I am looking into a large number of samples of deer and wild rodents. The tick borne pathogens, inside each genus, are too similar to differentiate and cloning will increase the number of samples greatly. I have been using primers for Ehrlichia, Anaplasma, Babesia and Theileria species but these primers are not specific and they cross reacted with other strains. In turn, the sequencing of the nested PCR positive samples failed (may be due to coinfection with multiple pathogens which were picked up with the same primer). I am thinking of switching to RLB but I don't know how sensitive and specific this method is in comparison to the nested PCR, cloning and sequencing.
RLB is time labor intensive and time consuming. If you are trying to detect multiple infection of ticks with Borrelia, Anaplasma, and Babesia, you need not clone PCR products. You can choose distinct markers for each. We have multiplex qPCR methods that test for Borrelia, Anaplasma, Babesia, plus a tick DNA control in just two reaction tubes. We tested thousands of ticks for many different public health agencies using these methods.
I agree with Estrada-Pena.
But generally, PCR-sequencing and if necessary, phylogenetic analysis are best way I think
I would vote for PCR as well, You can find LOTS of reliable protocols, markers, primer-sets. No need of clonning, if PCR works.
Please, spare your self the time and concentrate on PCR. You'll get the appropriate primers. Good luck.
I think that PCR is more suitable for your objectives, the only time consuming in this case is primer design, but is not impossible. Regards
PCR, perhaps consider RFLP, once you have generated sequences from your samples of all the possible parasites, that is, if product sizes are similar when doing the PCRs, this may help to discern different species or different parasites without sequencing each sample. It will be faster if you have to screen a large number of samples.
Thank you so much Stephen Rich, RLB will take 4 days to detect Anaplasma phagocytophillum, Ehrlichia chaffeensis, Babesia microti, Theileria spp and lyme Borrelia in about 40 samples.
If you recommend me to use multiplex qPCR method I hope if you can guide me to the species specific markers for these pathogens.
Thanks
Agustin Estrada-Peña, Thank you for your reply. Indeed there are several primers have been published before but when I tried it I found that it is not species specific for example when I used the anaplasma phagocytophillum primers with rodent samples it attached to the Candidatus neoehrlichia mikurensis. Another example, when I was searching for Ehrlichia chaffeensis in deer the primers attached to Ehrlichia muris, ewingii, ruminitium and Candidatus neoehrlichia shimanensis.
However, I'm looking for Anaplasma phagocytophillum, Ehrlichia spp, babesia microti, Lyme borrelia and theileria spp. and also I need to differentiate between them phylogentically.
Oh definitely PCR-RLB
We have Anaplasma/Ehrlichia/Babesia/Theileria/Borrelia/Rickettsia spp. RLB running routinely in my lab. (We have probes for all your listed pathogens. We even have more probes for other pathogens, but those are not really relevant to your question.)
Basically it takes a bit of setup time to get everything working. (For example get the miniblotters, membranes etc.) But it is a robust technique designed for field studies which can be used as a diagnostic test as well. And an addition to your reference about time needed for the RLB, it doesn't take 4 days. It takes one day to do DNA extractions + PCR (overnight) and the next day you do the RLB and you finish before 14:00. So it's one and a half day and if you get familiar with the technique 2 or more RLBs, depending on your equipment, at the same time is easily done. (You don’t need to do a nested PCR by the way, single PCR with a touchdown protocol is sufficient.)
Talking about the sensitivity, the RLB is generally speaking more sensitive than conventional PCR + running your products on gel. (Products that cannot be seen on gel, still give a clear signal after RLB.) There are a few papers that describe the sensitivity of the RLB if you want to get some exact numbers. qPCR is more sensitive compared to the RLB however, but the problem there is that you go for species specific PCR so it’s not really a technique that can be compared to the RLB if you want to screen samples which hold “unknown” pathogens. On the other hand, multiplex qPCR is possible, but hard to optimize and run with a “large” amount of probes. (RLB can have 43 probes on one membrane depending on the miniblotter of course.) Especially as it has, to my knowledge, not really been done before so it will take a long time setting it up, optimizing validating etc.and the RLB is already a tested and proven method for screening with this large amount of probes.
Phylogenetic differentiation is in theory possible with RLB, but only after you came across different (sub)species and were able to design a probe to distinguish between them. But for this part of your question I would suggest to screen large with RLB and only go for sequencing with the samples that you found to be positive for your pathogens of interest and use the sequence information gained this way for phylogenetic analysis as there is no real substitute for full sequence data when it comes to this. (Might be even needed to go for MLST for certain pathogens.)
If you want more information, you can contact me. And if I may be so rude: http://www.utrechtsummerschool.nl/index.php?type=courses&code=M20. We give a RLB summer course in the beginning of August this year.
Thank you Michiel Wijnveld so much for your detailed reply.
I would consider the PCR-RLB approach too. More sensitive and specific compared with PCR alone, plus the advantage of screening a huge number of specimens for multiple pathogens at a go.
Thank you so much Mr Githaka, I wish we start running RLB soon.
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