Norovirus (NoV) and sapovirus (SaV) of family Caliciviridae are major causative agents of acute gastroenteritis. This condition has become a global public health concern, with an estimated 1 billion cases of diarrhea occurring each year . It is also a major contributor to childhood morbidity and mortality, being responsible for approximately 3 million deaths worldwide in children <5 years old . Although many types of viruses, including NoV, SaV, rotavirus and adenovirus cause viral gastroenteritis, a recent study suggested that NoV and SaV are the most prevalent pathogens in gastroenteritis patients .
No reliable culture methods are currently available for detection of NoV and SaV, despite recent research efforts [4-6]. Among the detection methods for NoV and SaV reported thus far, real-time or conventional RT-PCR appear to be used most frequently for clinical samples [7, 8]. Although both types of PCR have good sensitivity and specificity, they are capable of detecting only one species at a time, making it difficult and expensive to run multiple samples rapidly. Multiplex PCR, on the other hand, can simultaneously detect two or more viruses in a single reaction, while also minimizing working time and reducing the risk of contamination compared with multi-step procedures. Moreover, multiplex PCR is advantageous for handling large numbers of clinical specimens. Recently, multiplex real-time RT-PCR has been used for detection of NoV (GI and GII) in clinical samples; however, a highly sensitive, quantitative method for the simultaneous detection of NoV and SaV remains to be established .
To address this need, we have established a quantitative and highly sensitive triplex real-time PCR method for the simultaneous detection of NoVGI, NoVGII and SaV. Stool specimens obtained from five pediatric patients who were subsequently confirmed to have NoVGI, NoVGII and SaV were stored at −80°C until use. The samples were centrifuged at 3000 g for 30 min at 4°C and the supernatants used as previously described . Viral nucleic acid was extracted using a QIAamp Viral RNA Mini kit (Qiagen, Valencia, CA, USA). The RT reaction mixture was incubated with random hexamers at 37°C for 15 min, followed by incubation at 85°C for 5 s using a PrimeScript RT reagent kit (Takara Bio, Otsu, Japan). Monoplexreal-time PCR amplification was performed as previously described . Triplex real-time PCR amplification was performed with a LightCycler 2.0 (DX400) (Roche Diagnostics, Mannheim, Germany) under the following conditions: 95°C for 5 min to activate DNA polymerase, 45 cycles of amplification with denaturation at 95°C for 45 s, and annealing and extension at 60°C for 45 s using a QuantiFast Multiplex PCR Kit (Qiagen). Amplification data were collected and analyzed with LightCycler version 4.1 (Roche Diagnostics). A 10-fold serial dilution of standard cDNA plasmids was used as a standard curve assay for quantification of copies. The PCR procedures for the amplification of various viral genes, including those of NoVGI, NoVGII and SaV, were performed as described previously [7, 11]. The primers and probes for real-time PCR are shown in Table 1.
Table 1. Primers and probe for PCR used in this study
|NoV GI||COG1F||CGY TGG ATG CGN TTY CAT GA|||
| ||COG1R||CTT AGA CGC CAT CATCAT TYA C|| |
| ||RING1-TP(a)||HEX-AGA TYG CGA TCY CCT GTC CA-BHQ|| |
| ||RING1-TP(b)||HEX-AGA TCG CGG TCT CCT GTC CA-BHQ|| |
|NoV GII||COG2F||CAR GAR BCN ATG TTY AGR TGG ATG AG|||
| ||COG2R||TCG ACG CCA TCT TCA TTC ACA|| |
| ||ALPF||TTT GAG TCC ATG TAC AAG TGG ATG CG|| |
| ||RING2AL-TP||Texas Red-TGG GAG GGS GAT CGC RAT CT-BHQ|| |
|SaV||SaV124F||GAY CAS GCT CTC GCY ACC TAC|||
| ||SaV1F||TTG GCC CTC GCC ACC TAC|| |
| ||SaV5F||TTT GAA CAA GCT GTG GCA TGC TAC|| |
| ||SaV1245R||CCC TCC ATY TCA AAC ACT A|| |
| ||SaV124TP||FAM-CCR CCT ATR AAC CA-MGB-NQF|| |
| ||SaV5TP||FAM–TGC CAC CAA TGT ACC A-MGB-NQF|| |
To prepare the control plasmids, the capsid gene (position 5291–5375, 85 bp for NoVGI; 5003–5100, 98 bp for NoVGII; and 5078–5181, 104 bp for SaV) for each genotype was amplified by PCR using the appropriate primers [7, 11]. The products were cloned into a pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA, USA) and purified with a High Pure Plasmid Isolation kit (Roche Diagnostics) according to the manufacturer's instructions. The concentrations of the plasmids were determined by measuring absorbance at 260 nm. The DNA sequences were confirmed by sequencing using the Big Dye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) . Data are expressed as mean ± SD. Statistical analysis was performed using Dunnett's test in SPSS version 12.0 (SPSS, Chicago, IL, USA). Values of P < 0.05 were considered significant.
First, to optimize the primer and probe concentrations and the sensitivity and specificity of the real-time RT-PCR program, the Ct values derived from the amplification plots for the primers (300–700 nM), probes (100–300 nM), and program parameters (denaturing, 30–45 s; PCR, 45 s) were assessed. The optimized triplex real-time PCR conditions derived from these results are shown in Table 2. In addition, to investigate the linearity and sensitivity of the present assay, 10-fold serial dilutions (107–101 copies) of NoVGI, NoVGII and SaV plasmids were prepared. A representative standard curve of the standard plasmid is shown in Figure 1a–c. Good linearity was obtained for 102–107 copies/reaction, and there was almost no difference in Ct value between monoplex and triplex samples. A good coefficient of multiple determination (R2 = 0.990) was obtained with 102–107 copies/reaction. The monoplex real-time PCR method used in this study is well established and reliable [7, 11]. Thus, these results suggest that the reliable measurement range of the present assay is 102 copies/reaction.
Table 2. Components and conditions of multiplex PCR assay
|Reagent||Volume (µL)|| || |
|2 × QuantiFast Multiplex PCR Master Mix||10|| || |
|20 × primer/probe mix†||1|| || |
|H2O||4|| || |
|Template cDNA||5|| || |
| ||95°C||5 min|| |
|PCR conditions||95°C||45 s||]45 cycle|
| ||60°C||45 s|
Figure 1. Performance of monoplex and triplex real-time PCR against a positive control plasmid. Plots of cycle threshold (Ct) versus serial 10-fold dilutions of the control plasmid. Performance of monoplex and triplex real-time PCR for (a) NoVGI, (b) NoVGII and (c) SaV.
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Next, monoplex real-time PCR and triplex real-time assays were compared by using stool samples containing NoVGI alone, NoVGII alone, SaV alone, or all three viruses (NoVGI + NoVGII + SaV). As shown in Figure 2, detection of NoVGI, NoVGII and SaV alone was comparable to that obtained with monoplex real-time PCR. In addition, each virus was detectable in the mixed sample (Fig. 2). No significant differences in quantitative values between the two methods were noted.
Figure 2. Comparison of viral loads determined by monoplex and triplex real-time PCR. Five stool samples (NoVGI alone, NoVGII alone, SaV alone and mixed sample (NoVGI + NoVGII + SaV)) were assessed.
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The present triplex real-time PCR assay was developed to minimize working time and the costs associated with simultaneously identifying and quantifying NoVGI, NoVGII and SaV, with the overall aim of improving detection of the causative agents of viral gastroenteritis infections. The present method reduced both the time and cost by half. Especially during winter outbreaks, such detection requires a rapid assay with high sensitivity and specificity. The present method should prove useful for confirming the causative agents at reduced cost. As NoV and SaV exhibit wide genetic diversity, the present primers and probes for a highly conserved genome region were used [7, 11]. However, because the choice of primer and probe sequences affects the accuracy of multiplex real-time PCR, and although our triplex real-time PCR method is fast and highly sensitive, further refinement of the primer and probe sequences might be warranted. In addition, one-step real-time PCR was not examined in this study. This method may be advantageous when screening large numbers of samples , and its application to the present method should be examined in the future.