PATHOGEN DISCOVERY TECHNIQUE

2015057405 趙偉光
CURRENT METHODS TO DETECT PATHOGEN

• 1. microbial cultivation
• 2. immunological assays
• 3. nucleic acid test
1. MICROBIAL CULTIVATION

•  a method of multiplying microbial organisms(bacteria,virus,fungi) by letting them

reproduce in predetermined culture medium under controlled laboratory conditions
• Widely used in laboratories
• Different system and mode of microbial culture
(a) Closed System: closed culture has no additional nutrients added to the system, and
waste products are not removed. 
(b) open system: continuous culture where periodically some of the bacterial culture is
removed and added to fresh sterile medium.
BACTERIA CULTURE

• There are several types of bacteria culture methods.

1.liquid culture
 desired bacteria are suspended in a liquidnutrient medium.
medium:  Luria Broth(for E.coli), Middlebrook 7H9 and Dubos Tween albumin broths(Mycobacterium species, )
Advantage: faster result can be obtained
Disadvantage: difficult to observed the contamination of the sample.
Mixed organisms cannot be separated.
2.Agar plate
concept: Petri dish that contains agar as a solid growth medium plus nutrients.
common types: (1) Blood agar plates: it is used to detect hemolytic activity, positive result
can be obtained if Streptococcus haemolyticus presents.
(2) Chocolate agar: blood agar plate in which the blood cells have been lysed by heating
the cells to 56 °C. it is used to cultivate Haemophilus influenzae and Neisseria meningitidis
• 3. Stab cultures
similar as agar plate except the agar is placed into test tube.
commonly used for short-term storage or shipment of cultures
VIRAL CULTIVATION

• Virus cultures require host cells in which the virus or phage multiply.
• It can be obtained from their appropriate eukaryotic host cells

• 1. Animal Inoculation
Suckling mice(less than 48 hours old) are most commonly used.
After inoculation, virus multiply in host and develops disease. The animals are observed for symptoms of disease and death.
 virus is isolated and purified from the tissue of these animals.
2. Inoculation into embryonated egg
method
• For inoculation, eggs are first prepared for cultivation, the shell surface is first disinfected with
iodine and penetrated with a small sterile drill.
• After inoculation, the opening is sealed with gelatin or paraffin and incubated at 36°c for 2-3 days.
• After incubation, the egg is broken and virus is isolated from tissue of egg.
• 3. Cell Culture
it is now routinely used for growing viruses.
•  the process by which cells are grown under controlled conditions.
• Cells are grown in vitro on glass or a treated plastic surface in a suitable growth medium
-the medium usually consists of phenol red, buffer, nutrients
Types
1.Primary cell culture: normal cells derived from animal or human cells.
2. Diploid cell culture :diploid and contain the same number of chromosomes as the parent cells
3. Heteroploid cultures:derived from cancer cells.
• Advantages
Relative ease, cheaper and sensitivity

disadvantage
The process requires trained technicians with experience in working on a full time basis
2. IMMUNOLOGICAL ASSAYS

• is a biochemical test that measures the presence  macromolecule or a small molecule in a

solution through the use of an antibody
• rely on the ability of an antibody to recognize and bind a specific macromolecule.
• Example
1. Enzyme-Linked Immunosorbent Assay (ELISA)
2.  Immunofluorescence Assay
3. Western Blotting Analysis
• 1. Enzyme-Linked Immunosorbent Assay (ELISA)
basic concept:  the target antigen is combined with antibody that is linked to an enzyme.
procedure:
• Advantages
(1) highly sensitive, specific
(2)easy to perform
(3) Generally safe and eco-friendly
• Disadvantages
(1) expensive to prepare antibody 
(2) Antibody instability :they are protein that requires refrigerated transport and storage.
• 2. Immunofluorescence Assay
basic concept:  purified antibodies (which are labeled with fluorescent dye) are
combined with target antigen.
procedure
Advantage
(1) Easy to operate
(2) Higher Dynamic Range: Easier to visualize rare and high abundant targets on the same
slide.

Disadvantages
(1)Lower sensitivity
• 3. Western Blotting Analysis
Basic concept: based on the principle of immunochromatography where proteins are separated into
polyacrylamide gel according to their molecular weight.
procedure
• Advantages
(1) Sensitivity
(2) Specificity
• Disadvantages
1. High Cost and Technical Demand
2. Prone to False Results: (a) false-positive results due to  antibody reacts with a non-
intended protein
(b)  false-negative result due to larger proteins are not given sufficient time to transfer
properly to the membrane. 
3. NUCLEIC ACID TEST

• technique used to detect a particular nucleic acid sequence and thus usually to detect and
identify a particular species or subspecies of organism.
• many NATs include a step that amplifies the genetic material, they are called  nucleic acid
amplification tests.
• 3 types of nucleic acid amplification tests
(a) Polymerase chain reaction (PCR)
(b) loop mediated isothermal amplification (LAMP)
• 1. Polymerase chain reaction (PCR)
common laboratory technique used to make many copies of a particular region of DNA
Procedure
Method to identify pathogen(bacteria) from the PCR result
 pure PCR product of the 16S gene is obtained, sequenced, and aligned against bacterial
DNA data base, then the bacteria can be identified.
Advantages
• (1) Allow faster diagnosis and identification while enhancing sensitivity and maintaining
specificity.
• (2) It allows enormous amplification of any specific sequence of DNA 
• 2. loop mediated isothermal amplification (LAMP)
it is  a single-tube technique for the amplification of DNA .
Principle
is a one-step amplification reaction that amplifies a target DNA sequence with high sensitivity and specificity under isothermal conditions. employs a DNA polymerase
with strand-displacement activity, along with two inner primers (FIP, BIP) and two outer primers (F3, B3) which recognize six separate regions within a target DNA
PROCEDURE
• Advantages of lamp
(1)Requirement of fewer operation steps
(2) Easy standardization
(3) an equipment free technique because it does not require costly equipments