In Vitro Detection of Dissimilar Amounts of Hepatitis C Virus (HCV) Subtype-Specific RNA Genomes in Mixes Prepared from Sera of Persons Infected with a Single HCV Genotype

Jorge F. Quarleri; María V. Bussy; Verónica L. Mathet; Vanesa Ruiz; Rubén Iácono; Ling Lu; Betty H. Robertson; José R. Oubiña

doi:10.1128/JCM.41.6.2727-2733.2003

DOI: 10.1128/JCM.41.6.2727-2733.2003

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FIG. 1.
Assessment of detection levels. Two different HCV types were analyzed by RFLP in each mixing experiment. The HCV types mixed are indicated at the right of each panel.
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FIG. 2.
Results of mixing assays. Scale and bar graphs show the quantitative contributions of each subtype (measured as percentages of pixels) to whole 5′ UTR RT-nested PCR products obtained by mixing different amounts of (sub)type-specific HCV RNAs. (A) Subtypes 1b (black bars) and 2c (gray bars). (B) Subtypes 1b and 3a (white bars). (C) Subtypes 2c and 3a.

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TABLE 1.

Serum sample characterization and primers used in RNA mixing assays

Serum sample	Viral load detected by:		Virus type determined by:				Primers used for RT-nested PCR (Set[s])
	Limiting dilution method (10⁵ PCR U/ml)	Roche Monitor version 2.0 (log₁₀ no. of genomic copies/ml)	Sequences analysis		5′ UTR RFLP	INNO-LiPA
	Limiting dilution method (10⁵ PCR U/ml)	Roche Monitor version 2.0 (log₁₀ no. of genomic copies/ml)	NS5B	Core	5′ UTR RFLP	INNO-LiPA
1	8	5.91	1a		1	1a	1 and 2
2	8	5.84	1b		1	1b	1 and 2
3	0.8	4.76	2c		2	2a or 2c	1 and 2
4	8	6.19	2c		2	2a or 2c	1 and 2
5	0.8	5.08		3a	3	3a	1 and 2
6	8	5.86	1b		1	1a	1 and 2
7	8	5.87	1b		1	1b	1 and 2
8	8	5.93	2c		2	2a or 2c	1 and 2
9	8	5.93	2c		2	2b or 2c	1 and 2
10	0.04	3.88		3a	3	3a	1 and 2
11	8	5.80	1b		1	1b	2
12	8	5.59	2c		2	2a or 2c	2
13	0.8	5.07		3a	3	3a	2
14	0.8	5.15		4a	3	3a	2

TABLE 2.

Combinations of serum samples used for mixing assays

Sample no. (subtype)	Type of mix for batch^a
	A					B					C
	1 (1a)	2 (1b)	3 (2c)	4 (2c)	5 (3a)	6 (1b)	7 (1b)	8 (2c)	9 (2c)	10 (3a)	11 (1b)	12 (2c)	13 (3a)	14 (4a)
1 (1a)		X	X	X	X
2 (1b)	X		X	X	X
3 (2c)	X	X		X	X
4 (2c)	X	X	X		X
5 (3a)	X	X	X	X
6 (1b)							X	X	X	X
7 (1b)						X		X	X	X		v	v
8 (2c)						X	X		X	X	v		v
9 (2c)						X	X	X		X
10 (3a)						X	X	X	X		v	v
11 (1b)								v		v		X	X	X
12 (2c)							v			v	X		X	X
13 (3a)							v	v			X	X
14 (4a)											X	X

↵ a Three independent batches of serum samples were designated A, B, and C. Sample numbers in each batch are shown, with corresponding subtypes listed in parentheses. Each letter in the table indicates a combination of the samples listed above and to the left. X indicates a combination of two samples from the same batch; v indicates a combination of two samples from different batches.

TABLE 3.

Quantitative contributions of each type of HCV to total 5′ UTR RT-nested PCR products obtained by mixing different amounts of type-specific HCV RNAs^a

Virus types mixed	Ratio of types in mix	Amount of product (percentage of pixels ± SD) from type:
Virus types mixed	Ratio of types in mix	1	2	3
1 and 2	90:10	48.7 ± 5.0	51.2 ± 5.0
	50:50	30.0 ± 3.3	70.0 ± 3.3
	10:90	12.7 ± 7.5	87.2 ± 7.5
1 and 3	90:10	53.3 ± 12.1		46.7 ± 12.1
	50:50	53.7 ± 15.6		46.2 ± 15.6
	10:90	25.2 ± 5.1		74.8 ± 5.1
2 and 3	90:10		90.5 ± 4.1	9.5 ± 4.1
	50:50		73.7 ± 8.2	26.2 ± 8.2
	10:90		59.7 ± 8.7	40.3 ± 8.7

↵ a Data shown are the averages of results from six (for mixtures of types 1 and 3 and 2 and 3) or eight (for mixture of types 1 and 2) independent experiments with 13 viremic serum samples. MMLV reverse transcriptase and 5′ UTR primer sets 1 and 2 were used for experiments. Reverse transcription performed by using Tth reverse transcriptase with another batch of five serum samples (not described in this study) rendered similar results. P values for comparisons between product ratios were as follows: for types 1 and 2 versus types 2 and 3, <0.0001 with all mix ratios; for types 1 and 2 versus types 1 and 3, not significant with a 90:10 mix ratio, 0.0060 with a 50:50 mix ratio, and 0.0044 with a 10:90 mix ratio; for types 2 and 3 versus types 1 and 3, <0.0001 with 90:10 and 10:90 mix ratios and not significant with a 50:50 mix ratio.