Journal of Clinical Virology 61 (2014) 286–288
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Rapid hepatitis B and hepatitis Delta virus RNA quantification from small-sized liver tissue samples Andrzej Taranta a,b , Magdalena Rogalska-Taranta a,c , Rodrigo Gutierrez a , Michael Peter Manns a,b , Michael Bock a , Karsten Wursthorn a,b,d,∗ a
Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School (MHH), Carl-Neuberg-Straße 1, 30625 Hannover, Germany German Center for Infection Research (DZIF), partner site Hannover-Braunschweig c ˙ Department of Infectious Diseases and Hepatology, Medical University of Bialystok, Zurawia 14, 15540 Białystok, Poland d ifi – Institut für Interdisziplinäre Medizin, Lohmühlenstraße 5, 20099 Hamburg, Germany b
a r t i c l e
i n f o
Article history: Received 27 April 2014 Received in revised form 7 July 2014 Accepted 28 July 2014 Keywords: HBV RNA HDV RNA Quantification Real-time PCR Multiplex qPCR FFPE
1. Reasons for developing the assay
2. Methods used in designing the assay
Hepatitis B and Delta viruses target human liver increasing the risk of serious liver-related health problems [1,2]. The quantification of HBV DNA and HDV RNA from patients’ serum is the main diagnostic method for monitoring the course of the infections [3,4]. Information on intrahepatocellular viral nucleic acid concentrations – among others – might contribute to the better understanding of the complex interaction between the two viruses. We have developed new multiplex quantitative real-time PCR (qPCR) protocols that require less nucleic acids isolated from formalin-fixed paraffin-embedded (FFPE) tissue compared to separate preparations, shorten hands-on time and save reagents. They enable the simultaneous amplification of the housekeeping gene -actin (ACTB) mRNA in combination with either HDV RNA or HBV pgRNA and S-RNA.
Fragments of HBV DNA (3170 bp), HDV cDNA (125 bp) and ACTB cDNA (905 bp) were cloned into the pUC19 backbone to get a uniform plasmid. Multiplex qPCRs with gene specific primers and TaqMan probes with different fluorescent dyes were established. Sequences of oligonucleotides were either newly designed (ACTB) or have been published before (pgRNA, S-RNA, HDV RNA) [4,5]. Respective sequences are listed in Table 1 together with the amplicons and probes.
∗ Corresponding author at: ifi – Institut für Interdisziplinäre Medizin, Lohmühlenstraße 5, 20099 Hamburg, Germany. Tel.: +49 40 28407600; fax: +49 40 2840760 222. E-mail address:
[email protected] (K. Wursthorn). http://dx.doi.org/10.1016/j.jcv.2014.07.016 1386-6532/© 2014 Elsevier B.V. All rights reserved.
3. Protocol RNA isolation, DNase I treatment and reverse transcription procedure are detailed in Supplementary materials. A reaction premix (18 l) consisting of 10 l LightCycler480 ProbesMaster 2× (Roche, Germany), primers and probes at the final concentration of 200 nM and 176 nM, respectively were mixed with 2 l of sample cDNA (Table 1). Quantitative PCRs were run on the LightCycler480 II with unique protocols (Table 2). 4. Validation data Both methods were tested with standard plasmid dilutions and cDNA derived from FFPE-preserved liver tissue of hepatitis B and
A. Taranta et al. / Journal of Clinical Virology 61 (2014) 286–288
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Table 1 Primers and probes used in HDV/ACTB and pg/S/ACTB qPCR protocols. Primer sequences, TaqMan labels and amplicon lengths are specified. HDV RNA specific oligonucleotides were designed by Mederacke et al. [4]. HBV pgRNA and S-RNA amplifying oligonucleotides originally by Malmström et al. [5].
Oligonucleotide name
HDV
HDV/ACTB qPCR
amplicon size [bp]
ACTB
71
pgRNA
pg/S/ACTB qPCR
99
S-RNA
88
83
5’-label-sequence-quencher-3’
Fwd1
TGGACGTKCGTCCTCCT
Fwd2
TGGACGTCTGTCCTCCTT
Probe
FAM-ATGCCCAGGTCGGAC-BHQ1
Rev
TCTTCGGGTCGGCATGG
Fwd
CCTTCCTTCCTGGGCATG
Probe
ATTO647N-TGTGGCATCCACGAAACTACCTT-BBQ
Rev
GTTGGCGTACAGGTCTTTG
Fwd
GGTCCCCTAGAAGAAGAACTCCCT
Probe
FAM-TCTCAATCGCCGCGTCGCAGA-BHQ1
Rev
CATTGAGATTCCCGAGATTGAGAT
Fwd
TCCTCCAAYTTGTCCTGGTYATC
Probe
ATTO550-ATGATAAAACGCCGCAGACACATCCARC-BHQ2
Rev
AGATGAGGCATAGCAGCAGGAT
Table 2 Protocols for the HDV/ACTB and pg/S/ACTB quantitative PCR on the LightCycler480 II (Roche, Germany). The following filter combinations were applied: 465–510 for FAM, 533–580 for ATTO550 and 618–660 for ATTO 647N. Overlapping spectra were compensated according to the manufacturer’s manual. HDV/ACTB qPCR
pg/S/ACTB qPCR
Step
Temperature [ C]
Time [s]
Step
Temperature [◦ C]
Time [s]
Initiation Cycling 45×
95 95 60 37
420 15 65 1
Initiation Cycling 45×
95 95 57 60 72 37
420 15 5 45 8 1
Cooling
◦
Cooling
Delta patients. Water and non-reversely transcribed RNA were used as negative controls. cDNA from primary human hepatocytes (PHH) was used for ACTB amplification. To determine the respective specificity and lower limits of detection (LLoD) and quantification (LLoQ) five independent runs in triplicates were performed. The threshold cycle (Ct ) values from ACTB in PHH were consistent throughout the runs with inter-run coefficient of Ct variation of 0.10% for HDV/ACTB qPCR and 0.18% for pg/S/ACTB qPCR. There was no probe excitation in the negative controls supporting the high specificity.
LLoD, LLoQ and coefficient of determintion (R2 ) are shown in Table 3. FFPE-embedded liver biopsies from 4 HBV monoinfected and 3 HBV/HDV co-infected patients at three different concentrations (native cDNA, 10-fold and 100-fold dilutions) were analysed with the novel qPCR methods. All seven samples were HBV RNA positive and all three Delta samples were positive for HDV RNA. The linearity of the dilutions was significant where applicable (R2 > 0.9; Supplementary table 1). Results of ACTB quantification were compared by Bland–Altman analysis and with the paired Student’s
Table 3 Lower limits of detection (LLoD) and lower limits of quantification (LLoQ) of the HDV/ACTB and pg/S/ACTB quantitative PCRs. qPCR
Amplicon
HDV/ACTB
HDV ACTB
pg/S/ACTB
HBV pgRNA HBV S-RNA ACTB
LLoD [cps/rxn]
LLoQ [cps/rxn]
Mean CV [%]
R2
p
4 4
40 40
0.92 1.68
0.9933 0.9956