Abour PCR sensitivity

Juan Pablo Martinez-Soriano jpms at IRAPUATO.IRA.CINVESTAV.MX
Thu Apr 18 17:47:41 EST 1996


In a recent discussion, Kevin mentioned:

>                               The work was carried out using purified
>virus and viral RNA rather than tuber material, however that will be the
>next stage.  The paper reports a 10-fold increase in sensitivity over
>ELISA. We have since increased that to 100-fold and believe that there is
>further room for improvement.

In recent experiments made at our lab, we have improved RT-PCR sensitivity
up to 1000-fold when compared with DAS-ELISA trying to detect Citrus
Tristeza Virus from FIELD samples. We have been using 2 sets of primers
(four oligos) that amplify two different regions of the viral genome but
yield same size fragments.
We have seen that sensitivity goes even way up if we combine ELISA and
RT-PCR. That is, RT-PCR made after ELISA.

>For many purposes it just doesn't make sense at
>the moment to use nucleic-acid based techniques when cheaper ones do
>the job just as well.
>
>However, post harvest tuber testing in potatoes may be an exception,

Well, you said it well. It depends on what is the bottom end of the
detection. In Mexico we are doing a massive survey where thousands of trees
need to be tested for CTV. It would be crazy to even thing to try RT-PCR on
each of them. We pool 5 to 10 samples (each coming from one tree) and do
ELISA on them, which was fine for some time. Suddenly, number of samples
and time became critical and we needed more fast and sensitive tools. We
know now that we can pool 100 (or more) samples and detect the presence of
one infected tree (even if 99 are healthy) with our RT-PCR. You can easily
see that cost is divided by 100 (or more) making the PCR test (per tree)
way cheaper than ELISA. Do not ask me why we do not do hybridisations.

See, potaoes are not exception anymore. Mexican citrus are along.

Best regards from sunny Mexico

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Juan Pablo Martinez-Soriano               jpms at irapuato.ira.cinvestav.mx
CINVESTAV Unidad Irapuato
Apdo. postal 629                               Phone [01152] (462) 51600
Irapuato, Gto, MEXICO                            Fax [01152] (462) 45996
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                                                *thanks to Gerard R Lazo





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