Multiplex qPCR for serodetection and serotyping of hepatitis viruses: A brief review

Table 2 Chemistries/Dyes used in qPCR assays

S. NO.	Class	Types	Structure	Mechanism of action	Advantages	Applications
1	DNA binding dyes	Ethidium Bromide, SYBR Green, SYBR Gold, YO-PRO-1, SYTO, BEBO, BOXTO, EvaGreen	Intercalating dyes	Bind to the minor groove of dsDNA during amplification	Inexpensive	Pathogen detection
					Easily available	Gene expression
						SNP detection
						Genotyping
2	Fluorophore labeled oligonucleotide	Primer probes
		Hairpins: Scorpions, Ampliflour, LUX	Loop based oligonucleotides	Bind to target during denaturation with emission of fluorescence	Inexpensive, Prevent formation of primer dimer, Less background signals	Pathogen detection
						Genotyping
						SNP allelic discrimination
						Mutation detection
		Cyclicons	Cyclic structure with reporter at 3’ end and quencher at 5’ end	Reporter and quencher in close proximity with energy transfer via FRET quenching. Their separation results in fluorescence emission during amplification	Inexpensive	Pathogen detection
					Less contamination	Genotyping
					Less background signals	SNP allelic discrimination
						Mutation detection
		Angler	Probe with DNA sequence bound to reverse primer through a HEG linker	During annealing step, DNA polymerase does extension of 3’ end reverse primer. Later on, SYBR Gold dye intercalates in dsDNA emitting fluorescence	Highly specific	Gene expression
						Pathogen detection
						SNP detection
						Genotyping
		Probes
		Hydrolysis Probes: TaqMan probes, MGB-TaqMan, Snake assay	Oligonucleotide with reporter at 5’ and quencher at 3’ end	Probe is degraded by 5’ to 3’ exonuclease activity of DNA polymerase generating fluorescence during extension	Design and synthesis easy	Microarray validation
						Pathogen detection
						SNP allelic discrimination
						Mutation detection
		Hybridization probes: Hybprobes, Molecular Beacon, HyBeacon, MGB Probes	A pair of oligonucleotides having reporter dye on first and quencher on second oligonucleotide	Binding to target during hybridization and annealing brings fluorophore into proximity producing fluorescence by FRET	Design and synthesis quick and easy	Microarray validation
						Pathogen detection
						Viral/Bacterial genotyping
						SNP allelic discrimination
						Mutation detection
		Nucleic acid analogues
		PNAs, LNAs, ZNAs	Intercalating/inserting dyes	Identical to conventional oligonucleotides	Resistant to nuclease and proteases activity	Discriminate between DNA and cDNA in prokaryotes
		Non-natural bases

All above details were collected from report published in Clinica Chimica Acta 2015; 439: 231-250[25]. SNP: Single nucleotide polymorphisms.