1. USE OF PEPTIDE NUCLEIC ACID-FLUORESCENCE IN
SITU HYBRIDIZATION (PNA-FISH) FOR DEFINITIVE,
RAPID IDENTIFICATION OF FIVE
COMMON CANDIDA SPECIES
Megan E. Reller et al.
J Clin Microbiol. 2007 November; 45(11): 3802–3803.

2. What is PNA ?
Peptide nucleic acid (PNA) is an artificially
synthesised polymer similar to DNA or RNA
invented by Peter Nielsen in 1991.
The phosphate ribose ring of DNA is
replaced with the polyamide
backbone in PNA

3. Structure and Properties of PNA
 PNA is made up of repeating N-2-aminoethyl-
glycine units linked by amide bonds.
 The purine and pyrimidine
bases are attached to the backbone
through methylene carbonyl linkage.
 Unlike DNA or DNA analogues
PNAs do not contain any sugar
moieties or phosphate group.
By convention PNAs are depicted like peptides,
with N-terminus at the left position and the C-
terminus at the right.

4.  PNA is different from DNA
in that the backbone of PNA
is acyclic, achiral and neutral.
 PNA can bind to complimentary
nucleic acid in both antiparallel
and parallel orientation. However,
the antiparallel orientation is
strongly preferred.

5. The neutral character of the PNA backbone is an important
feature that has many consequences :
 Lack of charge repulsion between DNA and PNA
increases the affinity.
 Mismatching of PNA/DNA is more destabilising than
the mismatching in DNA/DNA so there is increase in
specificity also.
 The lack of electrostatic repulsion between the two
strands in PNA/DNA duplex leaves the Tm
independent of salt conecentration.
 Peptide backbone are not easily recognized by
nulceases and proteases, thus extending the halflife of
PNA both in vivo and in vitro.

6. Applications of PNA
 PNA can bind to complementary sequences of mRNA and DNA
and can be used as Antisense and Antigene therapy respectively.
 Single nucleotide polymorphism assay can be improved with the
help of more specific binding of PNA primers with the DNA
segment to be assessed.
 PCR clamping: e.g. a few mutant cancerous cells may be present
among healthy cells in a tissue biopsy. PNA clamp complementary
to wild type can be used to block its PCR amplification while
allowing amplification of mutant type.
 It can be used in microarrays and biosensors
 It can also used as imaging probes in FISH.

8. PNA-FISH
 Use of PNA-FISH is dependent on the hybridization of PNA probes to
species specific regions of ribosomal RNA (rRNA), resulting in highly
sensitive and specific hybridization assay.
 Protocol steps require <90 minutes turn around time
(i) smear preparation
a. A drop of fixation solution is added to PNA-FISH slide
b. A drop of positive blood culture is added
(ii) Hybridization
PNA probe is added, which enters the cells and
binds to rRNA if present
(iii) Washing is done and unbound probe is removed
(iv) slide is read under fluoroscense microscope

10. Introduction and objective of study
 Candida species are important nosocomial bloodstream pathogens
and account for disproportionate morbidity and mortality.
 Although Candida albicans remains the most common isolate,
nearly half of all invasive infections are now caused by other
candida species.
 Since different species have distinct anti-microbial susceptibility
profiles, the prolonged time needed to identify organism to species
level (upto 5 days) precludes the early use of less expensive,
targeted therapy
 PNA-FISH is a diagnostic tool that uses fluorescence -labeled
probes that hybridizes to species specific rRNA sequence. It can
be used to hasten the identification of species.

11. A total of 238 slides were prepared and examined.
The slides were tested by PNA-FISH and cross
checked by the conventional method

12. Result and conclusion
 the five species specific probes were found to
be 99% sensitive and 99% specific.
 PNA-FISH can provide rapid (at least 24 hour
sooner) and accurate identification of candida
at the species level from colonies, thereby
enabling early specific treatment.
 Importantly, the detection of positive
fluoroscence by PNA-FISH provides a final
identification; therefore tedious confirmatory
testing is not necessary.

13. Technology is a way of organizing the
universe so that man doesn’t have to
experience it !!!!!!!!
Thank you !!!