why biochemical identification and DNA identification shows different results? - (Mar/25/2008 )
QUOTE (sanjiun81 @ Mar 27 2008, 12:24 PM)
i did screening on my bacteria isolated from different host... using both biochemistry and 16S.
I also did conventional and API for biochemical identification...
what i can say is: DNA identification (using 16S or other conserved gene) is more accurate then biochemical test. Biochemical test only served as a support to what you have for your DNA test.
different strains might have some differences in using the chemical/sugar provided in biochemical test. sometimes also depends on how much culture you put in... if you put less, you might not see the difference; sometimes, the changes is not obvious and you might need soemone who is experienced to interprate the result.
for me, it is not surprising if you get difference in biochemical test compare to DNA identification. Even though sequencing 16S or other conserved genes will still give you error... the result still more reliable compare to only biochemical test.
I also did conventional and API for biochemical identification...
what i can say is: DNA identification (using 16S or other conserved gene) is more accurate then biochemical test. Biochemical test only served as a support to what you have for your DNA test.
different strains might have some differences in using the chemical/sugar provided in biochemical test. sometimes also depends on how much culture you put in... if you put less, you might not see the difference; sometimes, the changes is not obvious and you might need soemone who is experienced to interprate the result.
for me, it is not surprising if you get difference in biochemical test compare to DNA identification. Even though sequencing 16S or other conserved genes will still give you error... the result still more reliable compare to only biochemical test.
Yeah, i thought the same about 16s too. But me prof cautioned me about doing 16s and then running biochem just to prove the result. that way, i'd be biased towards gettin the result. im facing that problem now and my reason of the discrepancy is that there're strains which behaves in a non-typical manner giving rise to different reactions.
On the 16s primers, if you're borrowing from other lab (which i did before), the make sure its for 16s and not some specific primers. Also, if you're extracting the PCR amplified bands from the gel, make sure its ~1.4 kb in size. I'm sure you sent in the right stuff for sequencing (bear in mind that I have heard cases in my lab whereby the one responsible for sequencing may mix up the sample).
-jchchye-
QUOTE (jchchye @ Mar 26 2008, 11:25 PM)
On the 16s primers, if you're borrowing from other lab (which i did before), the make sure its for 16s and not some specific primers. Also, if you're extracting the PCR amplified bands from the gel, make sure its ~1.4 kb in size. I'm sure you sent in the right stuff for sequencing (bear in mind that I have heard cases in my lab whereby the one responsible for sequencing may mix up the sample).
yup....
some closely related strains/isolates may only differ from each other by few nucleotides.
repeating sequencing to confirm is needed.
need to be extra careful.
-sanjiun81-
hi dreamchaser_jc:
you suggest to use Mannitol Salt Agar but because in API 50 CHL one of the carbon sources to utilize was D-Mannitol and my bacteria didn't react with that (shown negative reaction) so can we say the culture is not Staphylococci or do it on the MSA!?
phage434:
Thanks I found from your link (Lane, D. J. (1991) one of my primers was "1392r 5'ACGGGCGGTGTGTRC" (universal primer) and the forward one still couldn't found but I ask from the lab they said it is "68f 5'TNANACATGCAAGTCGAKCG" (1.5 kb)
when we look at the sequencing will see some characters that they are not nucleotide (e.g. R, N, or K and in some other primers we can see W, Y, M, S, D ...) what are they?
-leonardu-
QUOTE (leonardu @ Mar 27 2008, 05:02 PM)
hi dreamchaser_jc:
you suggest to use Mannitol Salt Agar but because in API 50 CHL one of the carbon sources to utilize was D-Mannitol and my bacteria didn't react with that (shown negative reaction) so can we say the culture is not Staphylococci or do it on the MSA!?
phage434:
Thanks I found from your link (Lane, D. J. (1991) one of my primers was "1392r 5'ACGGGCGGTGTGTRC" (universal primer) and the forward one still couldn't found but I ask from the lab they said it is "68f 5'TNANACATGCAAGTCGAKCG" (1.5 kb)
when we look at the sequencing will see some characters that they are not nucleotide (e.g. R, N, or K and in some other primers we can see W, Y, M, S, D ...) what are they?
you suggest to use Mannitol Salt Agar but because in API 50 CHL one of the carbon sources to utilize was D-Mannitol and my bacteria didn't react with that (shown negative reaction) so can we say the culture is not Staphylococci or do it on the MSA!?
phage434:
Thanks I found from your link (Lane, D. J. (1991) one of my primers was "1392r 5'ACGGGCGGTGTGTRC" (universal primer) and the forward one still couldn't found but I ask from the lab they said it is "68f 5'TNANACATGCAAGTCGAKCG" (1.5 kb)
when we look at the sequencing will see some characters that they are not nucleotide (e.g. R, N, or K and in some other primers we can see W, Y, M, S, D ...) what are they?
I see. In that case, i'm sure mannitol is not utilized then its not Staph....
Congrats, at least you know that your primers are correct then. One thing, how many sequencing reactions you requested for? What I did was I sequence using the forward and reverse primers on one strand and the same for another. That way, you could fill up the N's. But since you mentioned the 16s rDNA alignment has high similarity, then i suppose your sequence is clean.
The alphabets are names for the bases N: aNy, K: Keto. The good folks here point it to me when I asked them the same question.
Follow here: http://www.blackwell-synergy.com/doi/abs/1....1985.tb08977.x
Good luck.
-jchchye-
Thank you jchchye for the article, It was useful to me.
Leo
-leonardu-