Serological methods for detection of bacterial pathogens

3. PCR based methods
3
List of detection
techniques
2
Introduction
1
LAMP & Microarrays
4
Aggulutination Test
5
Ouchterlony double
diffusion test
6
Outline

4. definition
 Serological test have also been a important tool in the detection and
diagnosis of plant pathogenic bacteria. Serology is the study of reaction
between antigen and antibody.
 Production of antiserum, either polyclonal (PABs) or monoclonal (MABs),
is the first requirement for performing any of the several serological assay
techniques.
 Numerous serological techniques have been developed for identification
and characterization of plant pathogens.

5. 1
PCR based
techniques
2
LAMP
Microarrays
3
Agglutination test
LIST OF DETECTION TECHNIQUES
Alain 2017
4
Ouchterlony
double diffusion
test

6. PCR (POLYMERASE CHAIN REACTION))
• PCR is laboratory (in vitro) technique used to amplify
target nucleic acid (DNA) sequence into many copies
(million or billions) in the presence of primers
(olingonucletide primers).
• PCR was invented by Karry mullis et al. (1983).
• By using thermos stable enzymes Taq polymerase that can
amplify target nucleic acid

7. ESSENTIALCOMPONENTSOF PCR

8. DNA TEMPLATE
DNA polymerase sequentially adds nucleotides complimentary to template strand
at 3’-OH of the bound primers and synthesizes new strands of DNA
complementary to the target sequence.
The most commonly used DNA polymerase is Taq DNA polymerase (from
Thermus aquaticus, a Thermophilic Bacterium) because of high temperature
stability.

9. Primers
• Primers are synthetic DNA strands of about 18 to 25 nucleotides
complementary to 3’ end of the template strand. DNA polymerase
starts synthesizing new DNA from the 3’ end of the primer .
• Two primers must be designed for PCR; the forward primer and the
reverse primer. The forward primer is complimentary to the 3’ end of
antisense strand (3’-5’) and the reverse primer is complimentary to
the 3’ end of sense strand (5’-3’).
• If we consider the sense strand (5’-3’) of a gene, for designing
primers, then forward primer is the beginning of the gene and the
reverse primer is the reverse-compliment of the 3’ end of the gene.

10. • DNA polymerase sequentially adds
nucleotides complimentary to
template strand at 3’-OH of the
bound primers and synthesizes new
strands of DNA complementary to
the target sequence.
• The most commonly used DNA
polymerase is Taq DNA polymerase
(from Thermus aquaticus, a
Thermophilic Bacterium) because of
high temperature stability.

11. Steps involved in PCR
Denaturation
Annealing
Extension
A high temperature (94-
96°C) is maintained to
separate the two
complementary strands of
the double stranded DNA
into a pair a single standard
polynucleotide DNA.
• Two oligo nucleotide
primers which binds to their
complementary sequence in
the opposite strands of the
target single stranded DNA.
Temperature usually
maintained is 55-65°C.
The reaction mixture is
heated to 72°C which is the
ideal working temperature for
the Taq polymerase. The
polymerase adds nucleotide
(dNTP's) complimentary to
template on 3’ –OH of
primers thereby extending the
new strand.

13. PCR Based methods
I. RT-PCR
II. Nested PCR
III.Multiplex PCR
IV.RAPD ( Random Amplified Polymorphic DNA )
V. AFLP ( Amplified fragment length polymorphism )
VI. Bio-PCR
VII. SYBR green dye-based method
VIII. TaqMan PCR
Types of pcr techniques in bacterial pathogens

14. RT-PCR
• RT–PCR is a variation of PCR, or polymerase chain reaction. The two
techniques use the same process except that RT–PCR has an added step of
reverse transcription of RNA to DNA, or RT, to allow for amplification

15. Nested PCR
• Nested PCR usually involves two sequential amplification reactions,
each of which uses a different pair of primers. The product of the first
amplification reaction is used as the template for the second PCR, which is
primed by oligonucleotides that are placed internal to the first primer pair

16. Multiplex PCR
• Multiplex PCR is the simultaneous detection of multiple targets in a single
reaction well, with a different pair of primers for each target.

17. LAMP (loop mediated isothermal amplification)
Notomi et al. (2000) developed LAMP in collaboration with Eiken Chemical Co.,
Ltd, Japan
It is a simple, modified, cost efficient, nucleic acid amplification techniques that
amplifies target DNA at high specificity, specificity, selectivity and rapidity under
controlled isothermal conditions.
LAMP works under the principle of autocatalytic strand displacement reaction
where nucleic acid amplifies using BST (Bacillus stereo thermophillus) DNA
polymerase at constant temperature to produce millions of copies of DNA.
Primers
a) Forward inner primer (FIP)
b) Forward outer primer (FOP)
c) Backward inner primer (BIP)
d) Backward outer primer (BOP)
e) Forward loop primer
f) Backward loop primers

18. microarrays
• A microarray is a multiplex lab-on-a-chip. Its purpose is to simultaneously
detect the expression of thousands of genes from a sample
• A microarray is a laboratory tool used to detect the expression of
thousands of genes at the same time. DNA microarrays are microscope
slides that are printed with thousands of tiny spots in defined positions,
with each spot containing a known DNA sequence or gene.

19. Aggulutination test
• Agglutination tests are based on the presence of agglutinating antibodies that
can react with specific antigens to form visible clumps. In the agglutination
tests, the antibody - antigen reaction can be either a direct or passive agglutination
reaction.

20. Ouchterlony double diffusion test
• Ouchterlony double diffusion (also known as passive double immune diffusion)
is an immunological technique used in the detection, identification and
quantification of antibodies and antigens, such as immunoglobulins and
extractable nuclear antigens.