Droplet digital PCR (ddPCR) is a novel developed PCR technology providing the absolute quantification of target nucleic acid molecules without the need for a standard curve and regardless PCR amplification efficiency. Our aim was to develop a ddPCR assay for Hepatitis Delta virus (HDV)-RNA viremia quantification and then evaluate its performance in relation to real-time PCR methods. Primers and probe were designed from conserved regions of HDV genome to detect all the 8 HDV genotypes; the World Health Organization (WHO)-HDV international standard was used to calculate the conversion factor transforming results from copies/mL to IU/mL. To evaluate the clinical performance of ddPCR assay, plasma specimens of HDV-infected patients were tested and results were compared with data obtained with two real-time quantitative PCR (RT-qPCR) assays (i.e., in-house assay and commercial RoboGene assay). Analyzing by linear regression a series of 10-fold dilutions of the WHO-HDV International Standard, ddPCR assay showed good linearity with a slope coefficient of 0.966 and R2 value of 0.980. The conversion factor from copies to international units was 0.97 and the quantitative linear dynamic range was from 10 to 1 × 106 IU/mL. Probit analysis estimated at 95% an LOD of 9.2 IU/mL. Data from the evaluation of HDV-RNA in routine clinical specimen of HDV patients exhibited strong agreement with results obtained by RT-qPCR showing a concordance correlation coefficient of 0.95. Overall ddPCR and RT-qPCR showed highly comparable technical performance. Moreover, ddPCR providing an absolute quantification method may allow the standardization of HDV-RNA measurement thus improving the clinical and diagnostic management of delta hepatitis.

Clinical Application of Droplet Digital PCR for Hepatitis Delta Virus Quantification

Olivero A.
First
;
Rosso C.;Ciancio A.;Abate M. L.;Nicolosi A.;Armandi A.;Ribaldone D. G.;Saracco G. M.;Bugianesi E.;Rizzetto M.;Caviglia G. P.
Last
2022-01-01

Abstract

Droplet digital PCR (ddPCR) is a novel developed PCR technology providing the absolute quantification of target nucleic acid molecules without the need for a standard curve and regardless PCR amplification efficiency. Our aim was to develop a ddPCR assay for Hepatitis Delta virus (HDV)-RNA viremia quantification and then evaluate its performance in relation to real-time PCR methods. Primers and probe were designed from conserved regions of HDV genome to detect all the 8 HDV genotypes; the World Health Organization (WHO)-HDV international standard was used to calculate the conversion factor transforming results from copies/mL to IU/mL. To evaluate the clinical performance of ddPCR assay, plasma specimens of HDV-infected patients were tested and results were compared with data obtained with two real-time quantitative PCR (RT-qPCR) assays (i.e., in-house assay and commercial RoboGene assay). Analyzing by linear regression a series of 10-fold dilutions of the WHO-HDV International Standard, ddPCR assay showed good linearity with a slope coefficient of 0.966 and R2 value of 0.980. The conversion factor from copies to international units was 0.97 and the quantitative linear dynamic range was from 10 to 1 × 106 IU/mL. Probit analysis estimated at 95% an LOD of 9.2 IU/mL. Data from the evaluation of HDV-RNA in routine clinical specimen of HDV patients exhibited strong agreement with results obtained by RT-qPCR showing a concordance correlation coefficient of 0.95. Overall ddPCR and RT-qPCR showed highly comparable technical performance. Moreover, ddPCR providing an absolute quantification method may allow the standardization of HDV-RNA measurement thus improving the clinical and diagnostic management of delta hepatitis.
10
4
792
807
chronic hepatitis D; ddPCR; HDV
Olivero A.; Rosso C.; Ciancio A.; Abate M.L.; Nicolosi A.; Troshina G.; Armandi A.; Ribaldone D.G.; Saracco G.M.; Bugianesi E.; Rizzetto M.; Caviglia G.P.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2318/1855158
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