My colleague and I are planning to do a culture-independent study on identifying specific bacteria in a river system. We just have some questions before we undertake this study.
1. If we happen to sample pathogenic bacteria, do we need to work in a BSL-2 laboratory?
2. What is the general procedure for trying to identify specific bacteria? Do we need to perform DNA extraction, cultivation, etc.? We are planning to perform 16S rRNA metagenomic analysis and are scouting sequencing centers around our country.
Ciara Maria Ines Palma Del Rosario Look at work of Gyraite G, Katarzyte M, Schernewski G. First findings of potentially human pathogenic bacteria Vibrio in the south-eastern Baltic Sea coastal and transitional bathing waters. Marine pollution bulletin. 2019 Dec 1;149:110546. Article First findings of potentially human pathogenic bacteria Vibr...
It has most of important methods regarding identification pathogenic bacteria in water
The rules for different countries are different, so whether you need to use a higher Biosafety containment is going to depend upon where you are at, and what you are doing. I would strongly suggest you contact your biosafety office at your university for guidance.
But I think if you are not culturing the products and just sampling and extracting DNA, then you are likely ok. But for example disposing of the excess river water that is "contaminated" may require special disposal if there are hazardous pathogens.
- Sample collection: You will need to collect a representative water sample from the river system, taking care to minimize contamination during collection and storage.
- DNA extraction: The next step is to extract DNA from the water sample, which will require specialized reagents and equipment. The extracted DNA will contain the genetic material of all the bacteria present in the sample.
- PCR amplification: To target the 16S rRNA gene, you will need to perform PCR amplification of the extracted DNA, using specific primers that bind to the 16S rRNA gene.
- Sequencing: The amplified 16S rRNA gene will then be sequenced, typically using high-throughput sequencing technology such as Illumina or Ion Torrent.
- Data analysis: Finally, you will need to analyze the sequencing data to determine the presence and abundance of specific bacteria in the sample. This typically involves using bioinformatics tools to compare the sequenced 16S rRNA gene data against reference databases of known bacteria to identify the bacteria present in your sample.
It is not necessary to perform cultivation to identify specific bacteria using 16S rRNA metagenomic analysis, as this method allows for the detection of both cultivable and uncultivable bacteria. When scouting sequencing centers, it's important to consider their experience and expertise in processing environmental samples, as well as the type of sequencing technology they offer and their data analysis capabilities.
If you work "Culture-independent" where does the pathogenic bacteria cultivation come in? If you are worried about the few pathogenic bacteria in the water just work carefully, wear gloves, and clean after.
Other than this follow the protocol for water bacteria, there are many out there (e.g. Article Response of bacterial communities to variation in water qual...
).An issue might be filtering enough water to concentrate and capture bacteria, as filters tend to get clogged. We used a sandwich filter - GF/A over nitrocellulose filter, the GF/A removing large particles. If you can afford Anodisk for the finer filtration you might get better results as you can grind them for DNA extraction.
It is important to note that the choice of sequencing center and the specific protocols used for DNA extraction and sequencing can have a significant impact on the results of your study. It may be useful to consult with bioinformatics experts and other researchers in the field to determine the most appropriate protocols and analysis methods for your study.
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