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 | |
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] |