10 replies to this topic

[Adrian K]

Dear All,

As the title says it all: I was trying to develop a multiplex pcr which could be possible be done within 1 hour of time, and probably within 20 minutes if is possible. The multiplex pcr which i try to develop is for the use in clinical diagnostics to identify bacteria, using cultured broth or plate samples. My promising unoptimized trial was done in 25 minutes (initial protocol was 1hr 25 min), although with some bands become little faint (lower band) and some dimer appear which I have yet to troubleshoot. I had reported this to my PI but however, my PI was not very fond of this idea, and said 25 minutes or 1 to 3 hours doesn't really matters to her and asked me to forget about this idea.

How do you think?

My frustration was I don't see the reason why time doesn't matters in diagnostics. Was hogging the thermalcycler for long is a great idea when you can hastily done your PCR and get your result without (or maybe just slightly but not significant) reduce the data quality? How come my PI was discouraging? Weren't such optimization and improvement can contribute to a publication, perhaps a short communication? We need publications or else we are unable to present reports to grant committee for further grant extension, or subsequent grant application.

Your opinions are welcome. Thanks

Adrian

[lab rat]

Hello Adrian,

Are you using the Qiagen Fast Cycling kit?

Sorry I can't supply an answer to your questions regarding your PI.

regards,

lab rat

[Adrian K]

Hi lab rat,

Sorry, is not from Qiagen... my lab can't afford it...
Tried phusion flash and kapa 2G.
Adrian

Edited by adrian kohsf, 26 November 2009 - 12:54 AM.

[Maddie]

It matters a lot in Forensics. Say you have a sample and you catch the suspect. You can only keep him a day in jail I think. So, yes it can matter a lot. There is a guy at NIST that also developed a fast PCR assay. His name is Pete Vallone.

It also matters (in my community) because of the huge backlods the crime labs have.

[hobglobin]

It should also be court-proof, i.e. if the opposite side (or court-appointed appriasers) try to disprove it or try to raise doubts...

A fast thermocycler (fast heating and cooling, steep ramps) helps a lot to reduce times.. also the 10 minutes final elongation might be not necessary and all the denaturation cycles as short as possible...

Edited by hobglobin, 26 November 2009 - 01:06 PM.

[molgen]

I think that your PI wants to know what is new in this multiplex pcr that hasn't been dun before.
I recently came across this article that did the same sort of thing.

Doing the same just faster but less accurate is bad from the clinical side. Say you give a wrong answer to someone in sepsis, you can kill him. Better wait a bit more, and get a better answer.

[Adrian K]

The rapid multiplex PCR idea came when I observed the countless routine PCR had to be done by medical diagnostic laboratories and the heavy working load. So, I was thinking a faster PCR can reduce the overall and as well subsequent diagnostic time.

Yes, I do agree that accuracy is crucial, so as sensitivity and specificity. To diagnose a sepsis patient, most of the time direct blood PCR is not good enough as the cfu/ml is usually less than 1. Thus, as a golden standard, the blood should be (have to) culture overnight on plate media and use the visible colonies for detection and subsequent antibiotic sensitivity testing. A PCR by now is good enough to be done as we have far more colonies than required for the sensitivity. Serology or antibiotic susceptibility results is unable to be obtain by then, as the incubation have to be done for 2 more days. Furthermore a rapid pcr can use to confirm the diagnostic result by other methods which ambiguity can be easily happen (like API20NE).

The main "faster" effect is because of the different Taq and buffer, is not shorter PCR cycle (10-15 cycle) that many had thought (even my PI). That's the reason I don't understand why she couldn't take the idea...

How do you think?

[molgen]

Quote

The main "faster" effect is because of the different Taq and buffer, is not shorter PCR cycle (10-15 cycle) that many had thought (even my PI). That's the reason I don't understand why she couldn't take the idea...

I return to my original question:
What is new in this multiplex pcr that hasn't been dun before?

If it's only the speed of the reaction then why should she spend time and money on it?

[Adrian K]

Hi molgen, thanks for your kind reply...

Yes, is the speed of reaction, sensitivity and the need, and easy to perform which omitted the step of DNA isolation.

I was thinking come out a diagnostic and identification kit too.

The need is because conventional diagnostic takes too long (2-4 days), and most of the time the result is ambiguous and inconclusive.
Usually the admitted sepsis patient will pass away before the bacteria was being identified. In other words, the patient can't wait for any longer. Furthermore, a molecular diagnostic can be done in almost every where, just get the formula, do the PCR and get the result to determine whether it is a B.pseudomallei infection in a short while (from blood to plate culture to PCR outcome: less than 2 days).

I was thinking if this reason is not good enough, what else reason that I can justify in order to convince her? How do you think?

Edited by adrian kohsf, 01 December 2009 - 08:26 PM.

[molgen]

I'm sorry if I didn't make my self clear.

I cited an article and then asked if you did something different.
If nothing is different then your PI probably doesn’t want to spend time and money on it.
If there is a novel thing then try running it again by your PI with a better pitch.

[Adrian K]

Hi Molgen,

Thanks for keep me enlighten...I'll buy u a drink when you in Malaysia.

Compare with that article (although we aim at totally different organism), my pcr have 100% specificity of my target bacteria, and faster than conventional method which is not much reliable, although conventional method detect wide range of bacteria but lack of specificity which gives wrong diagnosis.

Anyway, I will try to convince her again.
If anybody can think of any better pitch, you are welcome to contribute....I need ideas....

Thanks...
Adrian