Development of a Hypersensitive Detection Method for Human Parvovirus B19 DNA

Abstract

A new detection method for human parvovirus B19 DNA was established using PCR coupled with a hybridization protection assay. The amplified product was detected using acridinium ester-labeled DNA probes. By this method, a few copies of B19 DNA were detected in human serum albumin.

Human parvovirus B19 (B19) is known to cause erythema infectiosum (fifth disease) in children (1) and chronic anemia in immunocompromised patients. Recently, Saldanha and Minor (14) reported that blood products from manufacturers around the world contained B19 DNA detected by the PCR method. This report drew attention to the risk of infection through blood products, especially in immunocompromised patients. Since B19 is nonenveloped and thermostable even at 80°C (9), inactivation of the virus is difficult if it is contained in a blood product. Therefore, a sensitive and rapid method of detecting B19 in blood products is required. We have attempted to establish a hypersensitive detection method involving the use of PCR (13, 15, 20, 22) with a hybridization protection assay (HPA) (2, 12).

To estimate the number of B19 DNA copies per assay, about 4.7 kb of B19 DNA (nucleotides [nt] 215 to 4891) was amplified from serum of an erythema infectiosum patient, cloned in plasmid pUC19, and propagated in Escherichia coli. HB101. The B19 DNA fragment was excised from the plasmid and purified. The concentration of the DNA was determined by measuring A₂₆₀. Three B19-positive plasma samples were supplied by H. Sato, Fukuoka Red Cross Blood Center. The concentrations of B19 DNA in plasma samples 1, 2 and 3 determined by the quantitative and competitive PCR method by H. Sato were 1.34 × 10¹⁰, 2.13 × 10⁹, and 2.60 × 10¹⁰ copies/ml, respectively.

Extraction of DNA from the plasma samples or human serum albumin (HSA) solution was performed by using an SMI-Test Kit (Sumitomo Metal Industries, Ltd., Tokyo, Japan) in accordance with the instructions supplied with the kit.

Two oligonucleotides primers were designed to amplify a 398-bp segment in the VP1-VP2 region of B19 DNA (17, 20) and named PVVPa 3187(+) (5′-CAA AAG CAT GTG GAG TGA GG-3′ [nt 3187 to 3206]) and PVVPb 3584(−) (5′-CTA CTA ACA TGC ATA GGC GC-3′ [nt 3584 to 3565]).

The reaction mixture (100 μl) for PCR amplification consisted of 10 μl of 10× Gene Taq Universal Buffer, 0.2 mmol of deoxynucleoside triphosphates, 2.5 U of Taq polymerase (Wako Pure Chemicals Industries, Ltd., Osaka, Japan), 15 pmol of each primer, and 10 μl of the template. Ten microliters of silicone oil was added to the reaction mixture. Amplification was performed as follows: 94°C for 3 min, followed by 25 or 40 cycles of 94°C for 30 s, 52°C for 30 s, and 72°C for 1 min, and finally 72°C for 5 min using a GeneAmp PCR System 9600 (Perkin-Elmer, Norwalk, Conn.).

For the HPA, two oligonucleotide probes were designed and named PARV3313(+) (5′-CTG CCA CAA TGC CAG TGG AAA GGA GC-3′) and PARV3348 (+) (5′-GCA CCA TTA GTC CAA TAA TGG GAT AC-3′). Both probes were labeled with acridinium ester (AE) as described by Arnold et al. (3). Ten microliters of amplified PCR products was mixed with 90 μl of TE buffer (10 mM Tris hydrochloride, 1 mM EDTA [pH 8.0]) and denatured by heating at 95°C for 5 min. The sample was then chilled on ice for 5 min. Fifty microliters each of AE-labeled probe and 100 μl of hybridization buffer were added, and the sample was incubated at 60°C for 15 min. To achieve hydrolysis of unhybridized probes, 300 μl of hydrolysis buffer was added and the mixture was incubated at 60°C for 10 min. After 5 min at room temperature, chemiluminescence signals from AE on hybridized probes were measured using a Leader I Luminometer (Gen-Probe Inc., San Diego, Calif.) and expressed as relative light units.

Ninety microliters of 5% HSA solution was spiked with 10 μl of a diluted plasma sample which contained 1, 10, or 100 copies of B19 DNA. DNA was extracted and served as the template for PCR. As shown in Fig. 1, the plasma sample containing a few viral copies exhibited high numbers of relative light units after 40 cycles of amplification and HPA. With 25 cycles of amplification and HPA, results were rather unstable. We therefore adopted 40 cycles of amplification for detection.

FIG. 1.

Detection of B19 DNA. B19 DNA was extracted from plasma samples which were previously determined to have B19 DNA contamination by quantitative and competitive PCR. PCR amplification was then performed for either 25 or 40 cycles. The horizontal axis indicates the B19 DNA concentration, and the vertical axis indicates chemiluminescence; the axes are log based. Symbols: □, specimen 1, 25 cycles; ▵, specimen 2, 25 cycles; ○, specimen 3, 25 cycles; ◊, negative control, 25 cycles; ■, specimen 1, 40 cycles; ▴, specimen 2, 40 cycles; ●, specimen 3, 40 cycles; ⧫, negative control, 40 cycles. RLUs, relative light units.

Neither human herpes simplex virus type 1, human cytomegalovirus (Sigma Chemical Co., St. Louis, Mo.), nor human hepatitis B virus (7) reacted with this system, which shows the specificity of the PCR-HPA method (data not shown).

To evaluate the variability of the method, spiking tests were performed using seven different samples of HSA solution and a plasma solution containing B19. The results showed that a single B19 DNA copy could be detected using this method in all of the HSA solutions tested (Table 1).

TABLE 1.

Recovery of B19 in 5% HSA solution

HSA lot no.	Virus concn (no. of copies/10 μl)
	100 copies	10 copies	1 copies	No DNA
1	172,064	158,338	21,934	816
2	188,676	161,383	29,680	870
3	191,064	161,383	23,910	879
4	172,053	140,827	14,455	854
5	181,441	142,992	30,929	807
6	187,419	148,016	27,424	707
7	174,069	160,466	19,347	718
Mean ± SD	180,969 ± 826.7	152,551 ± 8,507.9	23,954 ± 5,902.2	802 ± 69.9

When the viral DNA was spiked into a 25% HSA solution, efficiency of DNA extraction was variable because of the high viscosity of the solution and assay results were not reproducible. When a 5% HSA solution was used, this variability was not observed (data not shown).

In this study, the primers were designed within the conserved VP1-VP2 region of the B19 genes based on the DNA sequence in the GenBank database (3, 4, 5, 6, 17, 18). The HPA probes were also placed in the well-conserved region among various B19 genes for maximum detection. The sensitivities of detection were quite similar for three plasma samples and one DNA clone. We expect that we could detect almost any B19 DNA by this method.

With this detection method, we could detect a few copies of B19 DNA in contaminated blood products in only 6 h. In comparison, Southern blotting analysis using a radioactive probe (8) or a chemiluminescent probe requires about 30 h to perform (11, 22). Recently, the Red Cross Blood Center of Japan introduced a receptor-mediated hemagglutination test for large-scale screening of infectious B19 virus in blood donation samples. The method takes only 3 h to perform, but its sensitivity is as low as 10⁵ copies/ml (16, 19).

In conclusion, a new method for detecting B19 DNA has been established using PCR primer pairs and two newly synthesized probes for HPA. This method is specific and sensitive enough to detect a single copy of B19 DNA per assay in a 10-μl sample.