BIOL 325 DETECTION AND IDENTIFICATION OF MICROORGANISMS STUDY GUIDE

what is molecular genetics?

• studying the molecules that are mutated in genetic disorders like neuromuscular disease (ALS) which is the most common place where molecular genetics is used, muscular dystrophy, endocrine, and cardiovascular

• diagnosis of existing disease or predisposition

what is molecular oncology?

• looks at hematological cancers and solid tumor cancers

• markers and specimen collection will be different, but the tests have some overlap

• huge number of tests performed for oncology/cancers

• gene defects, expression profiles, etc

what is pharmacogenetics?

• fusion of genetics and drug metabolism

• identification of metabolic gene variants (slow, average, fast, and non-responders) to optimize drug therapy

• studying the genes that changes the metabolism of the drugs

what are genomics?

• uses genomic information (gene expression and gene pattern) for disease susceptibility, diagnostics classification, prognosis and optimal therapy

• identification and characterization of individuals

what are the five target microorganisms for molecular-based testing? give examples

it is everything from arthopods to prions which include:

• those that are difficult or time-consuming to isolate and culture

  • assay results need to be out as quickly as possible

    • e.g. M. tuberculosis and C.diff

• hazardous organisms

  • e.g. histoplasma and coccidiodes

• those without reliable culture methods/hard to culture

  • e.g. HIV and HCV

• high-volume tests

  • e.g. S. pyogenes, N, gonorrhoease, and C. trachmatis

• gene targets which analyze a particular gene

  • mecA for S. aureus (exacillin), van for enterccoccus (vancocyin), inhA for tuberculosis (isoniazid)

what are the 6 applications for molecular based tests in clinical microbiology?

• rapid/high-throughput (checking lots of patient samples at the same time) ID of the actual microorganism itself

• detection and analysis of resistance genes like antibiotics

• genotyping

  • identify what strain an individual has to change the therapy

• classification which allows to new infections

• discovery of new organisms

• epidemiology

  • tracks down individual that started the new infection

what are the four procedures for molecular based tests in clinical microbiology?

• traditional, real-time and reverse transcriptase PCR

• DNA sequencing

  • a bit more expensive and need to know what you are sequencing

• PFGE (pulsed field gel electrophoresis)

• Immunobased methods

  • ELISA, Latex Agglutination, immunofluorescence, immunoelectrophoresis

what are the 5 steps for specimen collection? explain them

• preserve viability/nucleic acid integrity of target microorganisms

  • most important part in molecular diagnostics because researchers only care about the DNA or the RNA integrity

• avoid contamination

  • if trying to culture a living cell, contamination isn’t as critical

• appropriate time and site of collection (blood, urine, and other)

  • some organisms that are analyzed will show immediately in their blood but might take weeks to show in their urine

• use proper equipment (coagulant, wood, dacron, plasma swab shafts)

  • different things adhere to different types of swab

• commercial collection kits can be used and are avaliable

what does the clinical and laboratory standards institute (CLSI) have?

has guidelines for proper specimen handling but it applies to handling the tests first then analyzing the sample

describe the necessary steps during specimen and sample preparation (written response question)

The necessary step for specimen and sample preparation is to consider the specimen type like if it is stool, plasma, or cerebrospinal fluid. Depending on what the specimen is, sometimes more rigorous lysis procedures are needed to penetrate the cell walls. Another necessary step is to consider the number of organisms in the sample. Another step is to inactivate inhibitors like acidic polysaccharides in sputum or polymerase inhibitors in CSF, inactive RNAses if analyzing gene expression, or inactivate hemoglobin in whole blood as they also inhibit DNA polymerase.

describe the three common steps of all PCR reactions (written response question)

The first step of all PCR reactions is the denaturing step where all of the sample is turned from double stranded to single stranded. The second step of all PCR reactions is the annealing step where the primers anneal to the target of interest and must be complementary to the strands. The third step of all PCR reactions is extension where the polymerase extends the fragment by reading the template and throws in the right complementary template until it falls off or time runs off.

what are the 7 techniques for direct study of microbial genome?

• PFGE

• MLST (multilocus sequence typing)

• MLVA (multilocus variable number of tandem repeat analysis)

• ribotyping which is the stability of 16S and 23S rRNA

  • have both conserved and variable regions

• rep-PCR → repetitive sequence base PCR

• DNA microarrays

• WGS

understand the procedure for PFGE (written response question)

Pulsed Field Gel Electrophoresis tests and compares other samples to the original outbreak strain to see if a mutation/genetic drift is going on in the strains or if an individual has the same strain. The other samples could be from wastewater or a patient. PFGE compares the fragment number and size, and a dot on the assay indicates that the fragment is different from the original outbreak strain, which also indicates the absence of a band. It is the gold standard for typing bacteria. There are guidelines for interpreting the PFGE patterns like if the strains are related, closely related, etc.

what are the guidelines for interpreting PFGE patterns?

• indistinguishable

  • genetic differences = 0 and fragment difference = 0

  • same strain as the outbreak strain

• closely related

  • genetic difference = 1 and fragment difference = 2-3

  • closely related to the outbreak strain

• possibly related

  • genetic difference = 2 and fragment difference = 4-6

  • possibly related to the outbreak strain

• different

  • genetic difference = more or equal to 3 and fragment difference is more than 6

  • unrelated to the outbreak

describe the various types of PCR control reactions (written response question)

Positive control is a positive template and is used to ensure that the procedure, reagents, and machine are working and typically comes with the kit. Negative control has no template or the expected result is zero. The reagent blank is a type of negative control where no template is present and whatever the nucleic acid was resuspended in, it is added to the PCR without anything. Amplification control is omnipresent meaning it is added into every test tube and has a template unrelated to the target. It has its own primer and template, should always work, and is used to make sure reagents, machines, and polymerase are always working. PCR also has internal controls that monitor the PCR steps. Homologous extrinsic controls for amplification and primer binding but sequence that is being amplified is different. Heterologous extrinsic controls for DNA extraction and amplification but the target is unrelated and different primers are used. Heterologous intrinsic is a nontarget that is naturally present DNA and is used to know if DNA was extracted beautifully, no contamination, and if the primers, PCR machine, and polymerase works.

what is the same and what put inside the target in homologous extrinsic? what is sent? why is it a great control?

• primer binding sites are the same as whats in the target but a foreign sequence is put inside which is typically a plasmid

• a manufactured plasmid with the primer kits is sent and there are primer binding sites in the plasmid

• modified version of target that maintains primer binding sites

• great control = makes sure primers and everything in the reaction works and is given a sequence of DNA that will amplify it

what is different in heterologous extrinsic? why is this spiked and what is the condition to where it can work?

• everything is different, such as primers and template, and the product mus be distinguishable from target

• spiking this during the extraction step to make sure the DNA is extracting well

• only works if the PCR product is a different size than the target because the same size will overlap

what is the method for heterozygous intrinsic? what does it not spike and what does it tend to be? what is looked for in this internal control? what is different in this in internal control?

• mulitplexed or run 2 separate reactions

  • need one rxn with correct primer and one with no primer control

• does not spike foreign or extra DNA and tends to be things that were always there like the gene that makes beta actin

• in the same DNA that was isolated, looking or a different target which tends to be called a housekeeping gene

• a different host sequence is used as a target in the same reaction

  • different primers to a gene in the same DNA

what are false positives in quality control? what are the 3 causes of this?

positive result from the patient but it is actually negative

• contamination like pipette was contaminated, the air had DNA from the prior isolated, or the same reagent blank was used

  • reagent blank should always be changed

• dead or dying organisms can still be amplified and be analyzed by PCR but not grown which might give a positive result even when the bacteria is dead but the infection as cleared up

  • retest by PCR 3-6 weeks after antimicrobial therapy

• detection of less than clinically significant levels

  • sometimes the PCR methods are too sensitive like MRSA can be cultured and PCR amplified to thousands of DNA which can leave to false positives

what are false negatives? what can cause them?

negative result when an individual is positive for the infection

• improper collection or specimen handling like losing the DNA

• extraction/amplification failure

  • check the internal controls

• technical difficulties with chemistry or instrumentation

  • check the method and calibrations of the instrument

what does validation of an assay mean?

results are sensitive, specific, and reproducible by FDA standards

how many methods have been FDA approved and what do they test for (6 things)?

• 32+ FDA approved methods

• tests for

  • bacteria

  • virus

  • viral types

  • mycoplasma

  • fungi

  • parasites (usually protists)

why should you buy a kit? what does the design of the primer have to be? what is in one species?

saves headaches

• the kit can target unique targets

  • many bacterial genes are conserved

• the kit will have primers for the sequence of interest

  • the design needs to be tailored to the sequence of interest like if it is DNA, RNA, or protein. the primers need to design labels, conditions, and reproducible (primer consistently produces the same result)

• in one species, they will have variable sequences

how many respiratory pathogens are routinely monitored in medical laboratories?

>12 respiratory pathogens

what are the 3 techniques for identifying viral infections?

• by presence/absence of viral antigens or antibodies

  • look for antigens of the virus or find the antibodies to the antigen that attack it

  • check antibody titers also means to ID recovery from virus

• molecular tests

• old school, culturing

  • requires more time, often leads to false negatives because growing and population enough cells to look at takes days

  • not all grow in culture

  • look for cytopathic effect, adding patient specimen to cell culture like how much virus is needed to kill a cell

name some common viral pathogens, how are they routinely tested for? viral load testing? antiviral resistance testing? (written response question)

HIV, EBV, HPV, and HCV are all routinely tested for using a molecular test. Influenza, MPV, Coronavirus, HCV, West Nile, and MMR are routinely tested for by using an RT-PCR test as they are RNA-based viruses. Viral load is often tested through quantitivate RNA testing. For antiviral resistance testing, a researcher has to know the sequence of a genome in order to sequence a known/particular area of a genome. The genes for antiviral resistance testing are highly mutable, so it may not have a kit.

what are the two mutations when monitoring of antiviral drug resistance?

• primary resistance mutation = specific for a particular drug and directly created the drug insensitivity problem

  • first mutation that happens, mutation occurs, and medication no longer works

• secondary (compensatory) mutation = try to recover the virus’s ability to replicate

  • mutation in the gene happened, and now the drug doesn’t work, so try to change another gene so it can replicate and proliferate

why is genotyping critical? what does CAP provide? what is HIV common for?

• critical to detect primary vs secondary resistance mutations

• CAP provides proficiency testing and independent controls

• HIV is common for error-prone replication of its reverse transcriptase and drug resistance

what are the four viral load measurements?

• sensitive (LLD) which is set by the DHHS and international AIDS society

  • used to detect the lowest limit of detection of the assay and sets the measurement of the level of sensitivity needed

• accurate which is the closeness to a standard of known value

  • spike a control with viral particles and it should constantly show what each particle output looks like

  • ability to determine true value

• precise = reproducibility of independently determine results

  • produces a consistent result

  • positive is always positive

• specific = controls should always give correct answers

  • negative is always negative

what are the assays for HIV and HBV?

RT-PCR, NASBA, and bDNA

what are the assays for CMV?

NASBA and hybrid capture

what is the assay for HSV, EBV, and HBV?

qPCR

how is multiplexing used? what is it used for?

• used because a lot of viral infections have the same phenotypes and you cannot tell them apart from each other

• 10 flu strains, MPV, rhinoviruses, adenoviruses and ASR NASBA for RSV

why is viral load testing important in HIV? what are the 3 methods used for determining the viral load? explain them

• should be determined prior to and for therapy

• amplicor-PCR which has high specificity but has limited dynamic range

  • viral load can vary in a HIV+ person

• NASBA which has a broad dynamic range, doesn’t detect all subtypes

  • will not pick up some mutant forms of HIV which needs a specific primer-probe sequence

• bDNA which has high throughput and dynamic range but false positive are common

  • does good at low levels but want to confirm it with another assay

what is the first and second step in HIV testing?

• first = identified in an EIA using antibodies specific for HIV

• second = viral load is determined for therapy with many methods

how many herpes viruses are there? how is it tested?

• 25 different Herpes viruses → CMV, EBV, HSV, VSV

  • often dormant and will only express in certain conditions

• the test must distinguish closely related viruses and it is amplification based

• needs unique assays to distinguishing them from each other so specific primers are needed to bind to unique sequences to amplify them

what is the tests for HCV?

• first test for antibodies by western blotting

• next test looks for an active infection

  • an antibody does not indicate an active infection because antibodies can stay even when the infection has fully cleared

  • need to look for an antigen to see if there’s an active infection

how many subtypes of HPV are there and what do some of them cause?

• over 200 HPV subtypes

• some cause cervical, anogenital, squamous cell

what is the most common microorganism that causes clinical infections and has large diversity? why?

• fungi/fungal infections

• lab personnel have high likelihood of lab-acquired infections because of the spores

what is the common detection method of fungal infections? what do thick walls require?

• lab smears and culture isolation

• thick walls require harsh chemical/physical isolation of NA but cannot be too harsh or else the nucleic acid will get destroyed

name some parasitic pathogens, how are they routinely tested for? (written response question)

Some parasitic pathogens are trypanosomes, plasmodia, toxoplasma, and giardia. These parasitic pathogens are routinely tested for with PCR-based molecular methods; however, the testing is limited because there is limited expertise in identifying them. Multiplexing is needed for patients with multiple intestinal parasites at the same time.

name some bacterial pathogens, how are they routinely tested for? (written response question)

Some bacterial pathogens are B pertussis, M tuberculosis, C pneumoniae, and Strep. The specimens are collected through nasopharyngeal, sputum, and blood. These bacterial pathogens are routinely tested for with 16S rapid as culturing takes too long. Another test that can be routinely done for bacterial pathogens is VNTR and MALDI-MS.

for urinary/genital pathogens, what are the first nucleic acids targeted by molecular methods? what are the various ways urinary/genital pathogens are collected? what is necessary for numerous mycoplasma?

• N. gonorrhoeae and C. trachomatis

• nasopharyngeal, sputum, blood

• for numerous mycoplasma, multiplexing is necessary

what are some key vocab terms involved in antimicrobial resistance? (written response question)

A key vocab term for antimicrobial resistance is the suffix -static, which refers to drugs that inhibit growth. Another key vocab term is the suffix -cidal which refers to drugs that kill organisms. In vitro susceptibility testing is another key vocab term used to determine the minimum inhibitory concentration needed to kill a bacteria.

what are antimicrobial agents classified by?

1. -static/-cital

2. mode of action

3. chemical structure

describe the genes that encode resistance to antimicrobial agents

• enzymatic inactivation of agent (bla)

  • bla gene gives some bacteria resistance to ampicillin so would test for bla before giving a patient ampicillin

• altered target (vanA, vanB, vanC) carried cell-2-cell on transposon and plasmids

• altered transport of agent in or out

  • porin genes which takes the drugs in through pores and spits the drug back out

• many genetic factors are acquired from other resistant organisms HUMANS select for resistance by using chemicals

what are the advantages of molecular detection of resistance to antimicrobial agents?

• in vitro susceptibility testing to determine the MIC and must give the patient the MIC

• rapid detection without culturing which is faster, more reliable, and safer then susceptibility testing

• mutated genes show stronger resistance like in MDR Tb

  • have to identify mutation if you know what kills the infection

• comparison of multiple isolates in epidemiological investigations

• multiplexing for the various van genes which is useful in VRE

does identification matter if you know what kills or inhibits it?

ID matters because by learning what mutations it has it helps predict future medications and how to treat

what is the proprietary PCR for rifampicin resistance in TB?

• takes days to week for ST to rifampin, streptomycin, etc

• purifies and concentrates TB from sputum

• isolates the gDNA by sonication

• amplifies rpoB gene using molecular beacons

• 90 min to identify TB and resistance to rifampicin

what is QuantiFERON-TB Gold (QFT)?

• it is a simple blood test that aids in the detection of M. tuberculosis and is the golden standard

• a bit more expensive but only needs one visit unlike the TB skin test

what is the process for QuantiFERON-TB gold when blood is drawn?

• the blood is drawn from the patient and antigens are added to the blood

• incubate the antigens with the persons blood and if the person has an acute TB infection, they will create interferon-gamma

• sample on ELISA well and results are analyzed

what is the process for QFT relating to antigen processing cells (APC)?

• APC is exposed to antigen and it takes up the antigen

• APC processes the antigen

• APC presents the antigen to antigen-specific T cell

• the antigen-specific T cell produces the interferon-gamma

• sample on ELISA well and the results are analyzed

why is the TB skin test not good?

it picks up the antibody reaction and anyone who has had TB in the past will have an antibody reaction even if there is no active infection and needs a second visit

what is stimulated with Mtb antigens?

lymphocytes are stimulated with Mtb antigens in the phlebotomy tube and no booster effect from repeated testing as in the case of TB skin test

what is the history of penicillin and methicillin?

• penicillin was discovered in the 1940s and developed resistance even back then, even when it wasn’t overprescribed

• methicillin is a close cousin of penicillin and was made to combat penicillin resistance but started gaining resistance after being exposed to all the mutations

what is PBP2 and how is it related to resistance?

• PBP2 is penicillin binding protein which is a mecA gene product

• it is the gene that was discovered to be responsible for resistance

• give an individual penicillin, PBP binds to it, and creates resistance

how is beta-lactam resistance detected? what do MALDI-TOF-MS peptide profiles distinguish?

• detected by amplification

• MALDI-TOF-MS peptide profiles can distinguish MRSA, VRSA, VRE

what is the problem with growing cultures?

often times, the incorrect conditions are used to try and grow cultures which won’t allow things to express which can lead to diagnosing things incorrectly

what is the definition of epidemic?

rapidly spreading outbreak of an infection disease that is isolated to a particular area

what is the definition of pandemic?

a disease that sweeps across wide geographical areas

what is the definition of epidemiology?

collection and analysis of environmental, microbiological, and clinical dara

what is the literature definition of molecular diagnostics? what is the use of it? what is the important part of it?

• the use of molecular methods for infection agents in order to study the distribution, dynamics, and determinants of health and disease in human populations

• used to track diseases

• the important part is it is often done to figure out if some infections are acquired independently or acquired from the hospital/same source

what is combined in molecular diagnostics? what aids?

• combines traditional mehtods with analysis of pathogen polymorphisms over time and place across human population and reservoirs

• aids in the study of host-pathogen interactions and infer hypothesis about host-to-host or source-to-host transmission

what is used to determine relatedness of multiple isolates in molecular epidemiology? what are the sources of genotype information?

• look for phenotypic similarity which requires culturing (serology, MALDI-TOF)

• genotypic → ribotyping, PFGE, analyzing targeting genomic or plasmid DNA

• species or strain-specific DNA sequences are the sources of genotype information

what are plasmids? what is the hope for them?

• plasmids are naturally found in bacteria and it is an extra chromosomal piece of DNA and is where scientists found them first

• small and easily to manipulate

• hope for them is to try to figure out what kind of infectious organism a person has

understand ribotyping (written response question)

Ribotyping is a common technique for bacterial characterization and identification of genotypes of rRNA. Ribotyping sequences bacterial ribosomal genes, more specifically 16s subunit rRNA gene, which are unique to each species, which in turn acts as a genetic fingerprint for different organisms. It is widely used in clinical diagnostics and the analysis of microbial communities in food, water, and beverages. Ribotyping can use old-fashioned methods such as blotting the digested gDNA with 16s and or 23s rRNA probes. More recently, it uses PCR primers to 16s or 23s rRNA genes to visualize on a gel with known standards with different bacterial strains to compare the sample to. It is rapid and specific.

what is the 16s and 23s rRNA gene?

• they are consistent within a particular species even with new strains and has areas that are not consistent but variable

• good to have both

what is the overall idea of ribotyping?

• can be used in different diseases like P. multocida and C. diff to see if strains are the same or different/have genetic drift on a gel or through MALDI-TOF

  • MALDI-TOF was able to see 10 ribotypes of C. diff in 95 different patients by identifying the peaks in the chart but also through a gel

understand the procedure random amplification of polymorphic DNA PCR (RAPD-PCR) (written response question)

RAPD-PCR is also called arbitrarily primed PCR and uses arbitrary 10-mer primers to amplify a target organism’s DNA sequence randomly. The 10-mer primers prime less spots; however, it should amplify any random sequence from some location in the target organism. When analyzed on a gel, RAPD-PCR looks at a strain like an outbreak strain and how it mutates as it exists and passes throughout a population by looking at any strains with genetic drift. It does not identify the organism and is overall trying to be quick and look at different patients to see what strain is infecting them.

what is RAPD able to do? what is it used for?

• RAPD is able to differentiate between genetically distinct individuals, although not necessarily in a reproducible way since it randomly primes

  • looks for regions that tend to be different between different species or genomes of bacteria (polymorphic)

• it is used to genotype microorganisms by comparing to ATCC strains which is a collection of cultured cells

what does “nearly all RAPD markers are dominant” mean? what is rarely detected?

• RAPD markers are the polymorphic regions and dominant means it tends to be common in human genes too

• cannot distinguish whether a locus is hetero or homo, just trying to ID the species

• codominant RAPD markers, observed as different sized fragments amplified from the same locus are rarely detected

what are the advantages and disadvantages of RAPD?

it is fast and inexpensive but it has low reproducibility

what are housekeeping genes? how was RAPD and multiplex PCR used with this?

• gene that all bacteria should have to survive

• RAPD and multiplex PCR looked at strains of Paramecium to see if any strains had genetic drift

  • is more sensitive when combined

understand the procedures for ERIC, REP-CPR (written response question)

ERIC stands for enterobacterial repetitive intergenic consensus, which is an interspersed repetitive element with 126 base pairs and mobile DNA particles. They are conserved, not transcribed, and flanked by inverted repeats. In ERIC, it utilizes primers that will bind to the enterobacteria sequences, and the gene next to it is looked at to distinguish it from the other different species of enterobacteria. REP-PCR stands for repetitive extragenic palindromic sequence PCR is 36 base pair fungi and has a similar process to ERIC. Primers will be designed to the REP sequence, and PCR will amplify a unique part of the sequence to see what strain it is.

what is amplified fragment length polymorphism (AFLP)?

• a highly sensitive method for detecting polymorphisms in DNA

• widely used to ID genetic variation in closely related species of plants, fungi, animals, and bacteria

• is a misnomer = results are not scored as length polymorphisms but as presence-absence polymorphisms

• reported to be faster than PFGE for strain analysis and may detect more polymorphisms than RAPD

understand the procedure for AFLP (written response question)

Amplified fragment length polymorphism digests gDNA with restriction enzymes and ligates adapters to the sticky fragment ends of the digested gDNA. The selected fragments are then amplified using primers that correspond with only the designed adapter of your choice and the restriction site sequences. Electrophoresis is done to separate all the restriction fragments from each other and a computer will be used to detect whether there is a presence or an absence of a band.

what does in silico PCR refer to? what does it mean and what can it do?

• refers to computation tools used to calculate theoretical PCR results using a given set of primers (probes) to amplify DNA sequences from a genome or transcriptome

• means that computers do analyses with databases to see what the theoretical results should be if the outbreak is the theorized bacteria

• gives an idea of what primers should be tested with, what REN should be tested and can figure out how to set up the experiment using the computational tools

what differs in software packages?

ease of use, efficiency, and cost

what are internal transcribed spacer elements?

• amplifies rRNA genes,→ the most conserved genes in the genome and a few variable sequences

• within an operon with intergenic spaces

• sequence or SSCP analysis

what is spa typing?

• looks for staphylococcus

• MRSA → VNTR (spa element) of repeat units 21-24 bp long each

• the gene that is being typed during spa typing is Protein A gene

• sequence Protein A gene or PFGE to figure out what kind of S. aureus an individual has

• needs to figure out if it is MRSA ASAP

what is multilocus sequence typing (MLST)

• looks at 7 locations and uses 7 primers for housekeeping genes, sequence all, and compare to a database

  • should be able to discern what species is being looked at

• highly unambiguous and portable, for characterizing isolates of bacterial species

• average of 30 alleles allows for resolution of 20 billion genotypes

• holy grail if you have no idea what is being looked at

what was the FDAs goal? what was it trying to do and what does ready mean?

• it was the WGS program

• trying to do is learn how to culture and have giant databases ready for particular pathogen

  • ready meaning knowing the conditions of the culture like how to culture it, what kind of media to grow it in, what temp is needed, and if oxygen is needed

what are the proactive applications of WGS technology?

• to protect consumers from foodborne illness around the world

• to evaluate persistence of pathogens in the environment

• to monitor emerging pathogens

• as a possible indicator of antimicrobial resistance

what is targeted amplicon sequencing? what does it use and what are bar codes?

• required a bit of knowledge for the sequence

• uses TARGET-specific primers followed by primers complementary to the target-specific primes with bar codes

• bar codes are identifiers like at the store to know what DNA you have

understand the procedure for whole genome sequencing (written response question)

Whole genome sequencing does not require prior knowledge of the sequence unlike targeted amplicon sequencing. Whole genome sequencing shears large DNA like gDNA either enzymatically like restriction enzymes or mechanically to more manageable fragment sizes. After, primers are ligated onto the fragments with adapters and barcodes to prime the whole genome to sequence.

what is required to remove in WGS?

bioinformatic removal of human sequences is required (99% of the DNA)

what does typing capacity mean? what does discriminatory power mean?

• can type multiple species of a particular gene

• if the method can tell the difference between strains of a gene/species from each other

what are classical methods? what about molecular methods? what are tests designed for?

• culture, antibody, or antigen detection

• target, probe, and signal amplification

• designed for ID of viruses, determination of viral load (# of viral particles per ml of fluid) and genotyping by sequence analysis

what is linearity and flexibility?

serial dilution of standard curve approximates a straight line

accurate measurement regardless of sequence variations

what is viral genotyping?

• viral genes mutate frequently to overcome antiviral agents

• there are no RNA repair pathways making RNA highly mutable

• in viruses, DNA based genomes mutate more than human genomes but not as much as the RNA based genomes

• gene mutations are detected by sequencing

what is the process for genotyping for HCV?

• extract genetic material from HCV in the patients blood, the amplify the material to differentiate the unique genotypes and is a FDA approved method

• uses fluorescence to detect the amplified signal

what is the virochip?

made in 2008, it is a small glass slide that can carry more than 36,000 tiny fragments of every virus ever discovered and could be used in viral ID clinically

can be reused if taken care of

what were the two examples that utilized virochip?

• example one = used to detect herpes, polymavirus, parvovirus, papillomavirus, and anellovirus in vestibular schwannoma which is responsible for deafness and balance issues

  • five samples were analyzed on the chip and uses 46 tissue samples to indicate their presence

• example two = investigation of patients infected with pandemic H1N1 influenza

  • found that H1N1 is human based not swine based

what were the common PCR methods used in diagnosing fungal infections? why?

• real time PCR, multiplexing PCR, and qPCR

• had a high-performance output with more rapid results than culture tests and is the preferred choice of the ID of antifungal drug resistance and requires only a small amount of sample

what is the golden standard for Plasmodium (malaria)? what are the other methods that are used? which one is good for those in underdeveloped countries like Africa? what is essential?

• gold standard is microscopic exam but not always possible and not the most common

• rapid diagnostic test (RDT) = alternative for detecting specific malaria antigens from all four strains

• qPCR of 18s (eukaryote) rRNA/rDNA

• LAMP

  • best because the output is fluoresence that can be seen with the naked eye

• it is essential to utilize a sensitive technique such as Plasmodium 18s rRNA LAMP to increase the detection rate of Plasmodium

what is used for Chaga’s disease, Trypanosoma cruzi?

• PCR is used a research tool, but not diagnosis

• patients have low circulating parasite loads, sensitivity is low and highly variable

• real-time PCR can be used like to show the rising copy numbers that provide a sensitive indicator of reactivation

what do PCR assays provide for Chagas disease?

• diagnoses acute phase (infection) and early congenital Chagas disease

  • a pregnant women can easily give their unborn child Chagas disease

• monitor for acute infection in the recipient of an infected organ or after accidental exposure

• monitor for reactivation in the immunosuppressed T. cruzi-infected host

understand the procedure for multiplex PCR (written response question)

Multiplex PCR utilizes multiple primers in one reaction to amplify many sequences simultaneously. Multiplex PCR can be used to type multiple strains due to the multiple primers used. It generates bands on an agarose gel to be able to identify strains that have genetic drift or are the same as the outbreak strain.