biochemical tests 2

which tests are rapid?

which are overnight?

rapid:

non-agglutination screen tests (catalase, oxidase, spot indole, PYR, bile solubility, optochin)

MALDI-TOF spectrometry

automated systems (VITEK 2 ID) 4-6 hours

real time PCR (qPCR)

overnight:

traditional AST or extended vitek 2 runs 13-16 hr

traditional gel based PCR / post PCR analysis

Vitek 2

identifies bacteria (all types) and yeasts

64 wells that contain a test substrate

- measure metabolic activities: acid, alkaline, enzymes, growth

- photometric system detects changes in light (turbidity/colour)

- test reactions read every 15 mins

- results can take 4-6 hours, 13-16 hours

biocode generated and compared to database

vitek susceptibility cards

which bacteria does it test?

cards perform AST of non-fastidious aerobic or facultative org

- det susceptibility of strain to panel of antibiotics

- organism in contact w/ diff concentrations of diff antibiotics

- if organism grows = resistant, else susceptible

The lowest concentration of an antibiotic which prevents visible growth of bacteria is called it’s MIC.

what is the MIC

lowest concentration of antibiotic which prevents visible growth of bacteria.

is compared to known organism MIC in database and reported as susceptible or resistant

what is PCR

polymerase chain reaction.

technique where many copies of a specific target DNA region are made in a test tube rather than an organism

components of PCR

1. template of DNA to be amplified

2. pair of DNA primers

----- 2 - forward and reverse of DNA/RNA 20 pairs

3. DNA polymerase

---- (taq polymerase- bc doesnt denature at high temp

4: dNTPs

---- (adenosine, guanosine, thymidine, cytosine triphosphate)

5. Buffer to maintain pH and provide Mg2+

---- to create optimal conditions for taq poly.

---- maintains pH 8-9.5

---- includes salts like magnesium chloride and KCL

what are probes

short nucleic-acid sequences that are labeled for detection of a specific target sequence are called probes

qPCR

combines PCR amplification and detection into a single step

- products are assayed as they accumulate rather than at the end

- uses fluorescent reporter dye or probe labelled with fluorescent dye

difference between regular and real time PCR

qPCR is faster, does not require laborious post PCR methods for visualization of PCR products, and allows for quantitation of amplified product

2 reporter dyes to visualize product by qPCR

SYBR Green and Taqman probe

SYBR Green

- binds to any double strand DNA

- bound dye has stronger fluorescent signal than unbound

- less specific than taqman and binds to any ds dna including target and non-specific pcr

Taqman probe

- dna sequence specific to target dna

- quencher (on 3') and fluorescent dye (on 5'). when close together, no fluorescence

during pcr:

as DNA polymerase extends primer and encounters Taqman probe, the 5' activtity cleaves probe and causes dye to come apart from quencher and emit fluorescent signal

what are the 3 main phases of amplification curve

1. baseline phase: initial cycles of PCR, low levels of bg fluorescence only, little change

2. exponential phase: pcr product begins to amplify and doube

3. stationary or plateau phase: components get used up and no increase in production

what are the threshold line and Ct on a qpcr amp curve

threshold line: fluorescence level used to distinguish amplification signal from bg fluorescence. set by instrument

ct (cycle threshold) : cycle number where fluorescence in sample crosses threshold line. measure of concentration of target DNA in pcr reaction.

more target dna initially means lower ct needed

MALDI-tof principle

rapid ID of bacteria, fungus, and mycobacteria.

uses a laster to vaporize and ionize protein molecules, measures the molecules mass-to-charge ratios. generates mass spectrum using time of flight spectrometry

spectrum produced from unknown org is compared to known database. ID based on closest match

Maldi process

place single colony into well

overlay with 1ul of organic matric

air-dry and insert plate in analyzer

uv beam hits test spot on slide

excitation of matrix causes vaporization, matrix molecules and microorganism proteins are ejected from target surface (desorption)

protons transferred from matrix to proteins (ionization)

electrostatic field accelerates ions into flight tube and they race to the end where the detector is.

smaller ions reach detector faster than larger ones (TOF measured)

what does the matrix in Maldi do.

absorbs laser energy and protects the sample

which organisms does maldi not correctly identify

ones with mucoid capsules or spores

species too closely related

e.g e.coli & shigella, s.pneumoniae & s.mitis

oxidase test

tests whether organism can produce cytochrome C oxidase enzyme

reagent: 6% tetramethyl-p-phenylenediamine dihydrochloride

if organism produces cytochrome C oxidase, reagent oxidized to indophenol which turns a deep purple blue

DNAse test

test whether organism can produce Dnase to breakdown Dna for carbon and energy

medium has: DNA, peptones (nutrients), methyl green dye (indicator)

dye and dna bind and give agar blue-green colour at pH 7.5

if Dnase secreted, dna hydrolyses --> no longer bound to dye and clear zone is produced around bacterial growth

PYR test

detect enzyme L-pyroglutamyl-aminopeptidase (PYRase)

PYRase enzyme hydrolyses L-pyrorolidonyl-B-napthylamide to B-napthylamine

PYR is disk, if PYRase produced, deep violet colour develops when reagent added (para dimethylaminocinnamaldehyde) (same as spot indole)

Bile solubility test

tests whether organism will lyse in presence of bile salts

bile salts: sodium desocycholate and sodium taurocholate

trigger catalytic enzymes in strep pneumoniae that promote autolysis of cells

-- will disintegrate within 30 mins of surfactants

optochin Test

determine if organism is susceptible to chemical optochin (ethyl hydrocupreine)

optochin disk added to plate and incubated

if diameter of inhibition >=14 mm then susceptible = pneumoniae

else resistant = other AH strep