WO2017194808A1 - Procédé pour la détection, la quantification et/ou l'identification d'aeromonas salmonicida spp. salmonicida - Google Patents

Procédé pour la détection, la quantification et/ou l'identification d'aeromonas salmonicida spp. salmonicida Download PDF

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Publication number
WO2017194808A1
WO2017194808A1 PCT/ES2017/070293 ES2017070293W WO2017194808A1 WO 2017194808 A1 WO2017194808 A1 WO 2017194808A1 ES 2017070293 W ES2017070293 W ES 2017070293W WO 2017194808 A1 WO2017194808 A1 WO 2017194808A1
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spp
salmonicida
salmonicide
aeromonas
time pcr
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Spanish (es)
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Clara FERNÁNDEZ ÁLVAREZ
Santiago Fernández González
Ysabel Santos Rodríguez
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Universidade de Santiago de Compostela
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Universidade de Santiago de Compostela
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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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • 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

Definitions

  • the present invention relates to method and kit for use in the specific detection of Aeromonas salmonicida spp. Salmonicide in bacterial cultures, pure and mixed, and in fish tissue samples, using a polymerase chain reaction (PCR) procedure in real time with a fluorochrome.
  • PCR polymerase chain reaction
  • the procedure is applicable in the aquaculture sector for the control of infections in salmonid and non-salmonid fish, as well as for the detection of the pathogen in natural populations.
  • Furunculosis is a bacterial disease caused by a Gram-negative bacterium called Aeromonas salmonicida spp. salmonicide or also known as typical A. salmonicide.
  • Aeromonas salmonicida spp. salmonicide or also known as typical A. salmonicide.
  • the disease has a wide geographical distribution and affects salmonids and non-salmonids of fresh and marine water, both in natural populations and in production farms (Austin and Austin, 2007).
  • Furunculosis is a systemic disease with acute, chronic and subclinical manifestations, which causes large economic losses in the aquaculture sector worldwide (Austin and Austin, 2007).
  • other subspecies of A. salmonicida have been described, such as Aeromonas salmonicida spp.
  • the diagnosis of the disease is usually based on the isolation of the pathogen, from the tissues of the diseased fish, and its identification by conventional microbiological methods that involve the isolation of the pathogen and its identification by morphological, physiological tests. and biochemistry, using classical methods or multi-characterization test systems (Santos et al., 1993. Aquaculture. 116: 111-120). Phenotypic, serological and molecular studies have shown that Aeromonas salmonicida spp. Salmonicide constitutes a homogeneous group, unlike the other subspecies known as atypical Aeromonas salmonicida (A. salmonicida spp. achromogenes, A. salmonicida spp. masoucida). However, these methods present
  • the identification of A. salmonicide and its detection in diseased fish tissues can also be performed using methods that do not require isolation of the microorganism in pure culture such as the ELISA technique (Hiney et al., 1994. Dis. Aquat. Organ. 19 : 161-167), conventional PCR (Hiney et al., 1992. Appl. Environ. Microbiol. 58: 1039-1042; Gustafson et al., 1992. Appl. Environ. Microbiol. 58: 3816-3825; Miyata et al. ., 1996. Aquaculture, 141: 13-24), isothermal nucleic acid amplification (LAMP) (Kulkarni et al., 2009. J Rapid Methods Autom Microbiol.
  • LAMP isothermal nucleic acid amplification
  • Real-time PCR is a sensitive technique that allows quantification of the products generated in the PCR reaction, by fluorescence detection of the generated product (Higuchi et al., 1993. Biotechnology 1 1, 1026-1030). This technique has been widely used for the detection, quantification and identification of Gram-negative pathogenic bacteria that infect fish including A. salmonicida (Balcázar et al., 2007. J. Med. Microbiol., 56: 323-328; Keeling, 2013. J. Fish Dis. 36: 495-503). But none of these procedures allows differentiating between the subspecies of A. salmonicida.
  • Miyata et al. described a pair of primers using as a target a 512 base pair fragment (bp), obtained after random genome amplification (RAPD) of the reference strain ATCC14174 of A. salmonicida spp. salmonicide, using arbitrary sequences of synthetic DNA as reaction primers.
  • the conventional PCR method described by these researchers allows the differentiation of A. salmonicida ssp. salmonicide of the atypical subspecies masoucida and achromogenes.
  • this method is slow, tedious and expensive to require the extraction of DNA from bacterial cultures and the analysis of amplification products by electrophoresis.
  • the present invention relates to a method and kit for the detection, quantification and / or identification of Aeromonas salmonicida spp. salmonicide and, more specifically, for the detection, quantification and / or identification of specific DNA of Aeromonas salmonicida spp. salmonicide either from bacterial cultures, pure or mixed, or from fish tissue samples.
  • the invention is based on the amplification and quantification of DNA by real-time PCR.
  • the proposed method comprises the amplification of a 1 19 bp fragment belonging to a gene encoding an effector protein with serine / threonine kinase activity of the type III secretion system specific to Aeromonas salmonicida spp.
  • Salmonicide Said amplification includes SYBR Green fluorochrome which allows the detection of amplification products.
  • the continuous reading of the fluorescence during the process allows the pathogen to be identified based on the specific fluorescence peak. This procedure does not amplify the DNA of other subspecies of Aeromonas salmonicida, nor the DNA of host fish, being the primers designed highly specific and informative.
  • Aeromonas salmonicida spp. salmonicide from bacterial cultures, pure or mixed, and tissues as well as, their differentiation from the subspecies Aeromonas salmonicida spp. achromogenes and Aeromonas salmonicida spp. masoucida, main causative agents of atypical furunculosis. - The cost and time necessary for the diagnosis of furunculosis is reduced.
  • the detection of PCR products is fast, easy and objective, allowing an easy and fast diagnosis of the infection. It does not require the use of electrophoresis in agarose gels, ultraviolet light, or the use of toxic agents for the detection of the products obtained.
  • a first object of the present invention is a method for the detection, quantification and / or identification of Aeromonas salmonicida spp. Salmonicide in samples of bacterial, pure and mixed cultures, and in fish tissues comprising the following stages:
  • DNA from a bacterial culture, pure or mixed, or from fish tissues b) Perform a real-time PCR assay on the samples from stage a) using a pair of primers specific for Aeromonas salmonicida spp. Salmonicide identified by the sequences SEQ ID NO 1 and SEQ ID NO 2 and a fluorochrome. c) Detect, quantify and / or identify the amplified nucleotide sequences as a result of real-time PCR.
  • An embodiment of this invention is the process of the invention, where primers having an identity of 75% with respect to the primers identified by the sequences SEQ ID NO 1 and SEQ ID NO 2 are used.
  • a preferred embodiment is a process of the invention. where said identity is 85%.
  • a more preferred embodiment is a process of the invention where said identity is 95% and an even more preferred embodiment is a process of the invention where said identity is 99%.
  • the percentage of identity in a given sequence is calculated taking into account that 99% identity means that 99% residues of the complete sequence of the primers identified by the sequences SEQ ID NO 1 and SEQ ID NO 2 are identical to the residues of the determined sequence.
  • Another embodiment of the invention is a kit for detecting, quantifying and / or identifying Aeromonas salmonicida spp. Salmonicide in a real-time PCR assay, comprising:
  • the positive control is present in the kit to determine the correct functioning of the kit components.
  • a preferred embodiment is the kit of the invention where the fluorochrome is SYBR Green.
  • a preferred embodiment is the kit of the invention where the positive control is A. salmonicida spp. Salmonicide of the reference strain ATCC33658.
  • Another preferred embodiment is a kit of the invention wherein said deoxynucleotides triphosphate are dATP, dCTP, dGTP and dTTP at a final concentration 200 ⁇ .
  • reaction buffer is 50 mM potassium chloride, 10 mM TrisHCI pH 9.0 at room temperature, 2.5 mM MgCl 2, 20mM (NH4) 2 S0 4, 2 , 5 units of polymerase per 25 ⁇ _ reaction.
  • Figure 1 Dissociation curve of amplification products obtained using bacterial suspensions of A. salmonicida spp. Salmonicide showing a melting temperature value of 80.5-81.0 ° C, specific for the amplified 1 19 bp fragment.
  • the melting temperature is indicated on the abscissa axis and the derivative of the relative fluorescence units on the ordinate axis.
  • Specific primers were designed based on the 3735 bp sequence of the aopO gene of A. salmonicida spp. Salmonicide A449, located in plasmid pAsa5 (GenBank Accession no.DQ386862.1) which codes for a serine protein / threonine kinase type III of the virulence-linked secretion system (Dacanay et al., 2006).
  • the Pick Primer and Primer-BLAST tools of the Information Service of the National Biotechnology Center were used.
  • Bacterial infection was performed through an intraperitoneal injection to juvenile rainbow trout (Oncorhynchus mykiss) (10.0 ⁇ 0.1 grams) with a bacterial suspension of A salmonicide SK181 / 12, SK164 / 12, MT416 and T096 7.1, following the methodology previously described (Santos et al., 1991.J Appl Ichthyol 7, 160-167).
  • the fish were anesthetized by immersion in tricaine methane sulphonate (MS-222, Sigma) (60mgL "1 , Neiffer & Stamper, 2009) and injected intraperitoneally 0, 1 mL of bacterial suspension containing between 10 6 and 10 9 cells / mL (10 fish per dose) During the experiment, the fish were kept in a 100 liter tank with aerated fresh water. The dying fish were sacrificed by an overdose of anesthetics and blood samples were extracted aseptically, spleen and kidney for analysis using culture-based methods and bacterial characterization and real-time PCR analysis with SYBR Green.
  • bacterial suspensions were prepared in sterile water free of nucleases adjusted to an approximate final concentration of between 3 x 10 9 and 3 x 10 12 cells / mL (tube 7 of the MacFarland scale) .
  • the number of colony forming units (CFU) was determined by the plate counting method, using Tryptic Soy agar medium supplemented with 1% NaCl (TSA-1) and the colonies of bacteria produced were counted.
  • tissues From healthy fish (sacrificed by anesthetic overdose), tissues (kidney, spleen and blood) were obtained, homogenized with saline solution at a final concentration of 25% weight / volume, were inoculated with an equal volume of bacterial suspensions of the reference strain of Aeromonas salmonicida spp. Salmonicide ATCC33658 containing between 1.5 x 10 9 and 1.5 x 10 2 cells / mL and incubated for one hour at room temperature. The number of colony forming units (CFU) was determined by the plate count method. Homogenized non-inoculated tissues were also prepared for use as a negative control. The homogenates were used in the PCR reaction with or without prior DNA extraction.
  • CFU colony forming units
  • tissues of 40 cultured rainbow trout (10g ⁇ 1g) and 40 turbot (45g ⁇ 5g) with and without clinical symptoms of furunculosis and 20 turbot with symptoms of tenacibaculosis were processed as described above. of being used in real-time PCR assays and microbiological methods.
  • DNA was obtained from tissue homogenates using Dynabeads® DNA Direct TM Universal (Dynal).
  • the DNA obtained from bacterial cultures and tissue homogenates were stored at -20 ° C until use.
  • Example 4 Amplification of DNA fragments from pure and mixed cultures.
  • reaction mixture was used in the amplification: 12.5 pL of SYBR Green qPCR Master Mix Maximum (2x), non ROX, (Thermo Scientifics), 1 pL of each primer (SEQ ID NO 1 and SEQ ID NO 2) (400 nmoles), 5 ⁇ _ of the problem bacterial suspension or 2 ⁇ _ of purified bacterial DNA.
  • the reaction volume was completed with sterile distilled water to 25 ⁇ _.
  • the reaction mixture was subjected to amplification in a MiniOpticon thermocycler for real-time PCR, with the CFX Manager TM detection software (BioRad), under the following conditions: an initial incubation step at 95 ° C for 15 min, followed by 35 cycles consisting of a denaturation at 95 ° C for 30 seconds, and a hybridization at 62 ° C for 30 seconds.
  • the analysis of the melting curves of the amplified products was carried out by gradually increasing the temperature from 60 ° C to 100 ° C at intervals of 0.5 ° C for 10 seconds and continuous reading of the fluorescence to determine the Tm of the specific amplification product for A. salmonicida spp. Salmonicide In all positive samples for Aeromonas salmonicida spp. Salmonicide was detected an amplification peak with a melting temperature of 80.5-81.0 ° C ( Figure 1).
  • Example 5 Amplification of DNA fragments from fish tissues.
  • reaction mixture 12.5 ⁇ _ of SYBR Maximum Green qPCR Master Mix (2x) (Thermo Scientifics), 1 ⁇ _ of each primer (SEQ ID NO: 1 and SEQ ID NO: 2) (concentration 400 nm end) and 2-5 ⁇ _ of DNA extracted.
  • the reaction volume was completed with sterile distilled water to 25 ⁇ _.
  • the reaction mixture was subjected to amplification in a MiniOpticon thermocycler for real-time PCR, with the CFX Manager TM detection software (BioRad), under the following conditions: a first incubation step at 95 ° C for 5 min, followed by 35 cycles consisting of a denaturation at 95 ° C for 1 minute, hybridization at 62 ° C for 30 seconds.
  • the analysis of the melting curves of the amplified products was carried out by gradually increasing the temperature from 65 ° C to 95 ° C at intervals of 0.5 ° C for 10 seconds and continuous reading of the fluorescence to determine the Tm of the specific amplification product for Aeromonas salmonicida spp.
  • Example 6 Specificity and sensitivity of real-time PCR with SYBR Green.
  • Aeromonas salmonicida spp. salmonicide 40 strains
  • Aeromonas sobria 2
  • Aeromonas caviae 2
  • Aeromonas bestiarum 1
  • Aeromonas hydrophila 2
  • Aeromonas media 1
  • Aeromonas allosaccharophila 1)
  • Aeromonas popoffii 1
  • Aeromonas simiae 1
  • Aeromonas trota 1
  • Aeromonas eucrenophila 1
  • Aeromonas jandeii 1
  • Aeromonas pisc ⁇ cola 2).
  • the aopO gene was detected in all strains of Aeromonas salmonicida spp. Salmonicide, when bacterial suspensions were used directly or DNA extracted from them.
  • the melting curve showed a single peak with an average Tm value between 80.5 and 81.0 ° C.
  • No amplification was obtained when bacterial suspensions or DNA extracted from atypical subspecies (Aeromonas salmonicida spp. Achromogenes, Aeromonas salmonicida spp. Masoucida) ( Figure 3) or other non-taxonomically related bacterial species were used.

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Abstract

La présente invention concerne un procédé pour détecter, quantifier et/ou identifier Aeromonas salmonicida spp. salmonicida dans un dosage de la PCR en temps réel. Le procédé consiste à réaliser un dosage de la PCR en temps réel dans des échantillons de cultures bactériennes, pures ou mixtes, ou dans un ADN isolé de cultures bactériennes, pures ou mixtes ou dans des tissus de poissons, au moyen d'une paire d'amorces spécifiques d'Aeromonas salmonicida spp. Salmonicida, ainsi qu'à détecter, quantifier et identifier les séquences nucléotidiques amplifiées. L'invention concerne aussi un kit pour détecter, quantifier et identifier Aeromonas salmonicida spp. salmonicida qui comprend les amorces mentionnées antérieurement, des fragments d'ADN témoins, des désoxynucleótides triphosphate, un tampon de réaction, et un manuel d'instructions.
PCT/ES2017/070293 2016-05-11 2017-05-10 Procédé pour la détection, la quantification et/ou l'identification d'aeromonas salmonicida spp. salmonicida Ceased WO2017194808A1 (fr)

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ES201630606A ES2578367B2 (es) 2016-05-11 2016-05-11 Procedimiento para la detección, cuantificación y/o identificación de aeromonas salmonicida spp. salmonicida
ESP201630606 2016-05-11

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115651989A (zh) * 2022-05-31 2023-01-31 中国水产科学研究院黄海水产研究所 一种杀鲑气单胞菌杀鲑亚种pcr检测试剂盒及其使用方法

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Publication number Priority date Publication date Assignee Title
ES2697949A1 (es) * 2017-07-28 2019-01-29 Univ Santiago Compostela Procedimiento para la deteccion, cuantificacion e identificacion de tenacibaculum maritimum

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03280882A (ja) * 1990-03-28 1991-12-11 Fujisawa Pharmaceut Co Ltd 魚類病原性菌種の決定遺伝子ｄｎａおよびその用途
WO1993011263A1 (fr) * 1991-12-04 1993-06-10 Eolas (Trading As Bioresearch Ireland) - The Irish Science And Technology Agency Sonde pour aeromonas solmonicida et son utilisation dans des procedes de detection ou de determination de cet organisme
CN104357570A (zh) * 2014-11-11 2015-02-18 中国水产科学研究院淡水渔业研究中心 一种杀鲑气单胞菌杀鲑亚种快速检测引物及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03280882A (ja) * 1990-03-28 1991-12-11 Fujisawa Pharmaceut Co Ltd 魚類病原性菌種の決定遺伝子ｄｎａおよびその用途
WO1993011263A1 (fr) * 1991-12-04 1993-06-10 Eolas (Trading As Bioresearch Ireland) - The Irish Science And Technology Agency Sonde pour aeromonas solmonicida et son utilisation dans des procedes de detection ou de determination de cet organisme
CN104357570A (zh) * 2014-11-11 2015-02-18 中国水产科学研究院淡水渔业研究中心 一种杀鲑气单胞菌杀鲑亚种快速检测引物及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE DataBase WPI 13 July 2016 (2016-07-13), Database accession no. 1992-035928 *
DATABASE DataBase WPI 13 July 2016 (2016-07-13), Database accession no. 2015-22901A *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115651989A (zh) * 2022-05-31 2023-01-31 中国水产科学研究院黄海水产研究所 一种杀鲑气单胞菌杀鲑亚种pcr检测试剂盒及其使用方法

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