Verification of Infectious Disease Molecular Assays: A Self-Study Module

Nucleic acid amplification tests are increasingly performed in clinical and public health laboratories in the investigation of infectious disease. They are also used extensively in the Laboratory Response Network to detect agents of bioterrorism. The 2003 revisions to the CLIA regulations included requirements for the establishment and verification of performance specifications for molecular assays. This intermediate-level program will provide the learner with information on infectious disease molecular assay verification procedures and processes. The program begins by listing and defining the CLIA requirements, and continues by providing guidance and suggestions on how to comply. The program describes how one laboratory team established the performance specifications for an in-house developed assay and verified the manufacturer’s performance specifications for an FDA-cleared assay. It concludes with suggestions on how to report results from in-house developed assays that use analyte specific reagents (ASRs). Exceptions to the CLIA requirements that may apply to public health laboratories during public health emergencies will be outlined. The program is delivered using a documentary style. Learners will hear from experts in the field of molecular diagnostics who will answer the difficult questions that many laboratory scientists have about verifying molecular assays. Learners may test their comprehension and competency by completing a post test.

Molecular diagnostics links from UNC

The University of North Carolina School of Medicine, Dept of Pathology and Laboratory Medicine, has a nice collection of links for molecular diagnostics, including many tutorials.

Web resources for TB and Molecular Diagnostics

TB

• Francis J. Curry National TB Center – offers free training resources, including some online courses with CEUs http://www.nationaltbcenter.edu/index.cfm
• CDC Div of TUBERCULOSIS ELIMINATION (DTBE) – CDC’s main site for TB control http://www.cdc.gov/tb/
  *new* webinar on MDR/XDR-TB: http://www.cdc.gov/tb/CE/mdrandxdrtb/default.htm
• Global Health Facts — a project of the Henry J. Kaiser Family Foundation, with global statistics such as new TB cases, prevalence rates etc. http://www.globalhealthfacts.org/topic.jsp?i=12
• TB Education and Training Resources — a service of the Centers for Disease Control and Prevention (CDC), Division of Tuberculosis Elimination (DTBE), includes a comprehensive links page http://www.findtbresources.org/scripts/index.cfm
• Southeastern National TB Center – offers free training resources, including webinars with CEUs http://sntc.medicine.ufl.edu/

Molecular training sites

PCR

1. Click here to read about PCR and view an animation: http://www.dnalc.org/ddnalc/resources/animations.html (Estimated time, approximately 20 minutes)
2. This animation illustrates the difference between conventional PCR and Real-time PCR: http://pathology2.jhu.edu/molec/techniques_main.cfm## (Estimated time, approximately 10 minutes)
3. Real time PCR tutorial. This includes information on quantity and quality control efficiency curves: http://pathmicro.med.sc.edu/pcr/realtime-home.htm (Estimated time, approximately 20 minutes)

Sequencing
4. Click here to read about Sanger and cycle sequencing and view an animation: http://www.dnalc.org/ddnalc/resources/animations.html (Estimated time, approximately 10 minutes)
5. This interactive simulation will take you through the steps of PCR and sequencing a PCR product to identify bacteria. Click on Virtual Labs in the menu bar at the top of the page; then choose the Bacterial Identification Lab. The virtual lab requires that you have the Shockwave plug-in installed in your browser. You can click on Get Shockwave to download the latest version. Click on the picture to enter the lab and follow the interactive simulation. http://www.hhmi.org/biointeractive/index.html (Estimated time, approximately 40 minutes)

VNTR Applications

6. Click on this site to read an article about discrimination of Mycobacterium tuberculosis complex bacteria using novel VNTR-PCR targets: http://mic.sgmjournals.org/cgi/content/full/148/2/519?view=full&pmid=11832515 (Estimated time, approximately 10 minutes)

LUMINEX (LIQUID ARRAY)

7. Review this website to understand the Luminex process. Watch the short animation describing the basics, the instrument, and the benefits. You may also select applications from the menu bar at the top of the page to see a list of applications currently under development. http://www.luminexcorp.com/technology/index.html (Estimated time, approximately 5 minutes)

Verification of Infectious Disease Molecular Assays

8. Take this on-line self study course to learn more about the verification of infectious disease molecular assays. Click on “Enter Course” to begin. www.bttrain.org/molecular (Estimated time: approximately 45 minutes to complete)
9. View a lecture on introducing a New Molecular Assay Verification of Procedures in the Molecular Diagnostic Laboratory: http://wwwn.cdc.gov/nltn/pdf/2005/NLTN_031805.ppt#329,2,Difficulties with Verification of Molecular Assays (Estimated time, approximately 10 minutes)

Optional Materials

If you are new to molecular diagnostics, or you teach others who are new to molecular diagnostics, you may find these optional materials useful.
1. This is a comprehensive website on molecular genetics. If it has been a long time since your last genetics class, this will be a good review. The part of this website most applicable to the molecular diagnostics course is the section on the “Molecules of Genetics”. Click on Enter; scroll down on the right side of page to Molecules of Genetics, click on each of the links numbered 15-26 and read the Concept and then use the arrow to proceed through the Animation. http://www.dnalc.org/ddnalc/resources/ (Estimated time, approximately 40 minutes)
2. Persons not experienced with PCR will find these explanations useful. DNA primer – A good basic overview which also contains information on sequencing PCR products. Contains a glossary. Of special interest are the diagrams at the beginning of the glossary.
http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/ (Estimated time, approximately 20 minutes)
3. This site includes a number of tutorials on molecular diagnostic techniques including PCR: http://www.appliedbiosystems.com/support/tutorials/ (Estimated time, approximately 15 minutes)
4. Here is some additional information on databases. Click on GenBank on the left hand side; click on “Entrez” at the top to access the database (scroll down to view the databases). Also return to the homepage, and click on “Education” to view teaching resources and online tutorials. www.ncbi.nlm.nih.gov (Estimated time, approximately 10 minutes)
5. Click on this site and scroll down for more information on “Genetic Analysis in the Laboratory”:http://genetics.nbii.gov/portal/server.pt (Estimated time, approximately 20 minutes)
6.This site provides a “PCR and Multiplex PCR” guide to troubleshooting, standards, and applications: http://info.med.yale.edu/genetics/ward/tavi/PCR.html (Estimated time, approximately 10 minutes)
7.  Click on this site to access the Cambridge Healthtech Institute (CHI) glossary and taxonomy: http://www.genomicglossaries.com/content/sequencing_gloss.asp

Reference

8.  Review this site for more information on microarrays: http://www.ncbi.nlm.nih.gov/About/primer/microarrays.html (Estimated time, approximately 15 minutes)
9.  Review more information on Nucleic Acid Research Group (NARG): http://www.abrf.org/index.cfm/group.show/NucleicAcids.32.htm (Estimated time, approximately 10 minutes)
10. Click on this site for a VNTR demonstration to try out a microsatellite repeats finder: http://biophp.org/minitools/microsatellite_repeats_finder/demo.php?PHPSESSID=0add18017be98afc0b72b487ddcc8530 (Estimated time, approximately 10 minutes)