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Probe Structure for the Molecular Laboratory Professional

The purpose of real-time PCR is to perform efficient amplification of a target sequence and quantify the PCR products in “real time” by employing the use of a fluorescent reporter.  Fluorescent reporters can found in the form of DNA-binding dyes or fluorescently labeled primers or probes.  It is extremely important to understand the difference between DNA-binding dyes, and the various fluorescent primer and probe based chemistries.  The best way to grasp these theories is often to have a visual illustration of each of the different chemistries.

DNA-binding Dyes

  1. SYBR Green Dye – SYBR Green I is a fluorescent DNA binding dye that is commonly used as it binds to all double-stranded DNA.
    • SYBR Green is detected by quantifying the increase in fluorescence during PCR.
    • Advantages to using SYBR Green are that it is inexpensive, easy to use, and easily incorporated into the PCR reaction.
    • Disadvantages of using SYBR Green are that there is usually an increase in background and non-specific binding that can lead to detection of false positive results.
probe1
Image courtesy of: http://www.sigmaaldrich.com/technical-documents/protocols/biology/sybr-green-qpcr.html

 Fluorescent PCR Primer and Probe Based Chemistries

  1. Taqman Chemistry – Utilizes 5’ – 3’ exonuclease activity of Taq Polymerase (enzyme that copies DNA and necessary for PCR) to generate a signal.
    • The probe is composed of a single stranded DNA oligonucleotide which is complementary to the specific target sequence of the PCR template.
    • The probe has a modification to the 3’ end so that the polymerase cannot extend the sequence.
    • The 5’ end has the fluorescent dye and the 3’ end contains the quencher
    • During DNA synthesis, the exonuclease activity of the Taq Polymerase will degrade the probe, thus resulting in release of the reporter from the quencher.
probe2
Image courtesy of: https://es.wikipedia.org/wiki/TaqMan#/media/File:TaqMan_GX_cartoon.jpg
  1. Fluorescent Resonance Energy Transfer (FRET) – Energy is transferred between two light sensitive molecules.
    • Increase in target à More probes bind à Increase in fluorescence
    • The 5’ end is the donor (catalyst) and the 3’ end is the acceptor (fluorophore)
    • The energy is detected in the form of heat or fluorescence emission.
    • If probes bind, energy is transferred from donor to acceptor and generates the signal.
probe3
Image courtesy of: http://www.cdc.gov/meningitis/lab-manual/images/chapt10-figure01.gif

 

  1. Molecular Beacon – This type of chemistry measures the accumulation of product during the annealing phase of PCR.
    • Signal is detected only when probes are bound to the template before displacement by the polymerase.
    • A chemical modification prevents degradation during the extension step of PCR.
    • The 5’ end contains the reporter fluorophore and the 3’ end contains the quencher.
    • The amount of fluorescence is directly related to the amount of initial template available for binding and inversely proportional to the cycle threshold (CT) value.
    • During extension, the probe is displaced by Taq Polymerase and the hair-pin (non-fluorescent) structure is restored.
    • Unbound molecular beacon probe à reporter is too close to quencher à no signal is generated.
    • Beacon probe binds to target à reporter is separated à signal is generated.
probe4
Image courtesy of: http://www.bio-rad.com/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/real-time-pcr-detection-standard-pcr-primer-and-molecular-beacon.gif
  1. Scorpion – Scorpion probes use two PCR primers, where one serves as a probe and once contains a stem-loop structure.
    • The stem-loop structure contains a 5’ fluorescent reporter and a 3’ quencher.
    • The loop of the scorpion probe contains complementary sequence to the internal portion of the target sequence.
    • If the primer binds and extends, the reporter is separated from the quencher and a signal is given off.
probe5
Image courtesy of: http://www.bio-rad.com/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/real-time-pcr-detection-scorpions-pcr-primer-probe.gif

Understanding the various primer-probe chemistries including the interactions between the reporters and quenchers will provide some basic groundwork for those interested in pursuing a career in molecular biology.

 

L Noll Image_small

-LeAnne Noll, BS, MB(ASCP)CM is a molecular technologist in Wisconsin and was recognized as one of ASCP’s Top Five from the 40 Under Forty Program in 2015.

Your Survival is in an Undisclosed Location

Over on NPR, Nell Greenfieldboyce writes about secret bunkers filled with healthcare supplies. You know, for the upcoming zombie apocalypse. (Okay, or maybe the next flu pandemic). It’s an interesting look at the logistics that go into managing stockpiles. Also, the author points out a sobering thought: while so much planning goes into inventory, dispersing that inventory in the event of an emergency could prove to be an issue.

 

Laboratory and Hospital Ebola Response

Laboratories are currently scrambling to define and put into place procedures for dealing with processing and testing of samples from highly infectious patients. The CDC has guidelines for healthcare workers and for laboratories specifically (http://www.cdc.gov/vhf/ebola/hcp/index.html). They also are very willing to help. Because Dallas had actual cases of Ebola, our hospital in Dallas mounted a hospital-wide response, in which the CDC and Texas State and County Health Departments were involved early on and throughout. This blog post describes the plans we instituted.

It quickly became clear that we did not want to transport infectious material through the hospital if we could avoid it, keeping everything infectious isolated in a single area. The hospital cleared an ICU wing which contained two negative pressure rooms, and the laboratory used an ICU room two doors away to create a mini-lab. The entire ICU wing was closed off as an isolation zone. No samples will leave the isolation zone unless they are headed for the CDC or State lab, and those will be couriered directly from the isolation zone.

All testing that can be, will be done on the I-stat in the patient room, including electrolytes, BUN, creatinine, ionized calcium and blood gases. A meeting was held with the ICU physicians who will be treating patients, to ask what testing they could foresee requiring other than those available on the I-stat. Their final list included platelets, CBC and coag tests, and originally also asked for ammonia and liver function tests. The only test we could not provide for them was ammonia. We couldn’t find a way to perform ammonia on a whole blood sample and had decided not to centrifuge any samples due to the possible risks of aerosolizing the sample and additional risks associated with aliquotting samples.

For the coag tests, we chose to use the I-stat PT/INR. Knowing that PT/INR on the I-stat is not FDA approved for anything other than Coumadin monitoring, we performed a full CLIA validation of the PT/INR in order to be able to use it for Ebola patients. Using the I-stat this way causes the PT/INR to become a high-complexity test, therefore only those individuals with appropriate licensure, training and competency will be performing the test at bedside.

Testing other than what is available on the I-stat will be done in the mini-lab set up in the nearby ICU room. It will be performed by lab personnel in full PPE, including PAPR (powered air purifying respirators), 3 layers of gloves, etc, all within the isolation zone. Lab testing in the mini-lab will occur once a day, with a possibility of twice a day. We purchased an Abaxis Piccolo for performing the liver enzymes and a Sysmex pocH-100i for the CBC and platelets. Both these analyzers will be run in the mini-lab room. The piccolo will be run inside a biosafety cabinet (BSC) which was put in the room because the piccolo is not a closed system. Sample pipetting into the piccolo carousel will occur in the BSC.

As far as blood utilization, the plan is to perform a one time, ABO only, blood typing on admission of a patient. A blood bank technologist in full PPE will perform the ABO only blood type manually in the BSC in the mini-lab. This ABO only typing has also been validated on samples allowed to settle rather than being centrifuged. The plan is for any patients to receive type O-negative blood if transfusions are required. However if they should require type-specific blood products for any reason (i.e. shortage of O-negative), it was felt that performing the blood type early before viral titers are really high would be better than waiting.

To work in the isolation wing, personnel must don full isolation PPE, including PAPR, etc, with a multi-step system in place for both donning and doffing the equipment. A buddy system is used throughout, with training on all procedures being continuous. The lab personnel who have volunteered to staff the mini-lab have undergone the PPE training. All of this perhaps excessive care is being taken in order to protect all other patients, as well as all healthcare team members, both lab and non-lab. Although Ebola may never reach our hospital, we live in a world where global travel makes if very likely that we will see patients with this or other highly infectious diseases appear in our facilities. It’s important to be as prepared as possible.

 

-Patti Jones PhD, DABCC, FACB, is the Clinical Director of the Chemistry and Metabolic Disease Laboratories at Children’s Medical Center in Dallas, TX and a Professor of Pathology at University of Texas Southwestern Medical Center in Dallas.

 

The Beginning of the Fellowship Trail

Well, last week my fellowship applications became quasi-complete. All my letters were sent for molecular genetic pathology (MGP) programs although I did find out that several never received them. Most of the hematopathology programs I am applying to had received three out of four letters of recommendation. And some did offer interview invites before the third letter came in (but most programs want at least three letters to consider my application complete for review).  Basically, I’ve been receiving one to two interview invitations by email each day.

I thought to myself that I should’ve started this whole process earlier. I thought that I had started early enough.  I had asked for letters of recommendation (LoR) informally before I transferred from my previous program in June and then formally asked at the beginning of August. I had intended to submit my applications on September 1st and hoped that the letters would be in by then or shortly thereafter. Throughout this process, I did find out that some programs began accepting applications as early as July 1st and some had deadlines of September 1st. I hadn’t been able to research as much as I would’ve liked before I left Chicago because I was on our busiest surgpath rotation (with over an hour drive twice a day and I only got the last day off) and I was also in the middle of moving cross-country.

I encourage you all to be early birds – to put everything together and ask early enough so that your application is complete (ie – letters are written and ready to be sent) by sometime in July, August by the latest.  Remember that your letter writers are probably writing letters for other residents are well, especially if they are your program director since every program requires a PD letter.

After I sent my applications in early September, some programs responded with a thank you email but most did not respond at all and so I wasn’t sure if they had received my application materials. I would receive weekly emails from a couple of programs telling me that my letters were missing but that was the exception and not the norm. So I would suggest following up with programs to make sure about the status of your application. I had no idea that programs were missing some letters I believed had been sent until I received an email from one. So then I called or emailed all the others to find that other programs were just waiting for letters as well to send my application for review.

Some of this confusion could have been avoided. I think that some of this happened because I added on additional programs – I was overcompensating thinking that I might not have applied to enough programs – and my letter writers weren’t sure which programs they had already sent letters to. Avoid this scenario by researching your programs early. Then make ONE master list of places you are applying to for your letter writers to have to (e)mail their recs all at once (at their convenience).

In terms of personal statements, programs seem to like them short and sweet. Most had limits of 500 words or one page and a couple even had a limit of 250 words. I found it very helpful to have multiple attendings in my subspecialty of interest and fourth year resident friends (who had gone through this process last year) read my personal statement and give feedback. You can write the personal statement any which way you like but the advice I received that helped me most was to write it in 3-paragraph (not too many sentences) form: 1) why/how I decided to pursue the subspecialty, 2) what I bring to the table in terms of the programs I am applying to, and 3) what I am looking for in a program and my future as a practicing pathologist.

As for CV’s, there is no one accepted way (like there is in the business world) to write a pathology-oriented CV (or at least that I know of; enlighten me if you know better). I’ve been updating my CV since college so I already had the basic structure. I’ve had a lot of leadership positions so much of it had to be abridged or left out when I composed my CV for residency applications so I didn’t need to do much this time around either. Speaking with one MGP fellowship director, it was suggested that I include my lab based skills on my CV since I have significant research/wet lab experience and she would like to see where I would start from in terms of my knowledge base. I already had this information from one of my previous job resumes so I just added it on to the end of my CV. So, if you are applying for MGP and have some skills in the lab, then highlight them!

The tricky part I’m finding is scheduling interviews. It’s not as easy to get time off during residency especially with call/tumor board/conference schedules and other service duties as when we had residency interviews as a medical student. So plan ahead and ask for lighter months during the interview season when your chiefs/PD compose the schedules for the coming year. I’ve found that it helps to call programs that are near each other to ask about the review process timeline once I receive an invite in a city where I applied to multiple programs. Let them know the situation and ask politely when a decision will be made so that you can schedule interviews in the same city/area together (especially if they are on the opposite coast from where you live).

There hasn’t been any method to the madness when it comes to each program’s interview schedule. One program emailed last week and asked if I could interview during their first round this week but I’ll be leaving for ASCP Annual Meeting for the whole week in a couple of hours. Other programs gave me dates to choose from in either October or November. For those programs which adhere to the CAP suggested deadline of December 1st (which I’m finding to be rare), those interviews occur in Jan/Feb but since I’m on rotations like surgpath where it’s difficult to get time off, I most likely won’t be able to make these. And with limited vacation days and finances to interview for 2 consecutive fellowships, I’ll most likely not be able to attend all the places I’ve received invites from for interviews.

I’ll let you know how it goes from time to time and hopefully someone will find my experiences on the interview trail useful.

Chung

-Betty Chung, DO, MPH, MA is a third year resident physician at Rutgers – Robert Wood Johnson University Hospital in New Brunswick, NJ.

Cryptococcus gattii and Trees

Today I stumbled across this story on NPR about the source of Cryptococcus gattii (formerly Cryptococcus neoformans var gattii) infections in California. The short answer: trees. What’s notable about this discovery? A high-school student (Elan Filler) is a co-author of the paper.

You can read the paper in PLOS Pathogens.

You can read about this organism, diagnostic testing, and treatment on the CDC website.

 

Swails

–Kelly Swails, MT(ASCP), is a laboratory professional, recovering microbiologist, and web editor for Lab Medicine.

Confirmed Case of MERS-CoV in the United States

Today, the CDC and the Indiana State Department of Health announced the first confirmed case of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in the United States at a community hospital in Munster, Indiana. The patient is currently in good condition.

If you’d like more information about MERS-CoV and how laboratory professionals should treat specimens from suspected cases, see Lab Medicine’s MERS information page.