microbiology task 4

7/28/2019 Microbiology Task 4

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TASK 4

MICROBIOLOGY PHARMACY

pcr method, Api 20 e system, and nccls

WWRRIITTTTEENN BBYY:

IKA YULIA RIZKI

1111012053

Lecturer : Prof. DR. Marlina, M.Farm, Apt

PHARMACY FACULTY

ANDALAS UNIVERSITY

PADANG

2012


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PCR METHOD, API 20 E SYSTEM, AND NCCLS

A. Polymerase chain reactionDefenition

The polymerase chain reaction (PCR) is a scientific technique in molecular biology to

amplify a single or a few copies of a piece ofDNA across several orders of magnitude,

generating thousands to millions of copies of a particularDNA sequence.

Developed in 1983 by Kary Mullis,[1]

PCR is now a common and often indispensable

technique used in medical and biological research labs for a variety of applications.[2][3]

These

include DNA cloning forsequencing, DNA-based phylogeny, or functional analysis ofgenes; the

diagnosis ofhereditary diseases; the identification ofgenetic fingerprints (used in forensicsciences and paternity testing); and the detection and diagnosis ofinfectious diseases. In 1993,

Mullis was awarded the Nobel Prize in Chemistry along with Michael Smith for his work on

PCR.[4]

PCR machine

The method relies on thermal cycling, consisting of cycles of repeated heating and

cooling of the reaction forDNA melting and enzymatic replication of the DNA. Primers (short

DNA fragments) containing sequences complementary to the target region along with a DNA

polymerase (after which the method is named) are key components to enable selective and

repeated amplification. As PCR progresses, the DNA generated is itself used as a template for

replication, setting in motion a chain reaction in which the DNA template is exponentially

amplified. PCR can be extensively modified to perform a wide array ofgenetic manipulations.
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Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq

polymerase, an enzyme originally isolated from the bacteriumThermus aquaticus. This DNA

polymerase enzymatically assembles a new DNA strand from DNA building-blocks, the

nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called

DNA primers), which are required for initiation of DNA synthesis. The vast majority of PCR

methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined

series of temperature steps. These thermal cycling steps are necessary first to physically separate

the two strands in a DNA double helix at a high temperature in a process called DNA melting. At

a lower temperature, each strand is then used as the template in DNA synthesis by the DNA

polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of

primers that are complementary to the DNA region targeted for amplification under specific

thermal cycling conditions.

PurposeThe Gram stain is used to detect the presence of bacteria, yeast, and other cells in direct

smears prepared from swabs, aspirates, secretions, etc. from any part of the body where

infection is suspected. Direct smears are often made ofthroat swabs, sputum, genital swabs,

wounds, abscesses, cerebrospinal fluid (CSF), serous fluids, joint fluid, urine, and stool. Gram

stain is also performed to help identify colonies isolated from cultures. In addition to gram-

negative or gram-positive, organisms are evaluated for size, shape, arrangement, number, and

any special characteristics such as bipolar staining and the presence of spores. These

characteristics often point the way to the most efficient selection of biochemical tests needed

to identify the organism. The finding of organisms on direct examination of some specimens issufficient to establish a preliminary diagnosis and justify immediate antibiotic treatment

pending confirmation by culture or other means. The Gram stain is very useful in identifying

anaerobic bacteria by comparing the microscopic morphology and number of organisms to

culture results. Significant numbers of characteristic bacteria on Gram stain not appearing on

aerobic culture often signals the presence of an anaerobic infection
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.

The Gram stain will identify male patients with Neisseria gonorrhoeae genital infections

with a specficity approaching 100% and a sensitivity above 90%. In female patients, the

sensitivity and specificity are lower owing to the presence of other genital flora, but the test is

still sufficiently specific to justify immediate antibiotic therapy when symptoms ofpelvic

inflammatory disease are present. The presence of bacteria on Gram stain of concentrated CSF

is presumptive evidence of bacterial meningitis and reason to begin antibiotic therapy. The

Gram stain is positive in the majority of bacterial meningitis cases. Recovery of bacteria from

other normally sterile fluids including exudative plueral, pericardial, and abdominal fluid and

inflammatory joint fluid is also presumptive

PrinciplePCR is used to amplify a specific region of a DNA strand (the DNA target). Most PCR methods

typically amplify DNA fragments of up to ~10 kilo base pairs (kb), although some techniques

allow for amplification of fragments up to 40 kb in size.

A basic PCR set up requires several components and reagents. These components include:

DNA template that contains the DNA region (target) to be amplified. Twoprimersthat are complementary to the 3' (three prime) ends of each of the sense and anti-

sense strand of the DNA target.

Taq polymeraseor another DNA polymerase with a temperature optimum at around 70 C. Deoxynucleoside triphosphates (dNTPs; nucleotides containing triphosphate groups), the

building-blocks from which the DNA polymerase synthesizes a new DNA strand.

Buffer solution, providing a suitable chemical environment for optimum activity and stability ofthe DNA polymerase.

Divalentcations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilizedfor PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate

during DNA synthesis[7]
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Monovalent cation potassium ions.

The PCR is commonly carried out in a reaction volume of 10200 l in small reaction

tubes (0.20.5 ml volumes) in a thermal cycler. The thermal cycler heats and cools the reaction

tubes to achieve the temperatures required at each step of the reaction (see below). Many modern

thermal cyclers make use of the Peltier effect, which permits both heating and cooling of the

block holding the PCR tubes simply by reversing the electric current. Thin-walled reaction tubes

permit favorable thermal conductivity to allow for rapid thermal equilibration. Most thermal

cyclers have heated lids to prevent condensation at the top of the reaction tube. Older

thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of

wax inside the tube.

Prosedure

Typically, PCR consists of a series of 20-40 repeated temperature changes, called cycles,

with each cycle commonly consisting of 2-3 discrete temperature steps, usually three (Fig. 2).

The cycling is often preceded by a single temperature step (called hold) at a high temperature
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(>90C), and followed by one hold at the end for final product extension or brief storage. The

temperatures used and the length of time they are applied in each cycle depend on a variety of

parameters. These include the enzyme used for DNA synthesis, the concentration of divalent

ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers.[8]

Initialization step: This step consists of heating the reaction to a temperature of 9496 C (or98 C if extremely thermostable polymerases are used), which is held for 19 minutes. It is only

required for DNA polymerases that require heat activation by hot-start PCR.[9]

Denaturation step: This step is the first regular cycling event and consists of heating the reactionto 9498 C for 2030 seconds. It causes DNA melting of the DNA template by disrupting the

hydrogen bonds between complementary bases, yielding single-stranded DNA molecules.

Annealing step: The reaction temperature is lowered to 5065 C for 2040 seconds allowingannealing of the primers to the single-stranded DNA template. Typically the annealing

temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA

hydrogen bonds are only formed when the primer sequence very closely matches the template

sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.

Extension/elongation step: The temperature at this step depends on the DNA polymerase used;Taq polymerase has its optimum activity temperature at 7580 C,

[10][11]and commonly a
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temperature of 72 C is used with this enzyme. At this step the DNA polymerase synthesizes a

new DNA strand complementary to the DNA template strand by adding dNTPs that are

complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the

dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The

extension time depends both on the DNA polymerase used and on the length of the DNA

fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase

will polymerize a thousand bases per minute. Under optimum conditions, i.e., if there are no

limitations due to limiting substrates or reagents, at each extension step, the amount of DNA

target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.

Final elongation: This single step is occasionally performed at a temperature of 7074 C for 515 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully

extended.

Final hold: This step at 415 C for an indefinite time may be employed for short-term storage ofthe reaction.

Figure 3: Ethidium bromide-stained PCR products aftergel electrophoresis. Two sets of primers were

used to amplify a target sequence from three different tissue samples. No amplification is present in

sample #1; DNA bands in sample #2 and #3 indicate successful amplification of the target sequence. The

gel also shows a positive control, and a DNA ladder containing DNA fragments of defined length for

sizing the bands in the experimental PCRs.

To check whether the PCR generated the anticipated DNA fragment (also sometimes referred to

as the amplimer oramplicon),agarose gel electrophoresisis employed for size separation of thePCR products. The size(s) of PCR products is determined by comparison with aDNA ladder(a

molecular weight marker), which contains DNA fragments of known size, run on the gelalongside the PCR products (see Fig. 3).
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B. API-20E

The API-20E test kit for the identification of enteric bacteria (bioMerieux, Inc., Hazelwood,MO) provides an easy way to inoculate and read tests relevant to members of the Family

Enterobacteriaceae and associated organisms. A plastic strip holding twenty mini-test tubes is

inoculated with a saline suspension of a pure culture (as per manufacturer's directions). This

process also rehydrates the dessicated medium in each tube. A few tubes are completely filled

(CIT, VP and GEL as seen in the photos below), and some tubes are overlaid with mineral oilsuch that anaerobic reactions can be carried out (ADH, LDC, ODC, H2S, URE).

After incubation in a humidity chamber for 18-24 hours at 37C, the color reactions are read

(some with the aid of added reagents), and the reactions (plus the oxidase reaction doneseparately) are converted to a seven-digit code which is called the Analytical Profile Index, from

which name the initials "API" are derived. The code can be fed into the manufacturer's database

via touch-tone telephone, and the computerized voice gives back the identification, usually as

genus and species. An on-line database can also be accessed for the identification. The reliability
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of this system is very high, and one finds systems like these in heavy use in many food and

clinical labs.

\

Note: Discussion and illustration of the API-20E system here does not necessarily constitute any

commercial endorsement of this product. It is shown in our laboratory courses as a primeexample of a convenient multi-purpose testing method one may encounter out there in the "real

world."

In the following photos:

Note especially the color reactions for amino acid decarboxylations (ADH through ODC) andcarbohydrate fermentations (GLU through ARA).

o The amino acids tested are (in order) arginine, lysine and ornithine. Decarboxylation isshown by an alkaline reaction (red color of the particular pH indicator used).

o The carbohydrates tested are glucose, mannitol, inositol, sorbitol, rhamnose, sucrose,melibiose, amygdalin and arabinose. Fermentation is shown by an acid reaction (yellow

color of indicator).

Hydrogen sulfide production (H2S) and gelatin hydrolysis (GEL) result in a black color throughoutthe tube.


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A positive reaction for tryptophan deaminase (TDA) gives a deep brown color with the additionof ferric chloride; positive results for this test correlate with positive phenylalanine and lysine

deaminase reactions which are characteristic ofProteus, Morganella and Providencia.

In the first set of reactions:

Culture "5B" (isolated from an early stage ofsauerkraut fermentation) is identified asEnterobacter agglomerans which has been a convenient dumping ground for organisms now

being reassigned to better-defined genera and species including the new genusPantoea. This

particular isolate produces reddish (lactose +), "pimply" colonies on MacConkey Agar which

exude an extremely viscous slime as may be seen here; this appearance is certainly atypical of

organisms identified as E. agglomerans or Pantoea in general.

Culture "8P44" is identified as Edwardsiella hoshinae. The CDC had identified this culture (in1988) as the ultra-rare Biogroup 1 ofEdwardsiella tarda which may not be in the API-20E

database. This system probably would not be able to differentiate between these two

organisms. Note that 8P44 shows H2S production which is probably typical ofEdwardsiella tarda

Biogroup 1. Clinical laboratories usually run this test in Triple Sugar Iron Agar in which theorganism's fermentation of sucrose (with consequent high acid production) tends to negate the

H2S reaction, and as a result the organism is mis-characterized throughout the literature as

H2S negative even though it shows a positive reaction in KIA and other H2S-detecting media.

C. NCCLSNCCLS Changes Name to Clinical and Laboratory

Standards Institute

On 1 January 2005, NCCLS officially changed its name, becoming Clinical and

Laboratory Standards Institute (CLSI). Glen Fine, MT(ASCP), MS, MBA, the organizations

Executive Vice President, explains, "The name change does notrepresent a shift of our core

organizational mission; instead, it better reflects our expanded standards-development activities

and global membership base."

Between 1967 and 2005, NCCLS grew into the patient-testing community's leading

resource for standardized best practices. By the beginning of 2005, the organization had

developed into a truly global body, with over 4,000 of the healthcare world's corporations,

governmental bodies, and laboratories counting themselves as members or volunteers, and

contributing to the development of consensus documents on topics ranging from Molecular

Methods to Automation and Informatics.
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So, why change the name, and why now?

Extensive market research found that a name change was imperative for more accurate

representation of the organization, on several different fronts:

1. Brand identity studies conducted in 2003 showed that many people were confused by the "N" inthe organization's name, which had stood for "national" when NCCLS was known by its full

name, National Committee on Clinical Laboratory Standards.

2. With the development of documents on such topics as point-of-care testing and respiratorycare, the scope of the organization's work could no longer be accurately defined as the clinical

laboratory, but had now become the clinic andthe laboratory.

3. With a vast, global member and volunteer base, the term "committee" no longer fairlydescribed the diverse participation and worldwide reach of the organization's consensus

process.

Fine states, "Our documents are globally recognized as standards and guidelines for health

technologies, so a change to reflect this expanded worldwide role was necessary."

"Our organizational values will remain the same," he adds. "Only our name is changing.


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Reference

Barker, Kathy. 1998.At The Bench, A Laboratory Navigator. Cold Spring Harbor Laboratory Press, New

York.

Barrow, G.I. and R.K.A. Feltham. 1993. Manual for the Identification of Medical Bacteria. Cambridge

University Press, New York

Beishir, Lois. 1991. Microbiology in Practice : A Self Instructional Laboratory Course. Harper Collins

Publisher Inc., New York

http://www.bd.com/vacutainer/labnotes/Volume15Number1/nccls_changes_to_clsi.asp

http://www.dmidjournal.com/article/S0732-8893%2800%2900172-3/abstract

http://jcm.asm.org/content/39/4/1360.abstract
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microbiology task 4

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