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

Table 1 Conserved genomic regions used as templates for amplification of hepatitis viruses in qPCR assays

Virus	Conserved region	Ref.
HAV	5’ UTR	[4,15,20,22]
HBV	S-gene	[4, 19-21]
	X-gene	[15]
HCV	5’ UTR	[4,15,19-21]
HDV	Ribozyme-1	[20]
HEV	ORF2	[15,22]
	ORF3	[20]
HGV	5’ UTR	[20]

HAV: Hepatitis A virus; HBV: Hepatitis B virus; HCV: Hepatitis C virus; HDV: Hepatitis D virus; HEV: Hepatitis E virus; UTR: Untranslated region; ORF: Open reading frame.

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.

Table 3 Global status of multiplex qPCR developed for hepatitis viral infections with and without other pathogens

No.	Assay systems	Instruments used	Group of pathogens detected		Types of chemistries/detection methods used	Ref.
			Hepatitis viruses	Other pathogens
1	Multiplex real time PCR	Mx4000 (Stratagene)	HBV, HCV	HIV type-1, T. pallidum	TaqMan-LNA probe	[21]
2	Multiplex real time PCR	Light cycler 480 (Roche)	HEV genotypes	-	N.A.	[41]
3	Real time PCR assay	ABI 7500 (Applied Biosystems)	HAV, HBV, HCV, HDV, HEV	-	TaqMan Array card	[42]
4	Multiplex qPCR assay	Light cycler 480 (Roche)	HBV, HDV	-	TaqMan probe	[43]
5	Multiplex qPCR assay	ABI 7500 (Applied Biosystems)	HAV, HEV	-	Hydrolysis probe	[22]
6	Multiplex qRT-PCR	N.A.	HAV	Norovirus genotypes 1 and 2	TaqMan probe	[44]
7	Multiplex ligation dependent probe real time PCR	Rotor-GeneQ (Qiagen)	HBV mutants	-	TaqMan probe	[45]
					MLPA probe
8	Multiplex real time RT-PCR	N.A.	HEV genotypes	-	N.A.	[46]
9	Multiplex qPCR	N.A.	HBV genotypes	-	SYBR Green	[47]
10	Multiplex Real time PCR	N.A.	HAV	Norovirus, Rotavirus, Coxsackievirus	TaqMan probe	[48]
11	Multiplex Real time PCR	Light cycler 2.0 (Roche)	HAV, HBV, HCV and HEV	-	FRET probe	[15]
12	Multiplex RT-PCR	ABI 2720 (Applied Biosystems)	HCV	HIV type-1	SYBR Green I	[8]
13	Multiplex qPCR	N.A.	HAV, HEV	Entero and Adeno-viruses	N.A.	[49]
14	Multiplex Real-Time PCR Assay	CFX96 (Bio-Rad)	HAV, HBV, HCV	-	READ technology based fluorophore	[4]
15	RT PCR assay	Smart cycler II (Cepheid)	HBV, HCV	-	TaqMan probe	[50]
16	Duplex real time PCR	ABI 7500 (Applied Biosystems)	HBV variants	-	Hydrolysis probe	[51]
17	Multiplex RT PCR	N.A.	HCV subtyping	-	Electrophoresis	[52]
18	Multiplex qPCR	N.A.	HBV genotypes	-	N.A.	[53]
19	Multiplex qPCR	N.A.	HCV	HIV type-1	SYBR Green I	[54]
20	Duplex real-time RT-PCR	ABI Prism system (Applied Biosystems)	HCV variants	-	Hydrolysis probe	[55]
21	Multiplex real time PCR	N.A.	HAV	Norovirus genotypes 1 and 2	N.A.	[56]
22	Duplex real-time qRT-PCR	ABI Prism 7000 (Applied Biosystems)	HAV	MS2 bacteriophage	MGB-TaqMan probe	[57]
23	Multiplex TaqMan RT-qPCR system	MX30005P (Stratagene)	HEV	FCV	TaqMan probe	[58]
24	Multiplex real time PCR	ABI 7300 (Applied Biosystems)	HBV genotypes	-	TaqMan probe	[59]
25	Real time PCR	N.A.	HBV genotypes	-	TaqMan probe	[60]
26	Multiplex real time PCR	Mx3005P (Stratagene)	HEV	FCV	TaqMan probe	[61]
27	Multiplex RT PCR assay	ABI Prism 7500 (Applied Biosystems)	HCV	PDV	MGB hybridization probe	[62]
28	Multiplex qPCR assay	N.A.	HBV	B19, HHV-8, EBV, CMV, VZV	N.A.	[63]
29	Multiplex qPCR	N.A.	HBV, HCV	HIV type-1	SYBR Green I	[16]
30	Multiplex Real Time PCR	ABI 7500 (Applied Biosystems)	HBV mutants	-	LNA probes with SYBR Green I	[64]
31	Microarray multiplex assay	ABI Prism 7700 (Applied Biosystems)	HBV, HCV	HIV type-1	Oligonucleotide array labeled with Cy5 and Cy3	[65]
32	Real time multiplex PCR	N.A.	HAV	Entero and Adeno-viruses	Probes labeled with FAM, R6G, ROX, Cy5	[66]
33	Multiplex real time RT-PCR	LightCycler (Roche)	HCV	HIV type-1	SYBR Green	[67]
34	Real time multiplex PCR	icycler iQ (Bio-Rad)	HCV variants	-	TaqMan probes	[68]
35	Multiplex real-time RT PCR	ABI 7000 (Applied Biosystems)	HCV genotypes	-	Primer probes	[69]
36	Multiplex real-time qPCR	Mx4000 (Stratagene)	HBV, HCV	HIV type-1	TaqMan probes	[70]
37	Automated multiplex PCR	ABI Prism 7700 (Applied Biosystems)	HBV, HCV	HIV type-1	TaqMan probes	[71]