POLYMERASE CHAIN REACTION (PCR)
PCR is rapid diagnostic technique to detect the infectious agents even in small volume of samples. PCR is typically used for one of the following scenarios:
1. The patient presents with signs and symptoms that are most likely an infection but a definitive diagnosis cannot be made.
2. When a patient does not respond appropriately to therapy, or
3. For confirmation of a diagnosis when a patient or
4. When the patient's natural history does not coincide with his or her clinical presentation.
Acquiring Sample for PCR Diagnostics
• Corneal scrapings
• Infected corneal button
• Aqueous and vitreous fluid, etc.
Typically, ophthalmic samples for PCR are collected in three ways. From least to most invasive, these include swab samplings of the external eye, an anterior chamber paracentesis, or a vitreous tap. The type of biopsy taken must be guided by disease suspicion, media opacity, structural parameters of the eye, coexistence of pathologic conditions, and the experience of the ophthalmologist in taking a biopsy from the vitreous or the anterior chamber.
Collection
All samples should be collected aseptically and placed into a sterile container. For a given PCR reaction volume, 5 to 10 μl of the patient sample is sufficient for amplification. Approximately 50 μl from an anterior chamber paracentesis is sufficient and should be immediately capped in a 1 ml sterile syringe. For vitrectomy samples, approximately 50-100 μl of a pre-infusion aspirate is collected in a sterile tube and capped upon collection. Vitrectomy samples are collected pre-infusion to prevent dilution of the pathogen DNA.
Placement of swab depends on disease suspicion. For suspicion of conjunctivitis, the swab is placed in the conjunctiva without touching the patient's external skin. For a corneal ulcer, the swab should be placed along the periphery of the ulcer, where the causative organism is most likely to be located. Once the sample is collected, the swab should be placed in a sterile tube containing 0.1 ml of a balanced salt solution. In the laboratory, the swab is "milked" and subsequently prepared for PCR.
Yield is not necessarily greatest with more invasive samples, such as aqueous or vitreous biopsies. For example, herpetic retinitis and CMV are better diagnosed with aqueous samples, whereas ocular toxoplasmosis and delayed-onset endophthalmitis is better diagnosed with a vitreous biopsy. External disease can be ideally diagnosed with swab samples.
Storage
Once obtained, both aqueous and vitreous specimens should be quick-frozen on dry ice or in liquid nitrogen and should remain so until the laboratory receives the sample. Freeze-thaw cycles are deleterious to the sample, degrading nucleic acids, particularly RNA. Swab samples should be stored at –70°C until the laboratory is ready to process them.
General Procedure
Polymerase chain reaction consists of three important steps:
1. Extraction of DNA from the standard strains of bacteria/fungi/viruses and clinical specimens
2. Amplification of the extracted DNA by PCR
3. Analysis of the amplified products by agarose gel electrophoresis.
DNA EXTRACTION PROTOCOLS—FROM CLINICAL SPECIMENS
Principle
Two methods for isolating DNA from different ocular samples are:
• Phenol chloroform extraction method
• Kit method (protocols followed as per the manufactures instructions, e.g. Qiagen, Germany).
PHENOL CHLOROFORM EXTRACTION METHOD
The cells containing DNA are chemically or physically lysed to release DNA, which is expected to have the nucleotide sequence or the region of interest.
1. Fluid samples includes extraction of DNA from aqueous humor, vitreous aspirate, cerebrospinal fluid and other body fluids. The fluid specimens are centrifuged at 3000 rpm for 15 minutes and deposit is used for DNA extraction in case of purulent specimens the required quantity is taken as such.
2. To 50 μl of the deposit of the fluid specimen equal volume of the lysis buffer solution (2 mg/ml of lysozyme in 20 mM Tris-Cl; ph- 8). 1 mM EDTA and 0.5% SDS) is added and incubated at 37°C for 1 hour.
3. Proteinase K is added to a final concentration of 200 μg/ml and incubated at 55°C to 60°C for 1 hour.
4. This is followed by addition of equal volume phenol: Chloroform: Isoamyl alcohol (25:24:1) and then sample is mixed gently 3-4 times and microfuged at 14,000 rpm for 15-20 mins. After that aqueous layer is separated into another sterile microfuge tube. If the aqueous layer is still turbid, another phnol:chloroform:isoamyl alcohol extraction is done.
a. To the aqueous layer one tenth volume of 5 M Nacl and double the volume of 100% chilled ethanol is added and kept at –20°C for a minimum of 1 hour and a maximum of overnight.
5. To remove the salt from the precipitated DNA it is brought to room temperature, microfuged for 15 mins. The supernatant is discarded by just inverting the tube gently
6. Two hundred microlitres of 70% ethanol is added and is spun for 15 mins at 14,000 rpm. Supernatant is discarded and the pellet is again washed with 70% ethanol. The same step repeated thrice.
7. The tube is blotted on a blotting paper and mouth of the tube is covered with parafilm.Holes are made on the parafilm and tubes are kept at 37°C so that the ethanlol is completely removed by evaporation.
8. To the dried tube 30 μl of Milli Q water is added to dissolve the DNA and kept at –20°C
PCR CONDITIONS FOR OCULAR FUNGAL GENOME
PCR Primers
Primers specific for the Internal Transcribed Spacer Region (ITS) common to all medically important fungi as described by Lindsley et al is used.
Forward Primer ITS 1 : 5' TCC GTA GGT GAA CCT GCG G 3’
Reverse Primer ITS 4 : 5' TCC TCC GCT TAT TGA TAT GC 3’
The expected product is 601 bp in length. Primers can de custom synthesized by commercial companies.
PCR ASSAY
All the PCR reaction are carried out in a 50 μl reaction volume with 0.2 ml thin wall polypropylene tubes (Axygen Inc, CA, USA) using Biorad thermocycler (model PTC 200). The reagents added to the tube are 5 μl of 10X PCR buffer (15 mM MgCl2, 500 mM KCl, 100 mM Tris-Cl and 0.01% gelatin), 8 μl of DNTP (1.25 mM of each DNTP) 10 pM of each nucleic acid primer, 1 U Taq Polymerase. 10 μl of DNA template and sterile water to make up the volume to 50 μl. PCR Amplification conditions are an initial step at 95°C for 5 minutes followed by 35 cycles carried out at 95°C for 30 minutes.
All PCR reactions include appropriate controls to exclude the possibility of DNA contamination. Visualization of the PCR product is done by subjecting 10 μl volume of the amplified reaction mixture to electrophoresis in Tris-borate buffer on 1.5% agarose gel incorporating 0.5 μg/ml ethidium bromide. The gel is examined on a UV transilluminator (302 nm) and photographed. The criterion for positivity is the presence of a 601bp band after amplification (Fig. 9.1).
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| Fig. 9.1: 10 µl aliquots of amplified product with 2 µl loading buffer are run on a 1.5% agarose gel with ethidium bromide Lane 1 shows PCR reagents Lane 2 shows negative control Lane 3 shows sample Lane 4 shows sample spiked with positive Candida DNA Lane 5 positive control of Candida DNA showed specific band in 601bp product. |
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