin content and 11-KT content in the giant grouper serum.
  • the method is fast, efficient and highly accurate and does not cause damage to the specimen.
  • the size of 5S rRNA is between 130 and 150 nucleotides.
  • the maximum value of microcapillary electrophoresis is usually close to 140 nt, and the value of total RNA refers to the size of the area generated by all areas covered.
  • fish gonadal tissue can be sampled non-lethally by dissection or with a micro gonadal (or oviductor) sampling tool and a 1 mg to 100 mg section of micro gonadal tissue is taken.
  • patent document EP3620536 provides a set of epigenetic biomarkers useful for predicting sex in fish, said epigenetic biomarkers being specific cytosine-guanine dinucleotides (CpG) that are differentially methylated in males versus females.
  • CpG cytosine-guanine dinucleotides
  • Patent CN108998545 refers to the application of p ⁇ R-mmu- 31018127 of Cynoglossus semilaevis.
  • the sex tag piR-mmu-31018127 is derived from an exosome from the fish seminal plasma.
  • RNA sequencing analysis a significantly different in expression in two types of fish through screening, adopting as a candidate a piRNA biomarker that has an indicative function for the two types of fish. It is finally determined through verification by adopting quantitative measurements in real-time PCR (qPCR).
  • the tag piRNA is p ⁇ R-mmu-31018127 with a sequence of GCATGTGGTTCAGTGGTAGAATTCTCGCCG, and a kit based on the tag is developed.
  • the provided method is non-invasive and efficient with reliable identification result and the genetic sex of Cynoglossus semilaevis is determined by quantitative determination.
  • Patent document WO14129982 reports sex determination systems in fish using a convenient and precise sexing method and kit that can be used in red arawanas to establish the optimal sex ratio of the reproductive populations to be used during breeding. in captivity for conservation and production purposes, wherein the method and the kit comprises a primer having an oligonucleotide sequence of 5' TAACTCAAAAGTAGAATAGAACAATG 3' and a second primer having an oligonucleotide sequence of 5' AATTCAAGGGAACTGATGACTCTA 3' to identify the sex in one or more genomic DNA samples that can be isolated from a DNA source, such as muscle, gills, fins, mucus, feces or a combination of one or more thereof.
  • a DNA source such as muscle, gills, fins, mucus, feces or a combination of one or more thereof.
  • document MX2013001464 reports a method for obtaining a reactive strip for the recognition of the sex of lepisosteids that includes the purification of vitellogenin from plasma of specimens of males induced with estradiol, production, purification and labeling of antibodies and preparation of reagent strips for sexing other species.
  • Nitrocellulose or nylon strips with vitellogenin immobilized on their surface are used for sexing fish species that do not present sexual dimorphism, mainly lepisosteids, both in the laboratory and in the field.
  • Patent ES239881 1 reports a method for identifying sex in fish through the use of a molecule associated with 5s ribosomal RNA (5s rRNA), or a molecule involved in the transport of proteins that are involved in the regulation of transcription of the gene that encodes 5s RNA as a marker to identify sex in fish.
  • the invention also relates to a method for sexing a fish population comprising determining the expression level of a molecule associated with 5s rRNA, or a molecule involved in the transport of proteins involved in the regulation of gene transcription. which encodes 5s rRNA.
  • the specific primers are from the 5S rRNA gene, Gen Bank accession number AM706461 fw (SEQ ID NO: 1): 5'-CTTACGGCCATACCACCCTG 3' (SEQ ID NO: 2) and Rv 5'- GTATCCCAGGCGGTCTCC-3' (SEQ ID NO: 3) using qPCR reactions with complementary DNA (cDNA) at a concentration of 5 ng/pl.
  • document US5480774 provides a method to determine the genomic sex of a salmonid by detecting the presence or absence of the growth hormone pseudogene GH-PSI.
  • the pseudogene is detected by amplification of a specific selected subsequence of the pseudogene, or by duplex formation of a nucleic acid that hybridizes specifically with the pseudogene and with no other gene in the genome of the salmonid species.
  • the method is useful in several salmonids, particularly Oncorhynchus tshawytscha and Oncorhynchus kisutch.
  • a male-specific gene expression was found in the secretory organ, which assigns both a fry nutrition function and also a pheromone-like signaling function for local females.
  • a possible genetic system that determines the sex of male heterogeneity in this species that has homomorphic chromosomes for both sexes is inferred from genomic data.
  • RNA samples were then analyzed on a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), using the RNA 6000 Pico kit (Agilent Technologies).
  • double-stranded cDNA was obtained, using the cDNA Synthesis System kit (Roche), with the following four steps: denaturation of the RNA, synthesis of the second cDNA strand and purification of the double cDNA strands.
  • the irregular ends of the cDNA fragments generated by this process were repaired and adapters were incorporated into both ends of the cDNA strands.
  • cDNA fragments were quantified using a TBS 380 QuantiFluor fluorometer (Promega, USA). The size of the cDNA fragments was checked on the bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) using a high-sensitivity DNA chip.
  • MSR Male Specific Region
  • kits and a method for early sex determination of the paiche that can be applied at any stage of the life cycle of this species
  • the kit and method is a rapid, economical, sensitive and efficient molecular method using non-invasive sampling
  • the kit and method provide a notable improvement over current methods for sex identification in A. gigas and offer advantages mainly over known methods in relation to the speed and determination of the sex of the animal at any stage of its development. Lifecycle.
  • the present invention is directed to a kit and a non-invasive method for early termination of sex in real time for paiche (Arapaima gigas) that is based on real-time polymerase chain reaction (qPCR) to the non-invasive identification of the genetic sex of specimens of any age of Arapaima gigas, from the genomic DNA extracted from samples of mucus present in its gills or a small segment of its caudal fin, through its interaction with two pairs of primers (oligonucleotides ) developed in this invention for this purpose and designed on a male-specific genome region (MSR) and a set of primers designed to amplify the TATA-Box Binding Protein Like 1 gene (TBPL1_205) that acts as a control or housekeeping gene (HKG). ) of the technique.
  • qPCR real-time polymerase chain reaction
  • Figure 1 shows the dissociation curves obtained from the amplicons generated by molecular method according to the present invention through the real-time PCR assay for the peaks generated with the male-specific region marker MSR_107 and the reference gene TBPL1 in paiche Arapaima gigas where a sample with the sex-specific primers MSR_107 and the endogenous control TBPL1_205.
  • Figure 2 shows the dissociation curves obtained from the amplicons generated by molecular method according to the present invention through the real-time PCR assay for the peaks generated with the male-specific region marker MSR_129 and the reference gene TBPL1 in paiche arapaima gigas, where a sample is contacted with the sex-specific primers MSR_129 and endogenous control TBPL_205.
  • the presence of the dissociation curve for the MSR_107 and MSR_129 primers determines the male sex of the analyzed individual (first peak from left to right in Figure 2) and in turn the absence of this dissociation curve indicates the female sex of the analyzed sample. .
  • FIG. 3 shows the agarose gel for PCR products from male-specific regions. It is shown that the males (M) present two bands: one belonging to the male-specific region MSR_107 and the other band belonging to TBPL1_205, while the female (FM) only presents the band belonging to TBPL1_205.
  • FIG. 4 shows the agarose gel for PCR products from male-specific regions. It is shown that males (M) present two bands: one belonging to the male-specific region MSR_129 and the other band belonging to TBPL1_205, while the female only presents the band belonging to TBPL1_205.
  • Figure 5 shows the regions in A. gigas from which tissue was collected for DNA extraction.
  • the present invention refers to a non-invasive sexing kit using real-time PCR for paiche (Arapaima gigas).
  • a non-invasive sexing kit using real-time PCR for paiche (Arapaima gigas).
  • qPCR real-time polymerase chain reaction
  • MSR Male-Specific Genome Region
  • TBPL1 TATA-Box Binding Protein Like 1
  • the non-invasive early sexing kit by real-time PCR for paiche (Arapaima gigas) of the present invention allows the identification of A. gigas males by detecting the MSR using one of the direct MSR_107 primer pairs (TGGAAATCAGGGTGAAACTGT) and MSR_107 reverse (GTCGTCCCTAAGCTGGTTATTATC) or MSR_129 forward
  • CCTGTAATACACCTATACCATCC TATA-Box Binding Protein like 1
  • HKG TATA-Box Binding Protein like 1
  • Females are identified by the absence of the peaks at 76.6°C/77.5°C in the dissociation curve and the presence of the peak at 84.7°C of the HKG.
  • the non-invasive early sexing kit by real-time PCR of the paiche also includes sterile swabs, lysis buffer solution, aqueous NaOH solution for the extraction of genomic DNA and a master mix that It includes the reagents necessary for the qPCR assay which are buffer solution, enzyme, nucleotides, sex-specific primers, a positive control and a negative control.
  • the kit comprises one of the primer pairs MSR_107 forward (TGGAAATCAGGGTGAAACTGT) and MSR_107 reverse (GTCGTCCCTAAGCTGGTTATTATC) or direct MSR.129
  • the kit may further include other desirable materials/components including lancets, buffer solutions, e.g., Sample Buffer, diluents, filters, capillaries, needles and/or syringes, among others.
  • buffer solutions e.g., Sample Buffer, diluents, filters, capillaries, needles and/or syringes, among others.
  • the present invention refers to a non-invasive method of early sex determination using POR in real time for paiche (Arapaima gigas), which comprises the following steps: a) Providing a mucus sample of Arapaima gigas gills or a segment of the caudal fin; b) Extract genomic DNA and incubate in a thermal block; c) Prepare and perform qPCR by adding the genomic DNA extracted in step b) to a solution A and add a premix of one of the pairs of MSR107 or MSR 129 primers and HKG primers and do the reaction; and d) Analyze the dissociation curves.
  • step a) the sample is carried out by collecting a mucus sample using a swab from the gills of a specimen of A. gigas, which is then immersed in 1 ml of lysis buffer solution. In the stages of fry, juveniles or when it is not possible to collect mucus, a segment of between 2 mm and 4 mm maximum section of the caudal fin is cut and immersed in 1 ml of 96% ethanol.
  • step b) the extraction of genomic DNA is carried out by adding 180 ⁇ l of 50 mM NaOH and 0.2 mM EDTA to the sample in a lysis buffer solution, incubating in a thermal block for 5 minutes at 95°C. , to which 180 pl of 300 mM Tris-HCI pH 8.0 is then added.
  • step c) the preparation and performance of real-time PCR (qPCR) is carried out by adding 2pl of genomic DNA extracted in step b) to a solution A (1 Opl), which comprises the enzyme Taq-polymerase, the SYBR Green intercalating fluorophore of chemical formula C32H37N4S, dNTPs (deoxyrhbonucleotides triphosphate), buffer solution and Mg 2+ .
  • a solution A (1 Opl), which comprises the enzyme Taq-polymerase, the SYBR Green intercalating fluorophore of chemical formula C32H37N4S, dNTPs (deoxyrhbonucleotides triphosphate), buffer solution and Mg 2+ .
  • 1.5pl (0.1 uM) of a premix of one of the MSR107 or MSR 129 primer pairs and 1.5pl (0.05 uM) of the HKG primers are added.
  • the reaction tubes are placed in the thermal cycler equipment, which must have specific filters to detect the emission of the fluorophore used.
  • the reaction is started with 5 minutes at 95°C.
  • 35 amplification cycles are carried out, each cycle consisting of 15 seconds at 95°C and 60 seconds at 62°C.
  • the dissociation curve is carried out by increasing the reaction temperature from 60°C to 95° with increasing steps of 2.2°C/s.
  • step d) the analysis is carried out by evaluating the presence of the peaks corresponding to the melting temperatures (Tm) of the amplification products generated by the MSR107/MSR129 primer pairs as appropriate. , and the pair of HKG control primers.
  • Tm melting temperatures
  • the presence of two peaks at the corresponding Tm indicates that the specimen is male ( Figure 1).
  • the presence of a single peak, corresponding to the HKG, indicates a female specimen ( Figure 2). If there is no peak corresponding to HKG, the identification is indeterminate and the reaction must be repeated.
  • the non-invasive method of early sex determination by real-time PCR for paiche is implemented by real-time polymerase chain reaction on genomic DNA extracted from samples of mucus present in the gills of the animal or a segment of its caudal fin or any other tissue of the animal, through the step of putting said sample in contact with a pair of sex-specific primers that hybridize and amplify a specific genomic region of males ( MSR) and a pair of primers that hybridize and amplify a region present in males and females (HKG) and in this way with both pairs of primers present in the reaction tube and after completion of the reaction it is identified as male if the MSR amplification product and HKG are detected or females if only the presence of HKG is verified.
  • MSR sex-specific primers that hybridize and amplify a specific genomic region of males
  • HKG pair of primers that hybridize and amplify a region present in males and females
  • the non-invasive method of early sex determination by real-time POR for paiche comprises in step c) the sequences of the direct sex-specific primer pairs MSR_107 :
  • the non-invasive method of early sex determination by real-time PCR for paiche comprises in step c) the sequence of primer pairs to amplify the gene present in males and females.
  • TATA-Box Binding Protein like 1 HKG: forward TBPL1 (CTCTGGCTTGTAGATGACATTGG) and reverse TBPL1 (TTTGTTGCGTGTCTCTGTGG), which has a dissociation temperature (Tm) of 84.7°C.
  • the amplification products of the primers indicated above can also be differentiated by electrophoresis in agarose gel, polyacrylamide gel or a mixture of both.
  • the method according to the present invention in step c) has a characteristic qPCR temperature profile which comprises initial 5 minutes at 95°C, followed by 35 cycles of 15 seconds at 95°C and 60 seconds to 62°C, followed by the dissociation curve profile comprising the stepwise increase of temperatures of 2.2°C from 60°C to 95°C.
  • the present invention refers to the use of the non-invasive sexing kit using real-time PCR for paiche (Arapaima gigas) through the identification of A. gigas males by detecting the MSR using one of the forward MSR_107 primer pairs
  • the sex-specific primers that participate in the kit and in the method according to the present invention were synthesized by order of the company Integrated DNA Technologies (IDT, USA).
  • IDTT Integrated DNA Technologies
  • the LightCycler 480 SYBR-Green I Master kit (Roche) was used.
  • the final volume of the reactions was 20pl and each reaction consists of: 1 Opl of the master mix (master mix) LightCycler 480 SYBR-Green I Master (2X), 0.1 pM of the sex-specific primers MSR107, 0.05 pM of the primers endogenous control TBPL1 and 30ng of total genomic DNA, the difference in volume was made up using water for PCR.
  • a LightCycler 480 II real-time thermocycler (Roche) was used with an initial denaturation step at 95°C for 5 minutes and 35 amplification cycles consisting of denaturation at 95°C for 15 seconds followed by hybridization/extension at 62°C for 60 seconds.
  • the creation of the dissociation curves was carried out in the same LightCycler 480 II equipment, which consists of gradually raising the temperature from 60°C to 95°C with a temperature ramp of 2.2°C/ s.

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Abstract

La présente invention concerne un kit de sexage précoce non invasif par PCR en temps réel pour déterminer le sexe de paiche (Arapaima gigas) à n'importe quel âge qui comprend une des paires d'amorces MSR_107 directe (TGGAAATCAGGGTGAAACTGT) et MSR_107 inverse (GTCGTCCCTAAGCTGGTTATTATC) ou MSR_129 directe (GATAATAACCAGCTTAGGGACGAC) y MSR_129 inverse (CGCTGTAATACACCTATACCATCC) et une méthode non invasive de détermination du sexe par PCR en temps réel pour paiche (Arapaima gigas), qui comprend les étapes de fourniture d'un échantillon de mucus ou un segment de nageoire caudale, d'extraction d'ADN génomique, de préparation et de réalisation de qPCR avec un prémélange de paires d'amorces MSR107 ou MSR 129 et d'amorces du HKG et d'analyse de la courbe de dissociation pour chercher la présence des pics correspondants aux températures de dissociation (Tm) des produits amplifiés.
PCT/PE2022/050016 2022-06-27 2022-08-25 Kit de sexage précoce non invasif par pcr en temps réel pour paiche (arapaima gigas) Ceased WO2024005656A1 (fr)

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Application Number Priority Date Filing Date Title
CONC2024/0018107A CO2024018107A2 (es) 2022-06-27 2024-12-27 Kit de sexado temprano no invasivo mediante pcr en tiempo real para paiche (arapaima gigas)

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PE2022001334A PE20240047A1 (es) 2022-06-27 2022-06-27 Kit de sexado temprano no invasivo mediante pcr en tiempo real para paiche (arapaima gigas)
PE001334-2022/DIN 2022-06-27

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Cited By (1)

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CN119592703A (zh) * 2024-12-09 2025-03-11 中国海关科学技术研究中心 鉴别巨骨舌鱼的引物、探针、试剂盒、方法及应用

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ADOLFI MATEUS C., DU KANG, KNEITZ SUSANNE, CABAU CÉDRIC, ZAHM MARGOT, KLOPP CHRISTOPHE, FERON ROMAIN, PAIXÃO RÔMULO V., VARELA EDU: "A duplicated copy of id2b is an unusual sex-determining candidate gene on the Y chromosome of arapaima (Arapaima gigas)", SCIENTIFIC REPORTS, NATURE PUBLISHING GROUP, US, vol. 11, no. 1, US , XP093125756, ISSN: 2045-2322, DOI: 10.1038/s41598-021-01066-z *
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN119592703A (zh) * 2024-12-09 2025-03-11 中国海关科学技术研究中心 鉴别巨骨舌鱼的引物、探针、试剂盒、方法及应用

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