Here we are…
To be clear, before talking about optimization steps, tests, calibration curves, trueness, validation and inhibition, PCR needs a primer and probe system to work and ensuring targeted gene detection.
So, detection strategy is crucial.
Working in microbiology field with bacteria, viruses and others, I will obviously pick up examples from personal results collected for several years now.
Nevertheless, working on pretty confidential fields in a private company, I will rarely give precised details or goals of the results… I think you will understand…
Let is back to development strategy. Many ways are possible but I can see mainly 2 considering detection and quantification in microbiology field.
The first targets a genus for example, so it has to detect any forms, serotypes or genogroupes and gives a so called “consensus system” even excluding any other microorganism. The second one targets a precised microorganism excluding all the other, one of the most known is Legionella pneumophila.
Thus, considering the 2 strategies, a gene has to be found comprising conserved regions among the different targeted microorganisms (consensus, strategy 1) or a gene comprising mutations in order to discriminate targeted one from all the others.
It foresees many hours looking for good sequences in online database, to compare them and to chose the right one with the right software…or doing it manually with the alignment and operator feeling and knowledge.
Firstable, the NCBI website is inevitable for search of sequences and references.
The approach is simple but can also takes time if meticulous.
1) Search on targeted microorganism in PubMed and also PCR development or genome studies.
2) Find all complete or partial sequences of our target (consensus or specific one).
For example, if you look for Legionella pneumophila on NCBI genome, 45 complete sequences (CDS) will be found. All of them are not Legionella pneumophila because we can have similitude or partial identical sequence. Nevertheless, we can also found the complete Lp1 Philadelphia sequence for example.
3) Back to « Nucleotides » results (more than 3000 items), partial sequences can be also looked for.
4) Once good sequences are found, alignments has to be done to compare them. Many websites are dedicated to it or also softwares like BioEdit
- Multalin
- Clustal software
- A pretty good list is done on Wiki links but as I do not know all of them, be careful about it ("List of sequence alignment software")
5) When a sequence is interesting about alignments and locus, primers and probe has to be determined (next post). Basically, either you have a very powerful up-to-date software like PrimerExpress from ABI, Oligosys or Beacon Designer or you can use free online software like Primer3.
Parameters are complete to change, but you need to know absolutely what you do before changing any of them, because it can affect deeply the obtained results.
6) When primers are chosen, it is good to check with which sequences it can match. Indeed, goals of these oligos is to specifically amplify the target and not the others.
Thus, the use of BLAST (Basic Local Alignment and Search Tool) software permits to check matching one more time of our chosen primers to any other sequences available in the database. It is a good step to check whether sequences are good or not.
Go on "Nucleotide Blast" and then :
- Paste your sequences in the first window
- Name you search in « Job title »
- Indicate the database you want to match your sequence with (on my point of view, it was mainly versus "nucleotide collection nr/nt").
- Optimize your search (in general I work with Blastn)
The obtained results comprises the first 100 results
- Access number of the answer
- Description (name of the matched target)
- Blast scores
- Recovery percentage
- E-value (the smaller, the better..)
If your target is the only 100% found sequence, it is very good.
If not, have a look to the sequences and evaluate the amplification risk of a non-targeted microorganism
7) Another interesting step is to study the chosen sequences with FASTA comparison software.
It will align both sequences and being able to find mutations.
Then, this in silico evaluation is done, and time has come for primers and probes order. Considering probe strategy, it will be discussed in a next post.
Oligos company are present on the market. As for me, I have good results with MWG-Eurofins and Eurogentec.
I always start development with a 0.2µmole order for primers and probes. If you order only primers, you will get them in less than one week.
Next posts will be on primers testing.
Have a good amplification
