Molecular biology assay

Molecular Biology Assay Services – Accredited Nucleic Acid Analysis and Genetic Testing for Global Markets

Our internationally accredited laboratory provides a comprehensive molecular biology assay service that supplies pharmaceutical developers, clinical‑diagnostics companies, food‑safety laboratories, forensic investigators, agrigenomics researchers and academic institutions worldwide with the independent, traceable data they need to detect, quantify and characterise nucleic acids in any biological matrix. Every analysis is performed under the rigorous framework of ISO/IEC 17025, and each report bearing the ILAC mark is unconditionally accepted by regulatory authorities, notified bodies and supply‑chain partners in all major economies. The molecular biology assay platform covers the complete workflow from the nucleic‑acid extraction and the quality control, through the polymerase chain reaction (PCR), the quantitative real‑time PCR, the Sanger and the next‑generation sequencing, to the fragment analysis, the genotyping and the gene‑expression profiling, employing the harmonised protocols of the ISO/TS 21569, the MIQE guidelines, the CLSI standards and the pharmacopoeial methods. For a contract research organisation validating a biomarker signature, a diagnostic manufacturer certifying a PCR kit, or a food‑testing agency screening for genetically modified organisms, this service delivers the legally robust, defensible genetic data that underpin regulatory submissions, product certification and the protection of human, animal and environmental health on every continent.

Product Samples We Regularly Subject to Molecular Biology Assays

The nucleic‑acid extraction suites, the robotic liquid‑handling workstations, the thermal cyclers, the real‑time PCR and the digital‑PCR platforms, the capillary‑electrophoresis sequencers, the next‑generation sequencing systems and the bioinformatics pipelines in our facility accommodate an extraordinarily diverse range of sample types. The following categories represent the most frequently tested items:

Human and animal clinical specimens – whole blood, serum, plasma, buffy coat, dried blood spots, cerebrospinal fluid, bronchoalveolar lavage, urine, saliva, faecal samples and tissue biopsies
Microbial cultures and isolates – bacterial colonies, yeast and mould cultures, viral transport media, sputum, wound swabs and endotracheal aspirates
Cell lines and primary cell cultures – adherent and suspension cells, stem‑cell cultures, organoids and the transfected or the transduced cell populations
Formalin‑fixed, paraffin‑embedded (FFPE) tissue blocks and sections – tumour and normal‑tissue specimens from the pathology archives, processed for the extraction of the fragmented DNA and RNA
Food, feed and agricultural commodities – raw and processed foods, grains, seeds, flour, vegetable oils, animal feed and the swabs from the food‑contact surfaces
Environmental samples – soil, sediment, water, air‑filter extracts, wastewater and the swabs from the industrial and the hospital surfaces
Forensic and trace evidence – hair, saliva stains, semen stains, touch‑DNA swabs, bone fragments and teeth, processed for the short‑tandem‑repeat profiling and the mitochondrial‑DNA sequencing

Nucleic Acid Extraction, Purification and Quality Control – The Foundation of Every Molecular Biology Assay

Genomic DNA extraction from the fresh, the frozen and the FFPE tissues, the blood and the microbial cultures according to the internal validated protocols and the principles of the ISO 11063 (Soil quality – Method to directly extract DNA from soil samples, adapted for the clinical and the food matrices): the sample is lysed, and the DNA is purified by the silica‑membrane, the magnetic‑bead or the phenol‑chloroform method, yielding the high‑molecular‑weight DNA that is suitable for the downstream PCR and the sequencing. The concentration and the purity are measured by the spectrophotometry and the fluorometry, and the integrity is assessed by the agarose‑gel electrophoresis or the automated capillary electrophoresis. This molecular biology assay step is the critical quality gate that ensures the success of every subsequent analysis.
Total RNA and the microRNA extraction for the gene‑expression and the transcriptome‑profiling studies: the sample is homogenised in the guanidinium‑thiocyanate‑phenol reagent, and the RNA is purified on the silica columns or by the alcohol precipitation, with the on‑column DNase digestion to remove the contaminating genomic DNA. The RNA integrity number is determined by the microfluidic electrophoresis, and only the samples with a RIN value above 7.0 are accepted for the RNA‑seq and the quantitative PCR experiments.
Plasmid DNA and the recombinant‑vector purification for the molecular cloning and the gene‑therapy applications: the bacterial culture is processed by the alkaline‑lysis method, and the plasmid DNA is purified by the anion‑exchange or the size‑exclusion chromatography, yielding the endotoxin‑free, transfection‑grade DNA that meets the quality requirements of the FDA and the EMA for the production of the clinical‑grade vectors.
Extraction of the viral nucleic acids from the low‑titre clinical samples and the process‑intermediate samples: the viral RNA or the DNA is extracted using the carrier‑RNA‑supplemented, magnetic‑bead‑based methods that maximise the recovery of the minute amounts of the target, and the eluate is concentrated and tested for the presence of the inhibitors before the RT‑PCR or the PCR.

Polymerase Chain Reaction (PCR) and Reverse‑Transcription PCR – The Core Molecular Biology Assay for Gene Amplification

Conventional and the nested PCR for the detection and the characterisation of the specific DNA targets according to the internal validated protocols and the principles of the ISO 22174 (Microbiology of the food chain – Polymerase chain reaction for the detection of food‑borne pathogens): the target DNA is amplified with the genus‑specific, the species‑specific or the strain‑specific primers, and the amplicon is visualised by the agarose‑gel electrophoresis, the capillary electrophoresis or the hybridisation probe. The presence or the absence of the target sequence is reported, and the semi‑quantitative band‑intensity or the endpoint‑fluorescence data are provided, supporting the pathogen‑detection, the GMO‑screening and the species‑identification applications.
Reverse‑transcription PCR for the detection of the RNA viruses and the mRNA transcripts: the RNA is reverse‑transcribed into the complementary DNA using the random hexamers, the oligo‑dT primers or the target‑specific primers, and the cDNA is amplified by the PCR or the nested PCR, enabling the detection of the SARS‑CoV‑2, the influenza virus, the norovirus and the other RNA pathogens, as well as the verification of the gene‑expression at the qualitative level.
Multiplex PCR and the panel‑based screening for the simultaneous detection of the multiple targets: several primer pairs that are specific to the different pathogens, the GMO events or the genetic markers are combined in a single reaction tube, and the amplicons are differentiated by the size, the melting‑temperature or the fluorescent‑dye labelling, providing the cost‑effective, high‑throughput screening that is widely used in the clinical‑microbiology and the food‑authenticity testing.
Allele‑specific and the mutation‑detection PCR for the single‑nucleotide‑polymorphism and the somatic‑mutation analysis: the primers that are designed to discriminate between the wild‑type and the mutant alleles are used, and the presence of the specific mutation – such as the EGFR exon 19 deletion, the KRAS codon 12 mutation or the BRAF V600E substitution – is detected, supporting the personalised‑oncology treatment decisions.

Quantitative Real‑Time PCR (qPCR) – The Molecular Biology Assay for Gene Expression, Viral Load and GMO Quantification

Absolute quantification of the DNA and the cDNA targets by the SYBR Green or the TaqMan probe‑based qPCR according to the MIQE (Minimum Information for Publication of Quantitative Real‑Time PCR Experiments) guidelines and the ISO 20395 (Biotechnology – Requirements for evaluating the performance of quantification methods for nucleic acid target sequences – qPCR and dPCR): the sample is amplified in the presence of a fluorescent intercalating dye or a sequence‑specific fluorescent probe, and the quantification‑cycle value is converted to the copy number or the concentration using a standard curve that is constructed from the known amounts of the reference standard. The linear‑dynamic range, the amplification efficiency, the limit of detection and the limit of quantification are reported for each target, and the data are normalised to the endogenous reference genes or the sample‑input quantity, providing the accurate, reproducible quantitative results that are required for the viral‑load monitoring, the cytokine‑expression profiling and the GMO‑content labelling.
Relative gene‑expression analysis by the comparative quantification‑cycle method: the expression of the target gene is normalised to that of the stably expressed reference genes, and the fold‑change relative to the control group or the calibrator sample is calculated using the 2⁻ᵅᵅCᵪ method, providing the biologically meaningful data that the researcher uses to identify the up‑regulated and the down‑regulated genes in the disease, the drug‑treatment and the developmental studies.
Digital PCR for the ultra‑sensitive, absolute quantification of the rare mutations and the low‑abundance targets: the sample is partitioned into thousands of the individual reaction compartments on a microfluidic chip or in a water‑in‑oil emulsion, and the endpoint fluorescence of each partition is scored. The concentration of the target is calculated from the fraction of the positive partitions using the Poisson statistics, achieving the limit of detection down to 0.01 % of the mutant allele in a wild‑type background, which is essential for the liquid‑biopsy applications and the detection of the minimal residual disease.
High‑resolution melt‑curve analysis for the rapid genotyping and the mutation scanning: after the qPCR amplification, the amplicon is gradually heated, and the fluorescence is continuously recorded, generating a melt‑curve profile that is characteristic of the nucleotide sequence. The variant‑specific melt profiles distinguish the homozygous wild‑type, the heterozygous and the homozygous mutant samples without the need for the post‑PCR sequencing, providing the simple, cost‑effective genotyping method for the pharmacogenetic and the breeding‑selection programmes.

Sanger Sequencing and Fragment Analysis – The Gold‑Standard Molecular Biology Assay for DNA Sequence Determination

Determination of the nucleotide sequence of the PCR amplicons, the plasmids and the purified DNA templates by the dideoxy‑chain‑termination Sanger sequencing according to the internal validated protocol and the principles of the ISO 15189 (Medical laboratories – Requirements for quality and competence, adapted for the sequencing): the template is amplified in a cycle‑sequencing reaction with the fluorescently labelled dideoxynucleotides, and the fragments are separated by the capillary electrophoresis. The resulting electropherogram is base‑called, and the quality‑trimmed sequence is compared with the reference database using the BLAST algorithm, providing the definitive identification of the species, the gene variant and the mutation. This molecular biology assay is the reference method for the confirmation of the PCR and the qPCR results, the characterisation of the novel genetic elements and the validation of the gene‑editing outcomes.
Fragment‑analysis‑based genotyping of the short‑tandem‑repeats, the microsatellites and the amplified‑fragment‑length polymorphisms according to the internal validated protocols and the principles of the forensic DNA‑analysis guidelines: the fluorescently labelled PCR primers are used to amplify the polymorphic loci, and the fragment sizes are determined by the capillary electrophoresis with the internal size‑standard correction, providing the genetic fingerprint that is used for the human identification, the cell‑line authentication and the marker‑assisted selection in the plant and the animal breeding.
Mitochondrial DNA and the chloroplast DNA sequencing for the species identification and the phylogenetic studies: the highly variable regions of the mitochondrial cytochrome‑c‑oxidase I gene, the 16S‑rRNA gene or the chloroplast rbcL gene are sequenced, and the species identity is confirmed by the database match, supporting the food‑authenticity, the wildlife‑forensics and the biodiversity‑monitoring applications.

Next‑Generation Sequencing (NGS) – The High‑Throughput Molecular Biology Assay for Genomics and Metagenomics

Whole‑genome, whole‑exome and the targeted‑gene‑panel sequencing on the Illumina, the Ion Torrent and the Oxford Nanopore platforms according to the internal validated protocols and the best‑practice guidelines of the American College of Medical Genetics and Genomics: the genomic DNA is fragmented, the sequencing libraries are prepared with the unique dual‑index adaptors, and the pooled libraries are sequenced to the depth that provides the required coverage for the detection of the single‑nucleotide variants, the insertions and the deletions, and the copy‑number alterations. The raw data are processed through the bioinformatics pipeline that includes the read alignment, the variant calling, the annotation and the filtering, and the clinically actionable variants are reported with the American College of Medical Genetics classification and the supporting evidence. This molecular biology assay is the cornerstone of the precision‑medicine programmes, the rare‑disease diagnosis and the cancer‑genome profiling.
RNA‑sequencing (RNA‑seq) for the transcriptome profiling, the differential‑gene‑expression analysis and the fusion‑gene detection: the total RNA or the messenger‑RNA is depleted of the ribosomal RNA, converted into the strand‑specific complementary‑DNA libraries and sequenced, and the read counts per gene are quantified, enabling the identification of the differentially expressed genes, the alternative‑splicing isoforms and the gene‑fusion events that are the therapeutic targets and the diagnostic biomarkers.
16S‑rRNA and the internal‑transcribed‑spacer (ITS) amplicon metagenomic sequencing for the microbial‑community profiling: the variable regions of the bacterial 16S‑rRNA gene or the fungal ITS region are amplified with the universal primers that carry the sample‑specific barcodes, and the amplicons are sequenced, generating the operational‑taxonomic‑unit tables and the taxonomic‑classification data that describe the composition and the diversity of the microbiome in the gut, the soil, the water and the food samples.
Shotgun metagenomic sequencing for the comprehensive characterisation of the complex microbial communities and the resistome profiling: the total DNA extracted from the sample is directly sequenced, and the unassembled reads are taxonomically classified and functionally annotated, providing the information on the metabolic‑pathway potential, the antimicrobial‑resistance genes and the virulence factors that is not available from the amplicon‑based methods alone.
Whole‑genome bisulfite sequencing and the reduced‑representation bisulfite sequencing (RRBS) for the epigenome‑wide DNA‑methylation analysis: the genomic DNA is treated with the sodium bisulfite, which converts the unmethylated cytosines to uracils, and the converted DNA is sequenced, providing the quantitative methylation status of the millions of the individual CpG sites, which is used to identify the epigenetic biomarkers of the ageing, the environmental exposure and the cancer.

Report Acceptance and Global Regulatory Compliance

All molecular biology assays described above are executed under the fully accredited scope of our ISO/IEC 17025 quality management system. Each test report that carries the ILAC mark is therefore automatically recognised by regulatory authorities, notified bodies, customs offices and supply‑chain partners in all major economies. For pharmaceutical developers, clinical‑diagnostics manufacturers, food‑safety testing laboratories, forensic institutes and agrigenomics researchers anywhere in the world, the report constitutes legally robust, internationally accepted evidence that the nucleic‑acid extraction, the PCR, the qPCR, the digital PCR, the Sanger and the next‑generation sequencing, the genotyping and the gene‑expression data have been generated in accordance with the applicable ISO, MIQE, CLSI and customer‑specified methods. The documentation can be directly used to support the regulatory submission for the drug or the diagnostic approval, the CE marking of the in‑vitro diagnostic device, the food‑safety certification, the forensic‑evidence presentation, the issue of inspection certificates according to EN 10204 or equivalent national standards, and the resolution of commercial and technical disputes concerning the genetic identity, the purity and the safety of any biological material.