Development of an assay for the detection and quantification of the measles virus nucleoprotein (N) gene using real-time reverse transcriptase PCR

The measles virus (MeV), genus Morbillivirus, family Paramyxoviridae, causes acute and highly contagious measles infection in humans (Griffin, 2007). The case fatality rate in developing countries is generally in the range of 1–5 %, but may be as high as 25 % in populations with high levels of malnutrition and poor access to health care (). Therefore, the World Health Organization has focused on the infection as an eliminative disease (WHO & United Nations Children's Fund, 2001; WHO, 2006).

A genetic detection method for MeV, i.e. reverse transcriptase nested PCR (nested RT-PCR), is widely used (Morita et al., 2007). While this method may be very sensitive, it can take more than 8 h to detect MeV. Moreover, cross-contamination during the RT-PCR processes may cause significant problems (Llop et al., 2000; Drago et al., 2004). To improve these problems, recently a real-time reverse transcriptase PCR (real-time RT-PCR) method using specific primers and probes has been applied for the detection of various virus genomes (Mackay et al., 2002). This method is highly sensitive and specific, and quantitative.

In this study, we developed a sensitive and quantitative assay for the detection of the MeV nucleoprotein (N) gene in clinical specimens (throat swabs) and viral suspensions derived from MeV-infected cells (Vero/SLAM cells).

Clinical specimens. Throat swabs were obtained from 22 patients with measles. The samples were centrifuged at 3000 g for 30 min at 4 °C and the supernatants were used in this study (Morita et al., 2007). The specimens were stored at –80 °C until used. Detailed data concerning patients, copies of the MeV N gene in throat swabs and genotypes are shown in Table 1.

Table 1. Patient data, copies of the N gene of measles virus in throat swab, and genotype

Viruses, virus propagation and RNA extraction. Five MeV strains (genotype A, CAM-70; genotype D3, MVi/Okinawa.JPN/31.01[D3]; D5, MVi/Okinawa.JPN/03.03[D5]; D9, MVi/Yamagata.JPN/3.04[D9]; and H1, MVi/Okinawa.JPN/14.03[H1]) and the 22 throat swab specimens were propagated in SLAM (signalling lymphocyte activation molecule, CD150)-expressing Vero cells (Vero/SLAM cells, kindly donated by Dr Y. Yanagi, Faculty of Medicine, Kyushu University) with Opti-MEM (Invitrogen). Vero and Vero/SLAM cells were maintained as previously described (Ono et al., 2001). Each virus suspension titre was 10⁶–10⁷ TCID₅₀ (0.1 ml)^–1 (tissue culture infective dose 50 %). MeV RNA was extracted from 200 µl of the throat swab specimen or the viral suspension using a High Pure Viral RNA kit (Roche Diagnostics). The extracted RNA was then suspended in 50 µl DNase/RNase-free water with 1 U RNase inhibitor (Applied Biosystems) µl^–1.

Design of primers and probe. We aligned nucleotide sequences of the N gene from various genotypes of the reference MeV strains (genotype A, B1, B2, B3, C1, C2, D1–D10, E, F, G1, G2, G3, H1 and H2), as previously described (WHO, 2005; Morita et al., 2007). Based on these data, we designed new primers and a TaqMan probe using Primer Express (R) version 1.5 software (Applied Biosystems) (Thomas et al., 2007) (Table 2).

Table 2. Primer and probe sequences used for MeV real-time RT-PCR

Preparation of control plasmid. To prepare the control plasmids, the N gene (position 1368–1616, 249 bp) in the following genotypes was amplified by PCR using the newly designed primers: genotype A, CAM-70 (GenBank accession no. U03650); genotype D3, MVi/Okinawa.JPN/31.01[D3] (AB435245); D5, MVi/Okinawa.JPN/03.03[D5] (AB435246); D9, MVi/Yamagata.JPN/3.04[D9] (AB186905); and H1, MVi/Okinawa.JPN/14.03[H1] (AB435247). The products were cloned into a pCR2.1-TOPO vector (Invitrogen) and purified with a High Pure Plasmid Isolation kit (Roche Diagnostics), according to the manufacturer's instructions. The concentration of the plasmid was determined by measuring the A₂₆₀. The DNA sequence was confirmed by sequencing, using a Big Dye Terminator version 1.1 Cycle Sequencing kit (Applied Biosystems) (Morita et al., 2007).

Synthetic RNA of the N gene. Synthetic RNA of the MeV N gene (full-length, genotype A, position 108–1685, 1578 nucleotides) was prepared using the T7 MEGAscript kit (Applied Biosystems) as previously described (Hummel et al., 2006). Synthetic RNA was treated with DNase and purified. The size of the synthetic RNA was determined by ethidium bromide staining on denatured agarose gel. The concentration of the synthetic RNA of the N gene was determined by measuring the A₂₆₀.

Procedures of quantitative real-time RT-PCR. The reverse transcription mixture contained 10 µl template RNA from the throat swab specimens, 4 µl random hexamer (20 pmol), 4 µl 5x RT-PCR buffer, 1 µl PrimeScript RT Enzyme Mix I (Takara) containing 10 units RNase inhibitor µl^–1 (Applied Biosystems) and 1 µl DNase- and RNase-free distilled water. The samples were incubated for 15 min at 37 °C then for 5 s at 85 °C. PCR amplification was performed with a 7500 Fast Real-Time PCR System (Applied Biosystems) under the following conditions: initial uracil-N-glycosylase (UNG) amplicon degradation (50 °C for 2 min) and denaturation of UNG at 95 °C for 10 min to activate DNA polymerase, then 50 cycles of amplification with denaturation at 95 °C for 15 s, and annealing and extension at 58 °C for 1 min. Amplification data were collected and analysed with Sequence Detector software version 1.3 (Applied Biosystems). A 10-fold serial dilution of standard cDNA plasmids (10⁷–10¹ copies of genotype A, D3, D5, D9 or H1) was used for the quantification of copies as a standard curve assay.

Genotyping of MeV. We performed genotyping of MeV isolated from the clinical samples. N gene amplification and nucleotide sequencing were performed as previously described (Takeda et al., 1999; Morita et al., 2007). Nucleotide sequences of the partial N gene of the MeV were analysed phylogenetically using the CLUSTAL W program available on the DNA Data Bank of Japan (DDBJ) homepage () and TreeExplorer (Version 2.12) (). Evolutionary distances were estimated using Kimura's two-parameter method and phylogenic trees were constructed using the neighbour-joining method (Saitou & Nei, 1987). The reliability of the tree was estimated using 1000 bootstrap replications.

Specificity assay. To examine the specificity of the real-time RT-PCR assay, eight enterovirus samples (echovirus types 9 and 18; coxsackie viruses A16, B2 and B5; enterovirus 71; and poliovirus types 1 and 3) and 18 respiratory virus samples (five strains of respiratory syncytial virus, five of mumps virus, four of human metapneumovirus, two of influenza subtype A and two of influenza subtype B) were tested. The titres of these viruses ranged from 10³ to 10⁵ TCID₅₀ (0.1 ml)^–1.

Optimization of primer and probe concentrations in the present assay
It is suggested that primer and probe concentrations affect sensitivity and specificity of real-time RT-PCR (Hashimoto et al., 2007). Thus we optimized the primer and probe concentrations as previously described (Hummel et al., 2006). The C_t (threshold cycle) values derived from the amplification plots for the present primer (ranging from 500 to 1100 nM) and probe (ranging from 100 to 350 nM) are shown in Fig. 1(a) and Fig. 1(b), respectively. In the present assay, 900 nM of both forward and reverse primers and 250 nM of probe showed the lowest C_t values. From these data, quantitative real-time PCR was carried out in a 20 µl reaction volume using a TaqMan Universal PCR Master Mix (Applied Biosystems) containing 2 µl cDNA, 900 nM each primer (MV-F and MV-R) and 250 nM TaqMan MGB probe (MV-T).

(9K): Fig. 1. Optimization of primer and probe concentrations in the present assay. Here, 107 copies of synthetic RNA of the N gene (full-length, genotype A) were used. The x-axis indicates the concentrations of primer or probe and the y-axis indicates the Ct value. Results are expressed as mean±SD from three independent experiments. Detailed procedures are described in the text.

Linearity, sensitivity and specificity of the real-time RT-PCR
To assess the linearity of the quantitative real-time RT-PCR, we prepared 10-fold serial dilutions (10⁷–10¹ copies) of cDNA plasmids (genotype A, D3, D5, D9 or H1), measured using newly designed primers and a probe. A representative standard curve and amplification plots using the standard plasmid (genotype A) are shown in Fig. 2(a, b). Good linearity was obtained from 10¹ to 10⁷ copies per reaction (R²=0.9996) using other genotype-cloned plasmids (genotype D3, D5, D9 or H1) (data not shown). Next, to address the issue of sensitivity of the present assay, we prepared 10-fold serial dilutions (10⁷–10¹ copies) of synthetic RNA of the MeV N gene (genotype A) and measured copy numbers as previously described (Fig. 3a) (Hummel et al., 2006). As a result, a good coefficient factor (R²=0.9987) was obtained from 10¹ to 10⁷ copies per reaction (corresponding to 5x10^–1–5x10⁵ copies µl^–1). The results suggest that the reliable measurement range of the present assay is 10¹–10⁷ copies per reaction. In addition, we prepared viral RNA (corresponding to 10¹–10⁵ copies per reaction) of some MeV genotypes (genotype A, D3, D5, D9 or H1) using a dilution series of the viral suspensions and quantified copy numbers of their N gene. Good linearities (from 10¹ to 10⁵ copies per reaction) as well as synthetic RNA were obtained for all genotypes (Fig. 3b). No other viruses such as enteroviruses, respiratory syncytial viruses, mumps viruses and influenza viruses (subtypes A and B) were detected with the present assay. The literature does contain some reports regarding the detection and quantification of MeV using sensitive real-time PCR methods (Hummel et al., 2006; Thomas et al., 2007). For example, Thomas et al. (2007) developed a quantification assay for the haemagglutinin (H) gene in MeV using a real-time PCR method and Hummel et al. (2006) developed a real-time RT-PCR method targeting multiple genes [nucleoprotein (N), fusion (F) and H genes]. In these methods, the sensitivity limits were two and ten copies per reaction, respectively (Hummel et al., 2006; Thomas et al., 2007). Thus these values were very similar to those of our method, even though the target gene and/or region of MeV were different. Taken together, the results suggest that our method is sensitive, specific and quantitative for the MeV N gene, and is applicable to the quantification of various MeV genotypes.

(14K): Fig. 2. Standard curve and amplification plots of quantification of the nucleoprotein (N) gene of MeV. A 10-fold serial dilution (107–101 copies per reaction) of cDNA plasmid (genotype A) was used. (a) The x-axis indicates the logarithmic concentration (log10) of the cDNA plasmid and the y-axis indicates the Ct value. (b) The x-axis indicates the Ct value and the y-axis indicates ΔRn (adjusted fluorescence intensity). Detailed procedures are described in the text.

(15K): Fig. 3. Linearity of the real-time RT-PCR assay. (a) A 10-fold serial dilution (107–101 copies per reaction) of synthetic RNA of the MeV N gene (full-length, genotype A) was used. (b) A 10-fold serial dilution (105–101 copies per reaction) of viral RNA of each genotype (genotype A, D3, D5, D9 or H1) was used. In Fig. 3(a, b), the x-axis indicates the logarithmic concentration (log10) of the synthetic or viral RNA and the y-axis indicates the Ct value. Results are expressed as mean±SD from three independent experiments. Detailed procedures are described in the text.

Isolation, genotype and quantification of MeV from throat swabs of patients with measles
MeV was isolated from all specimens and the genotype was confirmed as D5 by phylogenetic analysis (Table 1). In addition, throat swabs from patients with measles contained 3.9x10³–5.2x10⁶ copies ml^–1 of the N gene. No significant differences were found between intervals of onset of rash and sampling date and copies of MeV in the throat swab specimens. In these patients, the lowest number of copies of MeV from raw data was 15.4 copies per reaction (corresponding to 3.9x10³ copies ml^–1 in throat swab, multiplication factor 250), suggesting that this value represents a reliable zone of the standard curve in the present assay. In this study, we did not measure other MeV genotypes (D3, D9 or H1) in the throat swab specimens since we were unable to obtain them from patients with measles. Thus we quantified the A, D3, D5, D9 and H1 genotypes of the MeV N gene in viral suspensions derived from MeV-infected Vero/SLAM cells and some plasmid controls. As a result, each viral suspension contained 7.4x10⁷–2.0x10⁸ copies ml^–1. In Japan, over the past 10 years, domestic measles outbreaks have occurred in 1998, 2001, 2006 and 2007 (Kubo et al., 2002; Zhou et al., 2003; Nakajima et al., 2003; Morita et al., 2007). The predominant MeV genotype detected during these outbreaks was D5, although a small number of other genotypes including D3, D9 and H1 were also detected. Thus it may be important to confirm detection of these MeV genotypes in Japan. In the present study, in order to detect and quantify MeV genotypes and to prevent cross-reactions of the N gene in other paramyxoviruses such as mumps virus, respiratory syncytial virus and parainfluenza viruses, we targeted a specific region of the MeV N gene. However, the sequences of this region (the PCR target region) may be variable (Griffin, 2007). As such, it may be necessary to modify the primers/probe sequences in the future when mismatches are found to occur in them. Furthermore, we did not examine one-step real-time RT-PCR in this study, a method which may be advantageous when large numbers of samples are screened (Hummel et al., 2006). Thus in future work we will consider the need to introduce this technique in the present assay. The present results indicate that the newly developed assay may be applicable for the detection and quantification of MeV from clinical specimens (throat swabs) and viral suspensions. We thank Dr H. Yokoi and Ms T. Kitahashi for the helpful discussions. This work was partly supported by Research on Emerging and Re-emerging Infectious Diseases, Labour, and Welfare Programs from the Ministry of Health, Labour, and Welfare, Japan.

Footnotes

^,†, Hirokazu Kimura¹ †These authors contributed equally to this work.

The GenBank/EMBL/DDBJ accession numbers for the measles virus nucleoprotein gene sequences are AB447494–AB447515.