16s Rdna Pcr Assay
16s Rdna Pcr Assay - Related Videos
10:57
16S rRNA Sequencing: A PCR-based Technique to Identify Bacterial Species
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2023
Source: Ewa Bukowska-Faniband1, Tilde Andersson1, Rolf Lood1 1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 00 Lund, Sweden Planet Earth is a habitat for millions of bacterial species, each of which has specific characteristics. Identification of bacterial species is widely used in microbial ecology to determine biodiversity of environmental samples and medical microbiology to diagnose infected patients. Bacteria can be classified...
11:37
Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR
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Cited by 24 •
2016
A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. The methodology includes the co-extraction of DNA and RNA; preparation of DNA-free RNA; and 16S rRNA gene and transcript quantification via RT-q-PCR, including standard curve construction.
06:34
Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS
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Cited by 29 •
2016
Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.
11:22
Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing
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Cited by 65 •
2019
Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.
10:15
Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)
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Cited by 21 •
2011
An inexpensive, high throughput method for simultaneous detection of up to 43 molecular targets is described. Applications of mPCR/RLB include microbial typing and detection of multiple pathogens from clinical samples.
FAQs
Primers are designed to bind conserved regions flanking variable portions of the bacterial 16S rDNA locus. Conserved binding sites support amplification across different bacteria, while the intervening variable sequence provides information for characterization. This arrangement allows the assay to detect bacterial DNA broadly without making every amplified product equivalent for species-level identification.
Conserved regions provide shared targets that make bacterial amplification feasible across diverse organisms, whereas variable regions contain sequence differences useful for distinguishing bacterial groups. The balance between these regions determines whether results support broad detection, characterization, or more specific identification. Closely related organisms may still require additional testing because their 16S sequences can be insufficiently different.
Each cycle separates the DNA strands, allows primers to anneal to their target sites, and extends the bound primers to copy the selected locus. Repetition generates many copies from the original template, producing material suitable for downstream analysis. Gel electrophoresis can assess the amplified product, while sequencing can provide sequence information for bacterial characterization.
The targeted 16S rDNA regions may be highly similar among closely related bacteria, so an amplified sequence can support bacterial detection or group-level characterization without uniquely identifying one species. This limitation matters in infection studies where species-level assignment affects interpretation. Additional testing is therefore needed when the 16S result does not provide definitive discrimination.
A typical workflow uses a sample containing potential bacterial DNA, primers that flank selected 16S rDNA regions, and repeated PCR cycles of denaturation, annealing, and extension. The resulting amplicons are then examined by gel electrophoresis or analyzed by sequencing. These stages connect initial bacterial detection with characterization of the amplified genetic target.
The assay is particularly valuable when investigators need to identify bacteria from clinical or environmental samples, including organisms that are difficult to culture. It can help clarify possible infection sources and support characterization when culture-based recovery is limited. Because the method detects bacterial DNA, interpretation should also account for its limits in separating closely related organisms.
In immunology and infection research, the assay can identify bacterial components relevant to studies of host-microbe interactions and describe microbial communities present in a sample. Comparing detected bacterial profiles may help investigate infection sources or community composition. Its findings provide molecular evidence for these analyses, while species-level conclusions may require additional testing when sequences overlap.