Multiplex qPCR gives me the horn.

[MarkW]

To see why I'm excited you don't need to know the first thing about molecular biology, just that multiplex qPCR will do in 2-3 hours what currently takes us a minimum of five days, and will do it better. Start-up costs are in the region of £20K, with annual running costs around the same as our current systems.

I foresee a lot more time off for bike riding...

[ThePhatomfart]

Hi Mark , thats gone right over my head, you will have to explain it for me, I need my shoes off to count up to 19

[MarkW]

Ha ha!

OK, in very basic terms PCR (Polymerase Chain Reaction) is a molecular technique for exponentially amplifying tiny fragments of target DNA, which allows you (amongst other things) to say whether or not a certain organism is present in amongst a whole host of other organisms. It's a bit like finding a needle in a haystack. We spend a lot of time screening microbial pesticides for human pathogens, where we can be looking for a single viable pathogen spore among several billion spores of the test item, and currently that involves lengthy enrichment procedures followed by plating on selective media, incubation under various environmental conditions and then identification and enumeration in order to differentiate the contaminant from everything else in the sample, which takes days! PCR does away with all that, and allows us to identify whether a specific pathogenic organism is present in a sample or not in a couple of hours. Q-PCR goes a step further by doing it quantitatively and in real-time, which means we can say something about the amount of the organism in the sample as well. Multiplexing goes further still by allowing us to run up to five different pathogen screens in a single reaction, which speeds up our workflow even more.

We've been looking at bringing q-PCR into our lab for a while now, but given the type of work we do and the regulatory requirements of the studies we perform we had a number of regulatory and validation issues to address first.

[Guest]

PCR is often mentioned in police dramas.. particularly american ones.

[ThePhatomfart]

Think i got it , this process helps you to route out the bad guys that could cause humans harm, but with this you can do it faster, I know I'm being a bit simplistic but have I got it, I find it very interesting

[Guest]

Think i got it , this process helps you to route out the bad guys that could cause humans harm, but with this you can do it faster, I know I'm being a bit simplistic but have I got it, I find it very interesting

Not the only one mate

[MarkW]

You've got it!

As an example, the most widely used biopesticide worldwide is a bacterium called Bacillus thuringiensis. It's completely harmless to mammals, but once it gets in the alkaline conditions of the caterpillar gut it releases a potent endotoxin that kills the caterpillar stone dead. So this one is definitely a good guy as far as we're concerned.

A very closely related species is Bacillus anthracis, which is anthrax. That's not such good news from our perspective, and so one of the things we do is make sure that a product that is supposed to contain one doesn't contain the other. Separating them visually on agar plates is impossible - they look identical - so we either go through that whole faff I described earlier or we use PCR.

In PCR we take a primer (a sequence of DNA that is unique to the species you're looking for) and stick it in a thermal cycler with the test item. The thermal cycler unzips the two DNA strands, sticks the primer to the complementary bit of DNA and then starts replicating so that you get a measurable level of the target. If what you're looking for is in there you get a fluorescence signal that corresponds to the concentration of the target DNA; if it's not there you get nothing. Simples!

[ThePhatomfart]

Very clever that you can split DNA for that purpose, I suppose in the example of the caterpillar and leaving that part in which is harmless to humans ,but kills the caterpillars is of real worth to third world countries, my only problem now is trying to fit this into a conversation and trying to sound intelligent. Thanks for the explanation Mark

[MarkW]

No worries!

The DNA is only split to allow us to copy a specific part of it and identify the organism it came from though, not to 'engineer' the organism in any way.

Less developed countries certainly benefit from biopesticide technology, but you have to be careful what they make when they start their own production. This, for example, was isolated from a product we received for testing from Africa earlier in the year. Anyone want to guess what it is?

 

[ThePhatomfart]

I'd take a guess at E-bola

[MarkW]

Ha! Thankfully not: we can only handle organisms up to and including Biosafety Level 3 (that's stuff like hantavirus, HIV and bubonic plague). Ebola is a level 4 organism along with other really cool stuff like Marburg virus.

This was cholera. I briefly considered mass-producing it for people who wanted to lose weight, with a marketing campaign along the lines of Roger Mellie's best-selling diet book "Shit Yourself Slim".

[ThePhatomfart]

Bloody hell Mark ,I can do that with Guiness

[bonio]

This was cholera. I briefly considered mass-producing it for people who wanted to lose weight, with a marketing campaign along the lines of Roger Mellie's best-selling diet book "Shit Yourself Slim".

You're wasting your life away on bichemistry, mate. Think what kind a career you be having in an ad agency.

[Mr Fro]

I briefly considered mass-producing it for people who wanted to lose weight, with a marketing campaign along the lines of Roger Mellie's best-selling diet book "Shit Yourself Slim".

As a fellow scientist, I can say without doubt that you're on to a winner with this one.

While we're on Mellieisms, I think we should bring back "Thank w**k Arse Tits Monday".

Edited August 6, 2016 by Mr Fro

[MarkW]

As a fellow scientist, I can say without doubt that you're on to a winner with this one.

Since a good proportion of our customers could be expected to die on the toilet I thought an Elvis-themed advertisement would be good. Just need to come up with a suitable tag line...

[Mr Fro]

Since a good proportion of our customers could be expected to die on the toilet I thought an Elvis-themed advertisement would be good. Just need to come up with a suitable tag line...

Introducing the exciting new weight control formula: "I'm all shit up"!

[JRH]

As a fellow scientist, I can say without doubt that you're on to a winner with this one.

Since a good proportion of our customers could be expected to die on the toilet I thought an Elvis-themed advertisement would be good. Just need to come up with a suitable tag line...

 

Don't be cruel. Using Elvis

I'm leaving 1971.

Until it's time for me to go.

All shook up

But you would be better off with

Ring of fire. Johnny Cash

[MarkW]

This reminded me of an Elvis impersonator I knew when I lived in Stoke. I couldn't imagine how Elvis hits would sound with a Stoke accent, but I guess it could work:

Jaylouse Rock

Well that's o'rate

Artbreak 'otel (well since mar lady left me...)

American Trilogy (Live in Madison Square Garden version): Oh I wish I were in the lander cotton, ode times they onner forgotten, luke away, luke away, luke away Dixieland (sing eat mar mate... forky nell, thar's on thar own son...)

[ThePhatomfart]

Now Mark you forgot "me ducks" at the end of every sentence

[JRH]

Need the like button.

[OhJay]

You got a 5plex to work? bast*rd! What's your secret? I've got a 3 that play nicely together but adding anything else to it will then have them start competing and inhibiting each other. Titrated the primer concentrations and nothing can persuade it back to life so I've got to live with the 3 I use and any extras get their own run.

On the bright side I got a new rotor-gene out of work to run those extras

Made the mistake of mentioning it to one sales droid and he's been hounding me ever since about how they can support and help and he'll put me in touch with their tech guys if I start using their mixes...

I had to write an essay at uni trying to explain PCR to the uninitiated, borrowed my mum quite a lot to help me with that one

http://www.yourgenome.org/sites/default/files/illustrations/process/pcr_cycle_yourgenome.png

[ThePhatomfart]

Hi, have been watching this thread since Mark put it on, have found it really interesting , so much so that I have been reading up on the media, was looking at this, nearly all of it over my head but I'm getting the basic facts

http://thirdworld.nl/multiplex-qpcr-for-reliable-detection-and-differentiation-of-burkholderia-mallei-and-burkholderia-pseudomallei" onclick="window.open(this.href);return false;

[MarkW]

You got a 5plex to work? bast*rd! What's your secret?

Ha ha! We haven't even bought the machine yet!

Our bioanalytics study director had a couple of technical guys from Bio-Rad come to see us to find out what approach would work best for us. We liked the sound of 5-plex for its time-saving potential, but they did say that whilst running five targets simultaneously is possible it takes a lot of fiddling to optimise, so unless you're always looking for the same set of organisms it's not worth the effort and 3-plex is the way to go. I gave them the list of organisms we routinely look for in our pathogen screening and they thought 5-plex looked feasible. We shall see! But whatever we end up doing it'll be a vast improvement on agar plating.

In any case I'm just a simple entomologist - my role is to nod sagely and say "Absolutely" a lot whenever our study director is explaining this stuff to people.

[bonio]

In any case I'm just a simple entomologist

You mean an oxymorologist.