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suitable single-copy gene target for fungal qPCR


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#1 LabNerd

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Posted 17 November 2013 - 02:18 AM

Dear all

 

I have come across multiple fungal qPCR works that employ primers targeting the 18S region, however I am aware that (as referred to Rooney et al. 2005 in PNAS) rRNA genes in Eukaroya exists in multiple copies per genome. A lot of the qPCR papers only looked at Ct value vs copy number mainly after dilution curves (as an assessment of amplification efficiency) without addressing the multiple-copy per genome issue. 

 

I am trying hard to look for fungal qPCR primers which targets a gene conserved across different phyla, and that contains only a single-copy per genome, I found a paper (Black et al., 2013 in Journal of Biomolecular Techniques) investigating the single-copy gene benA (coding for beta-tubulin). However, as promising as it sounds, they used different primers for different fungal organisms. 

 

Does anyone know of a single primer pair that targets a single-copy gene with broad enough coverage to detect most members of Fungi?

 

Thank you very much!



#2 Trof

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Posted 17 November 2013 - 05:55 AM

Ribosomal DNA is present in mulitple copies in genome, but as long as the copy-number is fixed, it doesn't matter (I don't know if it is). Of course when used for quantification of Fungi, the copy number derived from a single-copy standard doesn't match the real "copy-number", but when comparing samples together and samples between laboratories using the same quantification system, it possible to compare.

 

rDNA is AFAIK used mainly because it's shows great conservation on the level of nucleotides. Many genes can have conserved protein sequence, but contain polymorphisms in DNA sequence. Unlike in humans and other popular model organisms, I don't think there is not big sequence coverage of the "most members of Fungi" so it's rather difficult to tell which site is really conserved.

 

You probably need to choose, depending on purpuse of your detection. If you want to detect all fungi possible with single primers, then you should stick with the rDNA. Or you want to precisely quantify fungi copies in sample and then you need a single-copy gene, but with different primers. I suppose different primers, if they are species-specific, also enable you to identify the exact species of the fungi, which could be an additional bonus if you need that. 

If the region of a conserved single-copy gene contains only a few polymorphic sites, and you want to detect them all, it's possible to design degenerate primers, but these are actually only mixtures of different primers in once. But depending on your purpose, this could also be on option.


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#3 LabNerd

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Posted 17 November 2013 - 06:06 AM

Thank you Trof for your valuable comments. We are trying to quantify fungal copy number in environmental samples, which contains a cocktail of different species and genera. A lot of the similar studies uses ITS region for quantification.

 

Comparing gene copy number in environmental samples, however, may be of little relevance, because different communities may have different species, some of which may have more copies of the target region than others. It's easier for bacteria because single-copy genes like rpoB can be used.

 

Either bacteria or fungus, I suppose it would be ok as long as one specifies that it's "gene copy number" that one is comparing between samples, and not biomass or cell concentration. However, whether "gene copy number" difference is relevant in environmental samples is up for debate.

 

Thanks again Trof ^_^



#4 Trof

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Posted 17 November 2013 - 06:23 AM

I know it's probably out of question for financial reasons, but for quatification and identification of enviromental samples, I think NGS probably with some sequence pre-capture would be great option (for future, maybe). With coverage high enough, NGS can be pretty well quantitative, and in the mixture of various bacteria, fungi and whatever gives the perfect picture of whole sample. I think they now even use NGS to identify even new species of ..whatever there is.

 

With primers to broad you may have a skewed picture of the quantity of relevant species, but with too specific ones you may miss something you didn't think of. Given also other problems with PCR inhibition and so, enviromental samples are always a great challenge.


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#5 LabNerd

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Posted 17 November 2013 - 06:30 AM

I know it's probably out of question for financial reasons, but for quatification and identification of enviromental samples, I think NGS probably with some sequence pre-capture would be great option (for future, maybe). With coverage high enough, NGS can be pretty well quantitative, and in the mixture of various bacteria, fungi and whatever gives the perfect picture of whole sample. I think they now even use NGS to identify even new species of ..whatever there is.

 

With primers to broad you may have a skewed picture of the quantity of relevant species, but with too specific ones you may miss something you didn't think of. Given also other problems with PCR inhibition and so, enviromental samples are always a great challenge.

 

 

Yeah we also use NGS to look at community profiles, but also wanted a more "conventional" DNA-based approach to come up with gene copy numbers.

 

I suppose there is always pros and cons to a method, and no one technique can explain everything sad.png


Edited by LabNerd, 17 November 2013 - 06:30 AM.





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