Box 1 Polymerase chain reaction (PCR)
In cycle 1 two primers anneal to denatured DNA at opposite sides of the target region and are extended by DNA polymerase to give new strands of variable length. In cycle 2, the original strands and the new strands from cycle 1 are separated, yielding a total of four primer sites with which primers anneal. The primers that are hybridized to the new strands from cycle 1 are extended by polymerase as far as the end of the template, leading to a precise copy of the target region. In cycle 3, double-stranded DNA molecules are produced that are precisely identical to the target region. Further cycles lead to exponential doubling of the target region. The original DNA strands and the variably extended strands become negligible after the exponential increase of target fragments.
Post-PCR Species Determination
Following panbacterial and panfungal PCRs, a second step is required to identify the species of bacteria or fungus. A variety of methods including sequencing of the amplified DNA, hybridization with specific probes, or restriction fragment length polymorphism (RFLP) have been used, of which sequencing is most common [10]. Sequence of the amplified DNA is compared with similar sequences in databases using NCBI BLASTn 2.2.26 program of the National Center for Biotechnology Information (NCBI, GenBank database). Usually, a score of 97% and similarity of 98% allow the genus recognition, and a score of 99% or more may assign a species name. More reliable is the identification based on phylogenetic analysis, which shows the phylogenetic distance between the analyzed sequence and the sequences in the databases. Phylogenetic analysis allows comparison with type strains available in the databases.
In hybridization technique, the amplified DNA obtained from the sample is denatured and transferred on a membrane (dot blot assay) to which are added labeled (radioactive isotopes, fluorophores, haptens like biotin or digoxigenin or an enzyme) specific probes. The probes are usually a short strand of oligonucleotide specific for hybridization with complementary sequence of either a species of organism or a group of organisms such as gram-negative or gram-positive bacteria. Conversely, in DNA chip technology, complementary sequences to signature genes of specific organisms or groups of organisms are dotted on the chip. To this is added multiplex PCR-amplified biotin-labeled denatured DNA (of the sample) that would hybridize to complementary DNA on the chip. Enzyme-labeled streptavidin in the next step would reveal the specific gene by color development on addition of the substrate [11].
In PCR-RFLP, the bacterial and fungal genome can be identified by their genetic fingerprint produced by the use of restriction enzymes on the amplicons. The number of fragments is proportionate to the number of restriction sites in the genome and is specific for particular species [1].
When species identification has been attempted, most studies have used sequencing compared to RFLP and hybridization [10, 12]. Value of PCR in the diagnosis of endophthalmitis caused by anaerobes such as Propionibacterium acnes has been well established [8]. Causative role of rare fungal species like Colletotrichum truncatum in endophthalmitis has been confirmed by sequencing of internal spacer regions of ribosomal DNA [13].
Aforementioned methods mostly identify mono-microbial infection. An entirely different approach through cloning 16S rRNA gene and next-generation sequencing has shown a polymicrobial etiology of infectious endophthalmitis [14]. The authors identified several novel bacteria associated with endophthalmitis.
Real-Time PCR
Recent studies have reported application of real-time qualitative or quantitative PCR in the diagnosis of infectious endophthalmitis. Most diagnostic applications of real-time PCR are qualitative. Quantitative PCR, the qPCR, determines the amount of DNA in the sample and can be absolute or relative. Real-time PCR requires a special thermocycler that measures fluorescence, which is produced in proportion to the amplification of the DNA, cycle by cycle (Box 2).
Box 2 Real-time PCR
Top—The template is amplified by primers and the amplicon allows for annealing of sequence-specific, labeled probes. As a new strand is synthesized, the probes are displaced, the label cleaved off, and a fluorescent signal proportional to the amount of the cleaved probe is generated. The fluorescence is measured and recorded at each cycle of PCR. Cycle threshold (Ct) is defined as the fractional PCR cycle number in which the sample fluorescence signal reaches a level above an assigned fluorescence threshold. The Ct value indicates the beginning of the exponential amplification of the template DNA or RNA and is proportional to the concentration of the sample.
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