Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction (PCR)
(Polymerase chain reaction based detection of fungi in infected corneas)
AIM: Our aim is to detect fungi in infected corneas with the polymerase chain reaction technique.
INTRODUCTION: Polymerase chain reaction ( PCR), a technique used to make numerous copies of a specific segment of DNA quickly and accurately. (1) This is , a process in which a solution that includes DNA is repeatedly heated and cooled in order to melt the DNA, anneal short DNA fragments called primers (typically artificially designed oligonucleotides) to the complementary DNA target, and enzymatically replicate the primer-bound sequences using temperature-dependent DNA polymerases such as Taq polymerase. PCR has a wide variety of applications and is considered a staple of a geneticist’s toolkit.(2)
Infectious keratitis causes extensive ocular morbidity worldwide. The true extent of visual impairment from this condition is thought to far exceed the recognised prevalence, particularly among agricultural workers in the developing world, where a “silent epidemic” of corneal blindness has been postulated.Fungal corneal infections in particular occur most frequently in inividuals who work in agriculture.This condition is also associated with diabetes mellitus and the acquired immune deficiency syndrome (AIDS)(3)
MATERIALS: - Human infected cornea - 250 µl of 1X magnesium free PCR buffer -sterile water -taq polymerase - 2% agarose gels containing ethidium bromide -PCR tubes - deoxyribonucleoside triphosphates - magnesium chloride - 1X PCR buffer
METHOD: Thirty patients submitted samples. Scrapings from an affected area of each infected cornea were obtained with a flame sterilised platinum spatula, and were streaked onto an agar plate. The platinum spatula was then rinsed in 250 µl of 1X magnesium free PCR buffer in a 1.5 ml vial, flame sterilised, cooled, passed again across the cornea, streaked across another agar plate, rinsed again in the same 1.5 ml vial, and again sterilised and cooled.
The PCR assay was optimised using dilute suspensions of fungal isolates in sterile, deionised water. The suspensions were overlaid with mineral oil and heated in thin walled PCR tubes for 20 minutes at 94°C to lyse the fungi. Reagent mixtures containing the appropriate primers were added after heat extraction of the sample DNA. The final reaction mixture contained 0.8 µM of each primer, 2.5 units of taq polymerase 0.25 mM deoxyribonucleoside triphosphates, 2.0 mM magnesium chloride, and 1X PCR buffer. For optimisation, PCR reactions were run in 50 µl volumes using thin walled 500 µl PCR tubes. Reaction tubes were heated for 3 minutes at 94°C, followed by DNA melting for 30 seconds at 94°C, annealing for 40 seconds at 57°C, and extension for 1 minute at 72°C. The same annealing temperature was used for all primers. For the second round of amplification, the amplified common segment was diluted 1/500 in sterile water, and 25 µl of this diluted product were used as template DNA for each of three nested reactions, each of which contained a species directed primer pair. The reaction product was resolved by electrophoresis using 2% agarose gels containing ethidium bromide, 0.375 µg/ml. A PCR result was considered positive if a DNA band of the predicted length for the primers used was present.
RESULT:
Diagram of polymerase chain reaction amplification scheme.
Agarose gel demonstrating sensitivity of PCR assay. Serial dilutions of quantified Aspergillus fumigatus suspensions were assayed with 30 cycles of primary and 30 cycles of species directed PCR amplification. Far left lane shows 100 bp DNA ladder, (200 bp fragment obscured by loading dye). Lane 1: Aspergillus fumigatus 500 fungal elements. Target 214 bp product DNA band visible, as predicted for A fumigatus directed primers. Lane 2: 100 elements, target DNA band present. Lane 3: 25 elements, target DNA band present. Lane 4: 5 elements, target DNA band present. Lane 5: 0 elements, no target DNA. Lane 6: template A fumigatus DNA, positive control.
DISCUSSION:
This study demonstrates that fungi can be detected in infected corneas using PCR techniques. Advantages of PCR as shown here include greater speed than culture methods, and the ability to analyse specimens far from where they are collected. The PCR assay used in this study requires 4 hours to generate results, significantly faster than the 2 days to 2 weeks required by any fungal culture technique. While fungal smears can be analysed by light microscopy in minutes, the effectiveness of this technique is more variable, and the results are not definitive. The ability of PCR based assays to detect or rule out the presence of fungi in less time would represent an advance in the management of ocular infections, and may also facilitate efforts to recognise and study fungal keratitis.
REFERENCES:
1) https://www.britannica.com/science/polymerase-chain-reaction
2) https://www.sciencedirect.com/topics/neuroscience/polymerase-chain-reaction
3) https://bjo.bmj.com/content/bjophthalmol/86/7/755.full.pdf
