Quantitative Detection of Hepatitis B Virus DNA in Two International Reference Plasma Preparations

DOI: 10.1128/JCM.37.1.68-73.1999

Article Figures & Data

Table 1.

Primers and conditions of nested PCR used for limiting-dilution assays by the three laboratories participating in phase 2 of the study

PCR and laboratory	Gene region	Position		Annealing temp (°C)	No. of cycles	Sequence reference no.
PCR and laboratory	Gene region	Forward primer^a	Reverse primer	Annealing temp (°C)	No. of cycles	Sequence reference no.
PCR 1
1a	S	392–416	750–726	65–57.4^b	30	14
1b	X	1275–1302	1577–1551	67.9–60.4^b	30	14
2	pre-S and S	2718–2738	1305–1285	55	35	8
8	S	194–213	706–687	55	40	27
PCR 2
1a		427–449	694–672	60.1/58.1^b	20/20
1b		1305–1328	1542–1522	63.5/61.5^b	20/20
2		2821–2843	191–168	60	35
8		305–324	433–414	55	40

↵a Numbering starts at the uniqueEcoRI site at position 3221/0.
↵b Laboratory 1 used a touchdown program for annealing temperatures from 5°C above the melting temperature of the primer down to −2.5°C for 15 cycles and then 15 further cycles at the lower temperature.

Table 2.

Phase 1 results from seven laboratories for Nin R1, R2, and dilutions A to D^a

Sample (dilution)	N
Sample (dilution)	Laboratory 1a	Laboratory 1b	Laboratory 2	Laboratory 3	Laboratory 4	Laboratory 5	Laboratory 6^b	Endpoint titer laboratory 7^c
R1	5.5 × 10⁸	5.5 × 10⁸	2.2 × 10⁹	4.3 × 10⁸	1.6 × 10⁹	4.0 × 10⁸	1.2 × 10⁷	1:1,000
A (1:64)	2.4 × 10⁷ (1:23)^d	3.2 × 10⁷ (1:17)	1.1 × 10⁸ (1:20)	4.3 × 10⁵^b (1:1,000)	3.0 × 10⁷ (1:53)	1.1 × 10⁷ (1:35)	8.2 × 10⁵ (1:14)	1:128
B (1:64,000)	1.1 × 10⁴ (1:50,000)	2.1 × 10⁴ (1:26,000)	4.8 × 10⁴ (1:46,000)	4.3 × 10^3e (1:100,000)	<5 × 10⁶	<2.9 × 10⁶	6.6 × 10² (1:17,900)	Negative
R2	8.6 × 10⁸	5.2 × 10⁸	4.7 × 10⁹	4.3 × 10⁸	2.0 × 10⁹	3.4 × 10⁸	2.7 × 10⁶	1:>100
C (1:32)	5.5 × 10⁷ (1:16)	1.3 × 10⁷ (1:39)	1.3 × 10⁸ (1:36)	4.3 × 10^7e (1:10)	5.2 × 10⁷ (1:38)	1.1 × 10⁷ (1:30)	3.4 × 10⁵ (1:8)	1:32
D (1:32,000)	3.0 × 10⁴ (1:28,700)	9.3 × 10^4b (1:5,600)	1.7 × 10⁵ (1:28,000)	1.4 × 10^4e (1:30,000)	<5 × 10⁶	<2.9 × 10⁶	1.2 × 10³ (1:2,390)	Negative
No. of replicates per dilution step	7	7	11–16	2

↵a Laboratories 1 to 3 used nested PCR and limiting dilution; laboratories 4 to 7 used reference samples.
↵b Data not considered for further evaluation because of fourfold or greater deviation from the true dilution factor.
↵c Endpoint titer of a hybridization assay.
↵d Values in parentheses are dilution factors determined by the laboratories.
e Due to low numbers of replicates, these data were not considered for calculation of the GMV.