Creative Biolabs, a leading contract research organization (CRO) with labs in the United States, provides standard and custom live biotherapeutics (next-generation probiotics) services. We have extensive experience in "fit for purpose" experiment development. Our mission is to advance projects by providing special, customized microbiological services.
Since health benefits conferred by probiotics are strain-specific, identification of the strain level is mandatory to allow the monitoring of the presence and the abundance of specific probiotics in a product or a gastrointestinal tract. The term strain-specific is very strict since the primers should bind exclusively to the specific sequence in a specific strain and no PCR amplification should be observed with DNA from any other strain in nature. Since the discovery of strain-specific genome sequences is very difficult, species- or subspecies-specific primers can be used to detect and quantify specific strains in the environment. Primers are arguably the most critical component of any PCR assay, as the properties of the primers control the unique specificity and sensitivity of this method.
Probing techniques are based on the hybridization of synthetically prepared oligonucleotides to specific target sequences on bacterial DNA. The specificity of the probe is largely dependent on the target sequence, although the stringency of the hybridization conditions and washings are also critical. Probing methods have concentrated on different regions of the bacterial ribosome. Highly conserved regions are targeted for universal, or domain-based, oligonucleotide probes, whereas different variable regions are used for genus-specific probes; highly variable regions are targeted for species-specific probes.
A good assay will not create primer dimers and be close to 100% efficient and exquisitely specific. When designing assays, the design process comprises a comprehensive workflow that demands careful consideration not just of the primers themselves but also of amplicon uniqueness, structure, and location, to bring about an optimal primer/amplicon combination for accurate quantification of nucleic acids.
Fig.1 Structure and mechanism of action of primer-probes. (Navarro, 2015)
The most important and challenging step is the identification of strain-specific sequences that can subsequently be targeted with specific primers or probes. Good primer or probe design principles are essential to prevent false positive or false negative results and to improve the sensitivity of PCR reactions. Creative Biolabs can design specific primers and probes for intestinal strains, traditional probiotics, and next-generation probiotics according to customers' goals and requirements, effectively promoting the detection, screening, and identification of target strains.
Probe-based analysis can provide quantitative information. Further advantages are the speed and high throughput of the 96-well-based real-time PCR system, as well as the omission of all post-amplification steps, thereby reducing labor and contamination risks. The reduced assay time and higher specificity compared to standard plate counts make PCR-based methods well-suited for the detection and quantification of probiotics. Conventional PCR combined with gel electrophoresis has been successfully used for the genus-, species- or strain-specific determination of the presence of probiotic organisms in the probiotic products or the biological samples (feces).
Creative Biolabs has extensive and deep knowledge and experience in the probiotic field to develop customized solutions. If you are interested in our gut probiotic primer and probe design services, please do not hesitate to contact us.
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