I'm at a molecular lab. My current entry was very generic because i didnt mention anything about the current outbreak. Now the situation is getting better so i think it is okay to touch on the touchy subject.
In the Molecular lab, our assays are base on Polymerase Chain Reaction (PCR). There are 2 kinds of PCR, Real Time PCR (RT-PCR) and the conventional PCR. I discuss extraction of Flu sample using RT-PCRin this entry. Will talk more about the probes on the next entry and after that will be conventional method.
As all may know, at the initial stage of the outbreak, everyone is very worried. Everyone with sore throat and fever wants to know if they are infected with the famous "swine Flu". Culturing of the virus takes a long time. The faster way to get the confirmation will be detection of the virus DNA. DNA works by having complementary base pair (A--T, G---C) the number of dashes indicates the number of bonds between the different complementary base pair.
Lets start on how we (the staffs) go about testing.
Test ordered: H1N1 PCR
Specimen Type: Swab in VTM media (If specimen is for PCr and Culturing)
Dry Swab (For PCR. swab nose then throat.
Protocol: If samples is from the campus (Within the hospital), Inform the lab by calling the lab, before despatching. This is a highly recommened step to do especially if it is a VIP sample or urgent sample.
From outside campus, liase with lab incharge. SHE/he will say either we receive from you or no we are not going to do it for you.
Once we receive the sample (In the lab), information will be matched from the order from and the specimen. If it matches (matches 99.9% of the time), swabs will be sent into the Biological Safety Cabinet (BSC). Order form will be keyed in.
2 tubes will be label, 1 2ml Tube and 1 1.5ml eppendorf tube. the 2ml tube will be for preextraction and the eppendorf tube will be needed for post extraction.
500ul (Micro litres) of PBS will be dispensed into the 2ml tube and swabs will be broken in the tube and tube vortexed. Swab will then be removed (this step is to dislogde the virus into the PBS solution). If it is in a VTM media, about 1.5ml of media is dispensed into the 2ml tube.
Extraction is done by a machine. it is able to run 24 sample at 1 time. For respiratory virus, for this case it is considered, the lab uses off board lysis (lysis will be done out of the machine.)
Lysis buffer will be dispensed into a different wells. 1 well for 1 patient. samples are added and incubated for 10mins. this allows the virus to be lysed. Magnetic beads are then added to the wells. The machine does extraction by using magnetic beads. DNA is -ve charge and the machine is able to manipulate magnetivity to extract DNA.
There is about 55ul of elute/DNA buffer. so 55ul of elute is transfered to the 1.5ml tube.
For real time PCR, it enables you to amplify and get the results at the same time.
Samples wil be added to cappillary tubes which contains a DNA master mix, (F-primer, R-primer, Taq Polymerase, dNTP and some other things which is from the commerical kit (TAQ MAN PROBE) which willl flouresce when to give a reading.)
The capillary tubes are put into the lightcycler. 50 cycles of PCR will be runned. PCR- Denaturation 95ºC, annealing 56ºC, elongation 72ºC. At the end of each cycle, a sensor will read the amount of light given out by the probes to give a reading. If it is a postive case, a sigmoidal curve will appear. Then we will bless him (just kidding). Positive or negative cases, reports will be sent out. If it is a urgent sample, we will call the consultant or doctor.
ALong with this test, a FLU A RT-PCR is runned on another machine as a confirmatory test. IF Flu A Pos, h1n1 pos = Good luck home quarrentine/ observation. If Flu A pos, h1n1 neg= no h1n1. If Neg Flu A, pos h1n1 = troubleshooting to be done by lab personnel, results inconclusive.
RT-PCR will enable the lab to get results in 4-6hours, unlike culturing which may take days. Therefore, molecular techniques is still more prefered in the campus.
Disclaimer: I will not be able to find out the quantity of reagents added. Above knowledge is base on my observation in the lab and hope i dont offend anybody.
Cheers,
Yeo Tiong Han
TG01
P.s. I will get pictures from the lab. Have to ask permission first. 15/08/09
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Hey man !
ReplyDeleteThanks for the interesting post. What are potential sources of variables that may affect the results ? What can be done to minimize them ?
Also, just out of curiosity, what precautions do you all take when you all handle h1n1 samples ? =)) lol.
Thanks !
Ng Tze Yang Justin
0703747F
Tiong Han.
ReplyDeleteWhy do you want to use Real-time PCR in diagnostic lab instead of just conventional PCR?
Li Yinliang Alex
0704894E TG02
Group 8
18 August 2009
hey TH,
ReplyDeleteis there any possibilities of PCR inhibitors in the reaction, and if so do you perform any control for it?
Cheers,
Yong Herng
0702243G
Hey Tiong Han,
ReplyDeleteWhat flourescent label do you use?
Yvonee
0703189A
p.s i know the answer for alex's qns and i'm like dying to answer LOL this is ridiculous -_-
Hey Yvonee,
ReplyDeletewhat do you mean by flourescent label? Basically, our results come out colour tagged by the machine. the mixture in the capillary tubes are COLOURLESS! It is the machine software that tagged the samples with colour. Virtually.
LOL!
To Alex:
Rt-PCR is alot faster, less labour intensive job. Faster as it combines gel and Amplification together, same for labour intensive.
To Yong herng and Justin:
TO Justin: Variables are inhibitors i think.
To both: We have recently received Tissues, ETT aspirate and BAL samples.
For tissue, we need to make sure that the tissue is Completely digested.
For the other 2 samples, we need to make sure that the viscosity is the same as WATER! so if it is like phlem, we have to digest the mucus with buffer (will check out what buffer, cant remember now. nt common samples)
As for other inhibitors, they include blood, wood from wooden swabs (Dont ask me where wooden swabs come from, Some pple use them and they are urgent, we cant reject, but will comment on the report.)
To Justin: we use to wear N95 mask (everyone in the lab wears that) but now just surgical mask. when handling samples at the BSC (Pre extraction) we wear yellow disposable gowns (use and Throw).
The buffer to digest the BAL and ETT is SDS.
ReplyDeleteCheers,
Tiong Han
P.s. Sry, above reply forgot to sign out.
How does the real time PCR works exactly? Time is required to heat, denature and reanneal. How exactly to get the result at the same time?
ReplyDeleteWhat does VTM stands for? IS VTM used specially for H1N1 virus?
Alvin
To Alvin,
ReplyDeleteVTM stands for Viral Transport Media. Regarding for H1n1 virus, i dont think so as it is a general transport media (to my knowledge).
During the PCR process, at the end of each cycle (reannealing) process. The light cycler will read fluorescence from the different capillary tubes. Also, the light cycler will plot a graph to show the degree of fluorescence. More DNA copies produced = to high fluorescence Level.
CHeers
Tiong Han
Hey Tiong Han,
ReplyDeleteWhat is the purpose of the VTM media? Are there other kinds of medias other than the VTM media for the culture? If yes, what makes VTM the desired media?
Felicia
0703345I
Hey Felicia,
ReplyDeletei have consulted my seniors on the purpose. It seems like they have no idea too. So here is my point of view, the purpose of using that media is just for transportation purpose. The scientific officers in the lab says it is okay to be transport in the media.
Basically, to put it in the media is to ease the workload on other labs which culture the virus.
As the name VTM stands for Viral Transport Media. so yea. (name says it all)
Cheers,
Tiong Han
Hope i answer your question