There are many different detection chemistries of qPCR. They include DNA binding dyes, hybridization probes, hydrolysis probes (66) and analogues of nucleic acids such as locked nucleic acids (LNA) probes (64) . These chemistries reliably detect and measures the products generated during each amplification cycle of the PCR according to the intensity of the fluorescence which correlates with the concentration of PCR products (66).
The greater the number of target sequence copies, the fewer the number of cycles necessary to produce a detectable fluorescence (67). The threshold cycle (Ct) is determined at the point at which the fluorescence first rises above the background. The Ct is a measure of the copy number of the RNA which can be determined from a standard curve of cloned RNA (68). The standard curve is obtained by amplifying known amounts of standards under the same conditions as the samples (69).
Hydrolysis probesHydrolysis probes are fluorescently labeled DNA oligonucleotides that are designed to bind downstream of one of the primers during PCR. The 5′-end of probe is labeled with a fluorescent reporter molecule while the 3′- end is labeled with a quencher molecule (66). Their mechanism is based on the 5′- to 3′- exonuclease activity of the Taq polymerase that degrades the bound probe during amplification. The cleavage of the probe separates the reporter molecule from the rest of the probe allowing the reporter molecule to fluoresce. When the reporter and quencher are in close proximity to one another fluorescence is suppressed (70). The hydrolysis probe is designed to increase specificity; it binds to a specific region of the DNA target (64).
These probes reduce the background fluorescence and have larger dynamic range because of the efficiency of reporter quenching (66).1.4.3. Analogues of nucleic acidsThe flaviviruses have high sequence diversity and therefore, designing primers and probes for a pan assay can be a major challenge.
However, Patel and co-workers designed degenerate primers and probes for the simultaneous detection of a broad range of flaviviruses. The probes in the Pan-Flavi assay were modified to contain LNA nucleotides to match all flaviviruses (58). LNA probes have higher melting temperatures because of the locked nucleotides, it is more stable and the probe length is shorter compared to TaqMan probes. Therefore, LNA probes has an increase sensitivity and specificity (72). PCR primers or probes modified with LNA nucleotides show very accurate mismatch discrimination and high specificity (73).