lab final - intro to molecular diagnostics (cls 607)

molecular lab areas

• Pre-PCR area

  • Reagent preparation

  • Specimen preparation

  • slight positive air pressure to keep stuff from flowing out

• Post-PCR area

  • Amplification

  • Detection

  • slight neg air pressure to keep stuff inside area

(PCR lab rules & regulations) work surfaces & workflow

• Always work in a one-way direction from pre-PCR to post-PCR to avoid carryover contamination from PCR products

• Post-PCR should be kept as far away as possible from pre-PCR to avoid aerosol contamination

• working surface in each area must be decontaminated with 10% sodium hypochlorite followed by 70% ethanol before performing assay procedures in that area

• Specimens must be stored separately from reagents so as not to contaminate open reagents

(PCR lab rules & regulations) attire & specimen handling

• Lab coats must be worn in all areas; the coat worn in post-PCR must never be worn in pre-PCR

• When handling material containing DNA/RNA or amplicon, always use a pipettor with aerosol-barrier tips (or a positive displacement pipettor). Post-PCR pipettors must never be used in pre-PCR

• Centrifuges should be kept at a distance from areas where master mixes are prepared

(PCR lab rules & regulations) glove safety

• Gloves must be worn at all times for operator safety as well as for control of contamination from one area to another

• Gloves must be changed before moving to the next work area

• Gloves worn in the specimen preparation area must never be worn in the reagent preparation area

• Gloves worn in post-PCR much never be worn in pre-PCR

preventing contamination: AMP erase

• When setting up PCR reactions, substitute deoxyuridine (dUTP) for deoxythymidine (dTTP)

• Any contamination amplicons will contain U rather than T

• When setting up subsequent PCR reactions, AmpErase (uracil-N-glycosylase, or UNG) is included as a reagent

• UNG cleaves the deoxyuridine in the contaminating amplicon, preventing the contamination from being amplified

• template unaffected since it doesn't contain deoxyuridine

• UNG is heat-inactivated during the first PCR cycle, so the desired PCR product is protected

negative controls

a known negative specimen that monitors for contamination

positive controls

• A known positive specimen (high, low) monitors for assay activity, contamination

• A known positive specimen + patient specimen monitors for inhibitors

internal controls

• Control target added to a patient sample prior to extraction (often present in extraction reagents in FDA-approved kits) monitors extraction efficiency and presence of inhibitors

• No template (reagent control) monitors for contamination

quality control

• Commercially available kits:

  • The number of kit-based tests (both with and without FDA approval) is rapidly increasing

  • Manufacturers provide reference samples and reagent sets that have been tested for accuracy, precision, detection limits, interfering substances etc

  • New lot numbers of kits must be analyzed with reference samples prior to use on patient samples

lab developed tests (LDTs)

diagnostic tests created and used within a single laboratory

• Must be proven effective by in-house validation studies, e.g., sensitivity, specificity, accuracy, precision

• 2024: FDA claimed regulatory authority over LDTs (makes approval more difficult)

• 2025: a federal court struck down this rule

FDA approved molecular tests (pic)

hereditary hemochromatosis

• Genetic condition that causes too much iron to be absorbed

• Iron overload—build-up of iron in tissues and organs

• Two common mutations in HFE gene:

  • C282Y & H63D

• Most likely to have symptoms C282Y homozygotes and C282Y/H63D compound heterozygotes

hereditary hemochromatosis general procedure

• DNA extraction & dilution

• 2 PCR reactions for amplification of C282Y & H63D

• restriction enzyme digest of C282Y & H63D

• run products & PCR reactions on gel electrophoresis

• analyze

interpretation of hereditary hemochromatosis results

• C282Y mutation ADDS a restriction enzyme site

  • homozygous C282Y = three bands

• H63D mutation REMOVES a restriction enzyme site

  • homozygous H63D = two bands

normal result for hereditary hemochromatosis

• C282Y = bands at 74 & 176 bp

  • band at 74 bp is diagnostic of normal allele

  • uncut DNA will be at 250 bp

• H63D = bands at 53, 55, 99

  • band 99 is diagnostic of normal allele

  • uncut DNA will be at 207

heterozygote result for hereditary hemochromatosis

• C282Y = bands at 29, 45, 74, 176 bp

  • band at 45 bp is diagnostic of mutant allele

• H63D = bands at 53, 55, 99, 152

  • band 152 is diagnostic of normal allele

homozygote result for hereditary hemochromatosis

• C282Y = bands at 29, 45, 176 bp

  • band at 45 bp is diagnostic of mutant allele

• H63D = bands at 55, 152

  • band 152 is diagnostic of normal allele

hepatitis C virus

• Causative agent hepatitis C, viral infection of the liver that can lead to liver disease

• Enveloped positive sense RNA genome

• Serology and/or PCR used for diagnosis

(HCV) how to handle RNA?

• RNA is more sensitive to degradation compared to DNA

• Rnases are very stable enzymes

• Only use supplies/reagents that have been treated to inactivated RNAses

• Ideally, have a set of pipettors dedicated to RNA work

• Aseptic technique

HCV RNA extraction kit steps

• For purification of viral RNA from plasma, serum, urine, cell culture media or cell-free body fluids

• General steps:

  • Lysis of viral particles

  • Buffering conditions adjusted so that RNA binds to column

  • Contaminants are washed away

  • RNA is eluted

how does the QIAamp viral RNA kit separate DNA and RNA?

buffering conditions are adjusted so that the viral RNA preferentially binds to the column

• PCR will use primers specific for the RNA

• minimizes human DNA/RNA by getting rid of human cells—serum is used instead of other cellular samples

two step vs one step vs end point reverse transcriptase PCR

• two step RT-PCR: reverse transcribe RNA to DNA in one reaction, then set up PCR

• one step: RT-PCR: reverse transcription and PCR all take place in one tube

• end point RT-PCR: only looking for end product (is product present or absent)

quantitative RT-PCR

amount of DNA generated in each cycle can be used to quantify RNA in original sample

HCV gel electrophoresis results

positive result--band at 298 bp

• CANNOT tell viral load

• rxn lane w/o reverse transcriptase should NOT have product

  • unexpected bands may mean contamination or nonspecific amplification