- Laboratory
Microbiological characterisation by molecular biology
Molecular biology techniques
Molecular biology techniques allow the detection, identification, typing and quantification of microorganisms based on their nucleic acids (DNA or RNA), regardless of their ability to grow in culture. This is especially useful for viable but non-culturable microorganisms, such as norovirus and Candidatus Liberibacter sp., among others; inhibitory matrices or situations where high sensitivity, specificity and speed are required.
AINIA has experience in the development and optimisation of different molecular biology techniques to ensure the quality and safety of products and facilities, which is why it includes a wide range of molecular biology techniques for the rapid detection of pathogens in its service portfolio.
Applications
There are different types of molecular biology techniques that enable the specific detection and quantification of genes and DNA and RNA fragments of microorganisms of interest. These microorganisms may be relevant due to their pathogenic or spoilage capacity, or for their benefits in health, human and animal nutrition, agriculture (biocontrol and biofertilisers), the environment (bioremediation), and industrial and biotechnological processes, where assessing their authenticity and concentration is key. The available techniques include:
- Conventional PCR enables qualitative detection of a specific gene or DNA fragment and is applied in colony confirmation, detection of virulence or resistance genes, and basic identification.
- Real-time PCR is based on the detection and relative quantification of DNA (copy of the target gene) and is applied in rapid microbiological control of pathogens and spoilage organisms in water, food, fertilisers, plant material, surface sampling devices (swabs, wipes, etc.), cosmetics and pharma.
- RT-qPCR (Reverse Transcription PCR) allows detection and quantification of RNA (viral, ribosomal or messenger) and is applied in the control of RNA viruses (norovirus, Hepatitis A), gene expression studies and microbial viability.
- Multiplex PCR focuses on the simultaneous detection of several genes or microorganisms in a single reaction and is applied in pathogen panels, detection of serotypes, resistance or virulence genes.
- Digital PCR (dPCR) enables absolute quantification of DNA without the need for a standard curve and is applied in the detection of low-concentration pathogens, complex matrices, and in monitoring and authentication of strains of interest in different products (infant formulations, probiotics, fertilisers, food supplements, starter cultures, etc.), assessing their concentration in absolute terms.
- Viability PCR (vPCR — Viability PCR) allows differentiation between live and dead microorganisms by PCR using DNA intercalating agents, enabling detection of only non-culturable cells and facilitating validation of disinfectant efficacy, rapid viability control, and analysis of VBNC (Viable But Non-Culturable) in cosmetics, pharma and food.
- REP-PCR is based on the amplification of regions between repetitive extragenic palindromic sequences (REP), generating genetic patterns to differentiate strains, and is applied in typing and differentiation of bacterial strains, epidemiological studies, quality control and traceability of strains in food, environmental or clinical microbiology, and comparison of genetic diversity within a species.
| Técnica | Principio | Objetivo | Ventajas | Aplicaciones en Microbiología |
|---|---|---|---|---|
| PCR Convencional | Amplificación de ADN mediante ciclos térmicos; detección final por electroforesis. | Detección cualitativa de genes específicos. | Simple, económica, muy específica. | Confirmación de colonias, detección de genes de virulencia o resistencia, identificación básica. |
| PCR en Tiempo Real (qPCR) | Amplificación y detección simultánea mediante fluorescencia (SYBR Green o sondas). | Detección y cuantificación de ADN. | Alta sensibilidad, rapidez, cuantificación absoluta/relativa. | Recuentos rápidos, control microbiológico en agua, alimentos, cosméticos y farma; P/A por Ct. |
| RT-PCR / RTqPCR | Conversión de ARN a ADNc (transcripción inversa) seguido de PCR o qPCR. | Detección o cuantificación de ARN. | Indica viabilidad/actividad metabólica; esencial para virus ARN. | Virus ARN (norovirus, SARS CoV-2), expresión génica, estudios de viabilidad. |
| PCR Multiplex | Amplificación simultánea de varios genes mediante múltiples cebadores. | Detectar varios microorganismos o genes a la vez. | Ahorro de tiempo y reactivos; análisis multigénico. | Paneles de patógenos, serotipado, genes de resistencia. |
| PCR Digital (dPCR) | Particionado de la muestra en miles de microreacciones y cuantificación absoluta. | Cuantificación absoluta sin curva estándar. | Extremada precisión; menos afectada por inhibidores; ideal para cargas bajas. | Matrices complejas, patógenos de baja concentración, estudios de mutaciones minoritarias. |
| PCR de Viables (vPCR) | Uso de colorantes intercalantes (PMA/EMA) que bloquean el ADN de células muertas. | Diferenciar viables vs. no viables. | Selectividad hacia células vivas sin cultivo; útil en matrices con conservantes. | Control rápido de viabilidad, validación de desinfectantes, estudio de células VBNC. |
| REP-PCR | Amplificación de regiones entre secuencias repetitivas palindrómicas extragénicas (REP). | Generar patrones genéticos (fingerprints) para diferenciar cepas. | Rápida, económica, buena resolución para estudios de diversidad y trazabilidad. | Tipificación y diferenciación bacteriana, estudios epidemiológicos, control de calidad y trazabilidad de cepas, comparación de diversidad genética dentro de una misma especie. |
AINIA’s competitive advantages
- Comprehensive microbiological testing services with technical advice to select the optimal methodology according to product and purpose (R&D, quality control, process optimisation).
- Availability of reference procedures, ISO standards, accredited by ENAC and CRBG (Biological and Genetic Resources Collection), to perform validation and verification of alternative methods based on molecular biology techniques in food, in accordance with ISO 16140.
- Agility in the design and optimisation of specific methods based on molecular biology techniques for the detection, quantification and specific typing of microorganisms of interest to the company.
- Extensive portfolio of services accredited by ENAC for rapid analysis of foodborne pathogens (Salmonella spp., Listeria monocytogenes, E. coli shiga toxin-producing STEC, pathogenic Yersinia enterocolitica, norovirus, Hepatitis A, etc.) and phytopathogens (priority pests such as Xylella fastidiosa, Candidatus Liberibacter sp., etc., in accordance with EPPO standards), among others.
- Integration into customised analytical plans together with other physicochemical services, providing flexible and tailored solutions for each client.
- Analytical agreements with positive release and weekend activity.
- Designated as official control for tests based on the application of molecular biology techniques (qPCR, RT-PCR, etc.)
MyLab Platform
Online access to sample traceability, real-time results, benchmarking against legal or customer specifications, and report and historical data download in Excel format.
Go to MyLab
Online requests
Agile and traceable request submission through the MyLab request module.
Automatic alerts
Notifications in case of non-compliance with legal limits or customer requirements.
Express service
Fast-track service with significantly reduced turnaround times.
Reports in English
Available at no additional cost upon request.
Specialised sampling team
Dedicated team for sample collection (surfaces, environments, designated facilities, etc.).
24h alert services
24h alert services to cover emergencies in the field of food safety.
Direct customer support for any technical or administrative queries.
(tel: 963 052 525 · mail: operaciones@ainia.es).
Key equipment
Application sectors
Food
Chemical
Cosmetic
Environment
Pharmaceutical
Agricultural
Healthcare
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Sonia Marco
Head of Microbiology Laboratory
