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Table 1 Summary of qRT-PCR for HDV

From: Hepatitis D: challenges in the estimation of true prevalence and laboratory diagnosis

Technique

Target site

Linear ranges

Advantage

Disadvantage

References

SYBR Green I qRT-PCR

A highly conserved sequence within the HDAg-coding region

1–106 copies

A sensitive method for the detection of HDV RNA at levels as low as a single copy

HDV is prone to mutations that can cause primer mismatch

[54]

TaqMan probe qRT-PCR

The ribozyme region of the genome

10–107 copies

Avoid post-PCR handing that can be a source of DNA carryover

For HDV-3 isolates, the forward primer will mismatch

[51]

Sybr green I qRT-PCR

The HDAg coding region

103–109 copies

Detect HDV genomic RNA, antigenomic RNA, and HDAg-encoding mRNA in an HDV cDNA-free RNA transfection system

The primers are designed according to HDV-1 and HDV-2

[55]

Hybridization probe qRT-PCR

Highly conserved regions

2 × 103–108 copies/ml

This approach facilitates a subsequent typing based on melting curve analysis

Only quantitative detection of HDV-1 and HDV-3

[43]

Cobas TaqMan-based in-house PCR

A conserved region that encodes the HDAg

3 × 102–1.5 × 108 copies/ml

This platform provides the automated extraction system, a minimal risk of contamination and a high degree of reproducibility

The DNA clone does not accurately monitor the expansion of RNA viruses

[52]

Hybridization probe qRT-PCR

A highly conserved region

103–108 copies/ml

High sensitivity and a wide dynamic range

A cloned cDNA does not permit assessment of the RT step

[44]

In-house qRT-PCR

The highly conserved ribozyme and s-HDAg regions

101–107 copies/reaction

This technology offers more options for primer design

This has proven challenging due to the high genetic diversity of the HDV genome

[45]

TaqMan probe qRT-PCR

The highly conserved ribozyme region of the HDV genome

7 × 102–7 × 106 copies/ml

A BMV RNA internal control which effectively monitors all stages of the assay

Using more than 10 fg BMV RNA per sample can reduce HDV assay sensitivity

[48]

Quantitect Virus kit

The conserved parts of the gene encoding the delta antigen

2.7 × 101–2.7 × 1011 copies/reaction

A one-step qRT-PCR can be automated for the accurate quantification of HDV in Europe in the presence of an encapsulated heterologous RNA used as an internal control

HDV-1 cannot be completely detected

[49]

Molecular beacon qRT-PCR

The ribozyme domain of HDV genome

13–13 × 1010 copies/ml

High sensitivity, reproducibility and wide detection range

A synthetic DNA has been used as standard

[46]

AgPath-ID One-Step kit

The ribozyme region of HDV genome

7.5 × 102–7.5 × 108 copies/ml

The positive control transcript controls for every step of the qRT-PCR

The clinical samples tested do not contain all HDV genotypes

[50]

One step EZ RT PCR kit

The conserved regions of HDAg

102–1011 copies/ml

A new protocol having armored RNA as external standard and GAPDH as the intrinsic internal control in single tube format

The extracted samples contain abundant DNA-natured GAPDH, while only a small amount of RNA-natured GAPDH

[60, 61]

The Eurobioplex HDV kit

HDAg coding region

2.75–8.5 log IU/ml

The kit exhibited good clinical agreement with the French National Reference Laboratory (FNRL) assay and detected all HDV genotypes

Tendency to underestimate HDV-5 to -8

[62]