Mls 402 - Final exam study guide

Molecular Diagnostics Final Exam Objectives
Chapter 11
1. Define RFLPs and discuss how they are used in parentage testing and human ID.
 RFLP is Restriction Fragment Length Polymorphism
 There are fragments that have many polymorphisms and they are identified
to help with human identification
 They will identify SNPs, insertions, and deletions and determine size variants
 Southern blotting with be used, because it is DNA
2. Interpret an RFLP assay when given unfamiliar data.
 Sure, the plus signs indicate the cuts, the heavy ones will be at the top
3. Describe short tandem repeat structure and nomenclature.
 STR are usually 2 to 6 base pair repeats
 They can be simple, compound, or complex
 Mononucleotide, dinucleotide, trinucleotide, tetranucleotide, and
pentanucleotide
 Tandem repeats (x units)
 Nomenclature: within genes, would mean gene name, but with non-gene
association STRs: D#S# system (DNA, chromosome, segment)
4. Explain allelic ladders and interpret slab gel and CGE for STRs using allelic
ladders
 Allelic ladders must not overlap in the same reaction.
 Allelic ladders will tell the weights of fragments of one allele and weights of
fragments of another allele of the same gene
5. Explain the use of STR for bone marrow engraftment monitoring.
 STR is used for chimerism testing: pre-transplant informative analysis and
and post-transplant engraftment analysis
 Informative analysis- checking for each (donor and recipients’) unique loci
 Engraftment analysis- checking to see if the person is fully chimeric, half-
chimeric, or engraftment failure
 STR are scanned to find informative loci (donor alleles differ from recipient
alleles)
 Locus for monitoring should have at least one recipient informative allele;
detect small amounts of residual recipient cells
6. Differentiate informative, non-informative, donor informative, and recipient
informative loci.
 Informative is when they are very different
 Non-informative is when they are the same, and they serve no use
 Yes, with the graph one can tell, there will be percent calculations
 Using these informative loci, peak areas are determined in fluorescence units
or from densitometry scans of gel bands
7. Define mitochondrial STRs and summarize their clinical use.
 HyperVariable regions of the mitochondrial genome are sequenced
 With unrelated individuals 8.5 bases are different
 It’s used for legal exclusion of individuals or confirmation of maternal lineage


Chapter 12
8. Identify the advantages and disadvantages of using molecular-based methods as
compared to traditional culture-based methods for all types of microorganisms.
 Advantages
 Quick than culture-based methods

,  Hazardous organisms can be detected more safely with PCR
 Reliable than most other methods
 High-volume tests are done easily than culture-based methods
 Antimicrobial testing
 Disadvantages
 Due to high sensitivity and specificity, proper quality control is critical
for molecular testing
9. Recognize the importance of proper specimen collection and preparation for
molecular detection of microorganisms.
 Viability/nucleic acid integrity has to be preserved
 Contamination is bad
 Maintain appropriate time and site of collection
 Use the right equipment
 CLSI has guidelines for proper specimen collection/handling
10. Explain the value of and interpret controls, in particular internal amplification
controls, in ensuring the reliability of PCR results.
 Sensitivity control: positivity at lower limit
 Amplification control: omnipresent template to target
 Reagent blank: no template present
 Same primer binding sites/sequence with non-target derived insert, controls
for amplification, and degradation of template
 Heterologous extrinsic: non-target derived control (different organism),
second set of primers, and control for extraction and amplification
 Heterologous intrinsic: present in the sample (human gene control), and
ensures that human DNA is in the sample
11. Discuss how false positive and false negative results may arise during molecular
testing for microorganisms.
 False positives happen because of contamination, or dead, dying, or less
than clinically significant levels of microorganisms
 False negatives happen because of wrongly collecting/handling the specimen
(like not having RNases), extraction/amplification control (check internal
controls), and/or technical difficulties with chemistry or instrumentation
12. Explain the process of PNA FISH and it’s applicability in the detection of
microorganisms.
 PNA FISH: Peptide Nucleic Acid Fluorescence In Situ Hybridization
 Ribosomal RNA (rRNA) in microbes are looked at, backbone is a peptide and
resistant to nuclease degradation
 rRNA is less reachable by DNA probes
 PNA FISH uses AdvanDX, bioMeriux
 Positive blood culture  gram stain  PNA FISH
 Rapid ID of positive culture (2.5 hours)

, 13. Compare and contrast the principles used in the COBAS AMPLICOR Analyzer and
the Tigris automated analyzer for the detection of CT and NG.
Detection of CT and NG
COBAS AMPLICOR Analyzer Tigris automated analyzer
 Specimen prep  Hybrid capture
 PCR amplification  Transcription-Mediated
o CT= cryptic plasmid Amplification
o NG= M * Ngo PII gene  Dual Kinetic Assay Detection
 Hybrid capture (HC) o Two acridium labeled
o DNA target probles
o RNA probe o 2 different targets
 Colorimetric detection detected at same time
o Light is measured as
photon signals in a
luminometer and are
reported as relative light
units
o Involves Luminescence
curves (remember pic)

14. Classify antimicrobial agents and discuss the importance of molecular testing in
the context of resistance genes using manual and automated methods.
 Antimicrobial agents classifications: Inhibit growth (-static) and Kill organisms
(-cidal)
 Mechanisms include enzymatic inactivation of agent, altered target, altered
transport of agent in or out, and acquisition of genetic factors from others
 Analysis of Resistant Genes
 Methicillin-resistant S. aureus
 Vancomycin-resistant enterococci
 Multidrug-resistant M. tuberculosis
 PCR
 GeneXpert System (Cephid) one way
 Molecular diagnosis: Target Amplification
 PCR, RT-PCR, Real-time, and TMA
 Molecular diagnosis: Signal Amplification
 bDNA, Hybrid capture
15. Explain the purpose and the importance of viral load testing for HIV, CMV, and
HCV.
 The purpose is to determine the number of circulating viral units/mL
 Not for diagnosis, performed only after proven positive for analyte with a
different method
 Acute infection: at baseline and as needed to follow therapy
 Chronic infection: asymptomatic and symptomatic phase
16. Interpret the clinical presentation of a viral load assay (non-responder, partial
responder, relapse, sustained responder, breakthrough).
 Breakthrough means they responded partially
17. Identify the genes used to quantify HIV.
 LTR, gag, pol, vif, vpr, env, nef, tat, rev
18. Explain viral genotyping
 Viral genotyping is when viral genes mutate to overcome antiviral agents