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CO2022007673A1 - Composition and method of detection of phytomonas staheli by rpa-lfa - Google Patents

Composition and method of detection of phytomonas staheli by rpa-lfa

Info

Publication number
CO2022007673A1
CO2022007673A1 CONC2022/0007673A CO2022007673A CO2022007673A1 CO 2022007673 A1 CO2022007673 A1 CO 2022007673A1 CO 2022007673 A CO2022007673 A CO 2022007673A CO 2022007673 A1 CO2022007673 A1 CO 2022007673A1
Authority
CO
Colombia
Prior art keywords
rpa
phytomonas
primers
detect
detection
Prior art date
Application number
CONC2022/0007673A
Other languages
Spanish (es)
Inventor
Cruz Maria Camila Rodriguez
Sierra Sandra Catalina Chaves
Jaramillo Carmenza Montoya
Hernan Mauricio Romero
Original Assignee
Corporacion Centro De Investig En Palma De Aceite
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Corporacion Centro De Investig En Palma De Aceite filed Critical Corporacion Centro De Investig En Palma De Aceite
Priority to CONC2022/0007673A priority Critical patent/CO2022007673A1/en
Publication of CO2022007673A1 publication Critical patent/CO2022007673A1/en
Priority to PCT/IB2023/055609 priority patent/WO2023233338A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • 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
    • 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
    • C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/34—Purifying; Cleaning
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Analytical Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Plant Pathology (AREA)
  • Sustainable Development (AREA)
  • Plasma & Fusion (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Se desarrolló un nuevo ensayo RPA-LFA para la detección de Phytomonas staheli que es la causante de la marchitez sorpresiva presente en la palma de aceite. Dicho método es simple de usar, proporciona resultados precisos, confiables, oportunos y permiten diagnósticos rápidos. Es un método simple de extracción de ADN que no requiere laboratorios profesionales ni procedimientos de extracción complicados, para facilitar la aplicación de este ensayo RPA-LFA en el campo. Dicho kit (3000), composición (2000) y procedimiento (1000) se constituye como un kit portátil que permite hacer las pruebas de diagnóstico directamente en las plantaciones de palma de aceite para detectar Phytomonas, sin la necesidad de llevar la muestra a un laboratorio que cuente con personal especializado para su identificación microscópica. El método de amplificación isotérmica que se utilizó en esta invención, el método RPA (Recombinase Polymerase Amplification) (Amplificación de Recombinasa Polimerasa), es una alternativa isotérmica de un solo tubo a la reacción en cadena de la polimerasa (PCR). Básicamente se trata de agregar una enzima transcriptasa inversa a una reacción de RPA, puede detectar tanto ARN como ADN, sin necesidad de un paso separado para producir ADN. Debido a que es isotérmico, RPA puede emplear un equipo mucho más simple que PCR, que requiere un termociclador siendo más rápido y eficiente en un rango de 35-42°C, y puede tolerar fluctuaciones en la temperatura; Esta invención opera mejor entre 37 a 42 °C. La mayoría de los ensayos de RPA se han centrado en la detección de patógenos relacionados con humanos o animales domésticos, así que esta invención proporciona una solución novedosa para detectar éste patógeno. En la presente invención se diseñaron las secuencias de los ácidos nucleicos de los iniciadores y la sonda. La invención (4000), se conforma de un Kit (3000) conformado por elementos y reactivos (3001), (3001.1), (3001.2), (3001.3), (3002), (3002.1), (3003), (3004), (3005), (3006), (3006.1), (3007), (3007.1), (3008), (3008.1), (3009), (3010), (3011), (3011.1). La invención (4000), también se conforma de una composición (2000) conformado por cebadores (primers) y sondas (2001), (2002), (2003). La invención (4000), también está conformada por un procedimiento (1000) junto con sus varias etapas (1001), (1001.1), (1002), (1002.1), (1003), (1004), (1005), (1006), (1007), (1008), (1009), (1010), (1011).A new RPA-LFA assay was developed for the detection of Phytomonas staheli, which is the cause of surprise wilt in oil palm. Said method is simple to use, provides accurate, reliable, timely results and allows rapid diagnoses. It is a simple DNA extraction method that does not require professional laboratories or complicated extraction procedures, to facilitate the application of this RPA-LFA assay in the field. Said kit (3000), composition (2000) and procedure (1000) is constituted as a portable kit that allows diagnostic tests to be carried out directly in oil palm plantations to detect Phytomonas, without the need to take the sample to a laboratory. that it has specialized personnel for its microscopic identification. The isothermal amplification method used in this invention, the Recombinase Polymerase Amplification (RPA) method, is a single tube isothermal alternative to polymerase chain reaction (PCR). It basically involves adding a reverse transcriptase enzyme to an RPA reaction, it can detect both RNA and DNA, without the need for a separate step to produce DNA. Because it is isothermal, RPA can employ much simpler equipment than PCR, which requires a thermocycler, is faster and more efficient in the 35-42°C range, and can tolerate fluctuations in temperature; This invention works best between 37 to 42°C. Most RPA assays have focused on the detection of pathogens related to humans or domestic animals, so this invention provides a novel solution to detect this pathogen. In the present invention, the nucleic acid sequences of the primers and the probe were designed. The invention (4000), is made up of a Kit (3000) made up of elements and reagents (3001), (3001.1), (3001.2), (3001.3), (3002), (3002.1), (3003), (3004) , (3005), (3006), (3006.1), (3007), (3007.1), (3008), (3008.1), (3009), (3010), (3011), (3011.1). The invention (4000), is also made up of a composition (2000) made up of primers (primers) and probes (2001), (2002), (2003). The invention (4000), is also made up of a procedure (1000) together with its various stages (1001), (1001.1), (1002), (1002.1), (1003), (1004), (1005), (1006 ), (1007), (1008), (1009), (1010), (1011).

CONC2022/0007673A 2022-05-31 2022-05-31 Composition and method of detection of phytomonas staheli by rpa-lfa CO2022007673A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CONC2022/0007673A CO2022007673A1 (en) 2022-05-31 2022-05-31 Composition and method of detection of phytomonas staheli by rpa-lfa
PCT/IB2023/055609 WO2023233338A1 (en) 2022-05-31 2023-05-31 Composition and method for detecting phytomonas staheli by means of rpa-lfa

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CONC2022/0007673A CO2022007673A1 (en) 2022-05-31 2022-05-31 Composition and method of detection of phytomonas staheli by rpa-lfa

Publications (1)

Publication Number Publication Date
CO2022007673A1 true CO2022007673A1 (en) 2022-06-10

Family

ID=81854988

Family Applications (1)

Application Number Title Priority Date Filing Date
CONC2022/0007673A CO2022007673A1 (en) 2022-05-31 2022-05-31 Composition and method of detection of phytomonas staheli by rpa-lfa

Country Status (2)

Country Link
CO (1) CO2022007673A1 (en)
WO (1) WO2023233338A1 (en)

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2438196B1 (en) * 2009-06-05 2016-12-21 Alere San Diego, Inc. Recombinase polymerase amplification reagents and kits

Also Published As

Publication number Publication date
WO2023233338A1 (en) 2023-12-07

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