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.

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

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.

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.

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.

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.

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

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.

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

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.

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