Can I do these tests at home? Parasites, chlamydia...

[Jordan Gallagher]

Hi! I'm new to this group. I am interested in doing the following types of tests at home, and would like to know if it's feasible? And what equipment I would need? At this point I haven't even bought a microscope yet.

Chlamydia and gonorrhea testing (nucleic acid or cell culture, from urine or swab)

Parasite testing from fecal and urine samples (giardia plus helminths / worms)

Any advice would be much appreciated, thanks!
Jordan

[Bryan Jones]

I'm not sure what the clinical standard is for these tests, but I believe you could test for all of these with a PCR based assay. The basic equipment you'd need to get started are:
Pipettor(s)
Thermocycler (i.e. "PCR" machine)
Gel box and power supply
Gel light box

Look for this equipment on ebay or your local university's recycling store. You'll also need a bunch of consumable like tips,  tubes, primers, polymerase, positive control DNA, agarose, DNA ladder, buffer salts, and dNTPs.

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[Jordan Gallagher]

Thanks Bryan! Sorry I'm such a newbie, but... Do I really need a lab space to do this? Meaning, a room in my house which is clean, sterile, temperature-stable? Or, if I'm careful (and maybe quick) can I get away with doing the PCR anywhere (let's say kitchen counter) as long as I clean the equipment and accessories thoroughly before and after use?

[Bryan Jones]

Best practices, especially if you are considering making decisions about your health based upon the results, would be to have a dedicated sterile/sanitized lab space. But there is really no reason why you couldn't do a PCR based assay on your kitchen counter. The PCR reaction itself needs to be tightly temperature controlled, but that's what the thermocycler does, fluctuations in ambient temperature of the room shouldn't be a problem.

On Mon, Jun 13, 2016 at 8:31 AM Jordan Gallagher <rapid...@gmail.com> wrote:

Thanks Bryan!  Sorry I'm such a newbie, but... Do I really need a lab space to do this?  Meaning, a room in my house which is clean, sterile, temperature-stable?   Or, if I'm careful (and maybe quick) can I get away with doing the PCR anywhere (let's say kitchen counter) as long as I clean the equipment and accessories thoroughly before and after use?

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[CodeWarrior]

as mentioned qPCR or rtqPCR should meet your needs. but you'll need pathogen specific primers. My understanding is you might have a hard time getting them. Modern DNA synthesis companies flag orders for sequences that match known pathogens as suspicious.

[hdsheena]

Giardia is detectable from fecal samples via microscopic examination if you do a floatation. It's pretty low tech, and you need to repeat it a few times to be sure you're catching a 'shedding' cycle, but with a decent scope, slides, coverslips and some floatation medium, you could do it in your kitchen (or maybe your bathroom....)

[Jordan Gallagher]

Thanks everyone! I have been dealing with giardia and its recurrence in my body due to lying dormant. It comes back a few times a year and I'm hoping to eradicate it with some herbal treatment, so it would be great to know if it's gone.

As for the chlamydia I was treated for it recently and need to be retested. But being of a DIY mindset I thought, why not test myself? A PCR test costs $250 each time in the lab, and false negatives are a possibility. So I was thinking to keep retesting myself and my partner to see if it appears to be still there.

But it sounds like the primers are the biggest obstacle to this idea. Plus there are 20+ strains of chlamydia trachomatis, from what I've heard, so if the primer is too specific, it will generate a false negative I think?

Also I have zero lab experience/skills, and reading about the NAAT process it seems daunting. Of course the thermocycler does a lot of the work, but as a lab novice/newbie I'd be clueless on the most basic steps of the process, unless I had someone walking me through it in person. It's fun to dream and imagine the possibilities, though.

[Gordana Ostojic]

PCR will amplify even smallest numbers of DNA, so I don't understand how it can be used for diagnostic purposes. I mean if you are sick from bacteria A, have million of them, and you have only 3 of bacteria B, and PCR does amplify bacteria B, wouldn't it lead to false conclusion that bacteria B is a culprit> 

[CodeWarrior]

qPCR doesn't just tell you what DNA you have but how much dna you have.

[Gordana Ostojic]

I understand that, but equipment-wise it is more complicated. DIY community does traditional PCR, at least I noticed that here, people mostly do pcr, gel electrophoresis for detection/verification. Maybe if one is really confident with the protocol, one can estimate original DNA amount by the electrophoresis line intensity, like compare to a known amount, but overall sounds very difficult.

[Bryan Jones]

You're right that qPCR is usually outside the scope of DIY bio, although the Chai Bioscience Open qPCR does bring it a lot closer ($3000 for the base model). You can do qPCR without the special florescence instrument though, like you suggested it would involve running bands of DNA on a gel and comparing the intensity. 
Traditional PCR would only test for presence/absence of the specific organism, it couldn't tell if that organism was the major cause of symptoms. That would be pretty useless if you are trying to test for a commensal organism that might be causing an opportunistic infection, but if you are healthy there should be zero giardia DNA in your urine, so I would think that a binary test should be sufficient.

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[CodeWarrior]

Surely the only hard part of doing qPCR is getting the fluorescent agent? A standard webcam with a coloured filter over it and a uv light in a dark room should work no? Especially if you have a negative controle to measure background illumination against.

[Bryan Jones]

That would be tricky. I'd be surprised if you could get the sensitivity & accuracy with a web cam and up light. But the bigger obstacle is that qPCR measures florescence between each of up to 40 cycles. So every couple minutes for a few hours you would have to pause your thermocycler, take out your samples, run to the dark room, take a picture, run back, put the samples back in the machine and unpause. Not impossible, but not exactly an efficient protocol.

On Tue, Jun 14, 2016, 4:09 PM CodeWarrior <code.w...@gmail.com> wrote:

Surely the only hard part of doing qPCR is getting the fluorescent agent? A standard webcam with a coloured filter over it and a uv light in a dark room should work no? Especially if you have a negative controle to measure background illumination against.

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[Daniel C.]

You couldn't rig up a 1080p webcam above the thermocycler and just have it take snapshots however often?

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[Gordana Ostojic]

How are they removing intercalated dye during or after the cycle? Also dyes are typically prone to bleaching under constant illumination and stability can be messed up during the thermal cycling. UV also requires careful setup, lots of things absorb the UV, including DNA. They probably use some other dye for intercalating.

[Bryan Jones]

The most common dye used is sybr green, which absorbs in the blue spectrum and, as the name implies, emits in the green spectrum.

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[CodeWarrior]

well actually I was suggesting you move the thermo cycler to a dark room and leave the camera running set up to take video. I'm guessing if the webcam  isn't sensitive enough you can just add more stimulating light.