lecture 4 Flashcards
what is rapid result testing
-smaller automated testing or susceptibility testing
-uses dehydrated substances to detect pre formed enzymes
-some are serological for AB AG detection directly from pt - throat swab for strep
-chromogenic media w/ culture
-rapid testing by automation
-rapid testing by molecular techniques - covid test
-increased accuracy and faster results within 4-6 hours reduces hands on time
how can results be detected :
Agglutination:
Fluorescence:
Chromogenic reaction:
Colorimetric:
Turbidity:
Agglutination: AG/AB reaction = clumping.
Fluorescence: Non-fluorescent to start, becomes fluorescent if enzyme breaks down substrate. tagging specimen
Chromogenic reaction: Dye - chromophore colorless to start if enzyme present becomes colored.
Colorimetric: Color produced due to pH or a chemical reaction (enzyme breaks down substrate).
Turbidity: Increased cloudiness if bacteria present.
Colorimetric or Turbidimetric methods can be qualitative or quantitative measurements.
how is turbidity measured
-Light-rays are scattered and absorbed by bacteria
in the solution rather than transmitted in straight lines through the sample.
• If measure scattered light = nephelometer
• If measures transmittance = turbidimeter
automated detection systems
-measure light change to detect positive or negative results
photometer- measures intensity of light by converting light into electrical current that changes based on the amount of light absorbed
spectrophotometer/colorimeter- measures concentration of a substance or intensity of color of a solution by how much specific colors are absorbed or transmitted
fluorometer: detects increasing levels of fluorescence absorbs UV light
how does a spectrophotometer work
- measures the amount of light that is absorbed or transmitted by the sample - quantitative measure of the concentration of matter in the sample – i.e. amount of bacteria r
Spectrometer:
-COLLIMATOR- a lens that transmits a straight beam of light (photons).
-The light passes through a MONOCHROMATOR (PRISM) splitting it into many wavelengths.
-the SLIT selects and transmits the wavelength you want measured & this wavelength of light is shined onto a sample
Photometer: detects number of photons or intensity of light transmitted or absorbed AFTER the wavelength of light passes through the sample in cuvette and converts into an electrical signal on a display
the more concentrated a solution (darker) is the higher the absorbance of light and lower the transmittance
what is the vitek2
-designed for the space program
-identifies bacteria(aer, ana, fast, facul, GP, GN) and yeasts
-64 wells with test subtrate
-measures metabolic activties, acidification, alkalization , growth in presence of inhibitory substances
-uses photometric system to detect changes in light - turbidity
-reading every 15 mins
-results from 4-13 hours
-biocode is generated and compared with known org biocodes
-has cards to perfrom susceptibility testing of non fast, aero, facul bacteria
-Antibiotic susceptibility testing (AST) determines the susceptibility of a bacterial strain to a panel of antibiotics.
-if bacteria grows in certain antibiotic then resistant if it cant then its susceptible
-the lowest concentration of an antibiotic that prevents VISIBLE growth is called MIC
- then this MIC is compared to known org MIC in database
what is PCR
-polymerase chain reaction - where copies of a specific target DNA region are made in a TEST TUBE rather then an organism
what is needed:
-dna to be amplified
-dna primers
-dna polymerase
-dNTPs
-buffer to maintain pH and provide MG
PCR COMPONENTS
Target DNA - is the DNA extracted from the specimen dont have to be the target sequence but if it is present then it will be used as a template for the new DNA
DNA Polymerase- enzyme in cells that use existing SINGLE DNA strands as a template to make new DOUBLE strands by reading the template and adding nucleotides one at a time
-TAQ POLYMERASE is used because it doesnt denature at high temps that are used in the PCR cycle
dNTPs (deoxyribonucleoside triphosphates):
-Four dNTPs (adenosine triphosphate, guanosine triphosphate, thymidine triphosphate and cytosine triphosphate)
- “building blocks” added to ss of DNA by Taq polymerase to make new ds of DNA
Primers (forward and reverse)
-2 short sequences of complementary DNA or RNA -20 base pairs
-forward or reverse bind to either end on target DNA strand
–taq can only add a few to already existing DNA strand - gives a starting point
3’ TO 5’ BINDS TO 5’ TO 3’
-LABELLED PRIMERS ARE PROBES
what part does the PCR buffer play and what are the next steps in pcr
PCR buffer: creates an optimal environment for the Taq to operate
-pH between 8-9.5 with salts like MGCL and KCL to help Taq
Next steps: extract, purify the DNA = template DNA
put your DNA and all ingredients in small tube into a thermocyler
what is a thermocycler
- a thermal block and holes to hold the PCR reaction tubes
-raises and lowers the temp of the block
-repeating steps of denaturation, extension, and annealing
before the cycle starts you may have to do a hot start and heat the block at 95C for 1-10 mins if you are using a dna polymerase that is heat activated
PCR STEPS and analysis
Denaturation - 98C 1-2 mins- increase temps to separate the DNA strands
Annealing 50-65C for 1-2 min - decreased temps allow primers to pair to comple DNA template
Extension 68-72C 10-15 min - polymerase extends the primer to form nascent DNA strand
Exponential amplification of target region about 2 bill copies in 30th cycle
ANALYZED BY
agarose gel electrophoresis
-NA or protein seperated based on charge, size and shape from neg to pos
-visualize with dye staining fluorescence
-compared with ladder - a ruled of base pair sizes
VISUALIZING OUR SAME PRODUCT BY REAL TIME PCR (qPCR)
-qPCR combines PCR amplification and detection into a single step
-amplification products are assayed as they accumulate instead of at the end
-positive result can be seen while still running
-uses fluorescent reporter dye or probe labelled with a fluo dye
-the thermocyler will also have a UV light source to excite the reporter and a camera to detect the fluor in EACH cycle
-two dyes
SYBR Green – what we used
Taqman
FASTER with quantitation of amplified product
what is qPCR-SYBR Green
-Fluorescent dye that BINDS to any double strand of DNA
-dna BOUND with dye has a stronger fluorescent signal than unbound – increased fluorescence is measured
Less specific than Taqman- will bind to any amplified double stranded DNA product target & non target
emission by BINDING
WHAT IS qPCR-Taqman Probe
-short sequence of DNA specific to a section of target DNA
-It has a fluorescent dye on its 5’ end and quencher on its 3’ end -when close and bound no fluoresce
-the probe binds to homologous region on target DNA
-as the DNA polymerase extends the primes on the target DNA and comes in contact with the probe. The probe cannot be extended, It will eat the probe because it has exonuclease activity causing the fluorescent dye to come away from the quencher and emit a fluorescent signal
what are the phases of a qPCR curve
-seen on a amplification curve
1-Baseline phase- 3-15 cycles of PCR when there is no change or low amplification to fluor signal (little background)
2.Expotential phase - 16-25 cycles in where the PCR product is starting to amplify and double
3.stationary or plateau phase - 26-38 cycles in components get used up and no increase in amplified product
