Real-time quantitative polymerase chain reaction analysis combined with reverse transcription (RT-qPCR) has been widely used to measure the level of RNA virus infections. Here we present a direct RT-qPCR assay, which does not require an RNA purification step, developed for the quantification of several RNA viruses, including dengue virus.
At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.
The genus Flavivirus of the Flaviviridae family comprises more than 70 enveloped, positive-stranded RNA viruses that are transmitted by mosquitoes and ticks. Importantly, flavivirus infection in humans often leads to severe clinical illness, such as hemorrhagic fever and encephalitis1. Indeed, a recent outbreak of the Zika virus (ZIKV), a flavivirus, has spread explosively throughout the Americas and has been shown to be associated with neurological complications, including microcephaly2,3. Flaviviruses, therefore, have significant clinical and economic impact on modern society.
An important flavivirus is dengue virus (DENV), a mosquito-borne virus widely distributed in the tropical and subtropical areas of the world. DENV is transmitted by Aedes mosquitos, including Aedes albopictus and Aedes aegypti, resulting in the global spread of dengue outbreaks4. Currently, the World Health Organization (WHO) classifies dengue disease as three categories: dengue without warning signs, dengue with warning signs, and severe dengue4. Although primary infection with one of the four serotypes of DENV (DENV-1 to -4) is often asymptomatic or self-limiting, epidemiologic studies have shown that the secondary infection of different serotypes increases the risk of more serious forms of dengue. It is worth noting that antibody-dependent enhancement of DENV infection caused by the non- or subneutralizing antibodies produced during the primary infection has been proposed as a potential mechanism of severe dengue which occurred as the secondary infection. However, no specific antiviral drug is available for DENV infection5.
For the development of antiviral agents against DENV, routine and robust assays, which are amenable to a high-throughput setting, are essential for detecting virus replication quantitatively. Conventionally, biological assays (e.g., plaque assay) for quantifying infectious viruses and immunological assays (e.g., enzyme-linked immunosorbent assay [ELISA]) for detecting viral antigens have been used to monitor DENV replication in vitro and in vivo6,7. However, these assays are often laborious and require several days to accomplish, which impedes the handling of large numbers of samples. In this regard, RT-qPCR is a reliable assay for detecting DENV infection: it is specific, sensitive, and relatively speedy7. In addition, the RT-qPCR technique has more advantages in a high-throughput format. Nevertheless, the typical procedure of RT-PCR for RNA viruses demands the recovery of viral nucleic acids from collected samples. Although various extraction methods can be employed for RT-qPCR, multiple steps are still needed to obtain purified viral RNA. Also, RT-qPCR assays usually require expensive spin columns or hazardous organic solvents, thereby making the preparation process more cumbersome. Since avoiding mishandling and/or cross-contamination between samples is a critical factor for the successful quantification of viral genomes, a less laborious and time-consuming method is preferred, particularly if the RT-qPCR is to be applied to high-throughput format.
Here, we report a simple RT-qPCR procedure for measuring DENV RNA in a culture supernatant of infected cells that does not involve viral RNA purification. This optimized RT-qPCR protocol is composed of two steps: i) the incubation of a supernatant of DENV-infected cell culture with a processing buffer, and ii) one-step RT-qPCR on a real-time PCR instrument. Accordingly, DENV RNA in a culture supernatant can be detected within 90 min (Figure 1). We also describe the application of the direct RT-qPCR to other RNA virus infections.
Today, viral nucleic acid detection by fluorescent-based real-time PCR is becoming a gold standard for the molecular diagnosis of pathogenic human viruses, due to its sensitivity and rapidity7. This is particularly important for confirming virus infection in the early phase of acute infectious diseases such as dengue fever17. Also, in the field of basic DNEV research, RT-qPCR assay is an indispensable tool for monitoring virus replication in in vitro culture, which will lead to an understanding of the replication biology of DENV and the discovery of antiviral inhibitors. In this study, the fluorescent-based real-time PCR assay was further developed by a combination of a processing buffer and a one-step RT-PCR reagent so that DENV RNA in the culture supernatant of infected cells was able to be quantified by RT-qPCR without purifying the viral RNA genome. When compared to routine assays to detect virus replication, such as plaque assay, ELISA, or conventional RT-qPCR employing RNA purification6,7, a simplified protocol of the RT-qPCR assay resulted in a great reduction of the time and steps necessary to quantify DENV RNA. This is also an important feature that has advantages in minimizing the contamination and loss of genetic materials. Nevertheless, it should be noted that the use of a clean hood is essential in the set-up of the IVT (step 2.1) and RT-qPCR (steps 4.1–4.4) reactions to avoid the cross-contamination of any amplicon-related products or RNase. It is also crucial to handle the culture supernatant, including infectious virus, inside a biosafety cabinet (step 3.3) to reduce a risk of laboratory infection.
Another significance of the direct RT-qPCR assay is that a wide range of viral RNA copies can be analyzed at the same time without dilution or concentration of the sample. When a serially diluted DENV 3'UTR RNA standard was used, the direct RT-qPCR protocol produced a linear standard curve over seven orders of magnitude, and the lower quantification limit was 500 RNA copies (Figure 2). For the detection of DENV in the culture supernatant of infected cells, 8 x 103 to 8 x 10-2 PFU viruses in a 10 μL RT-qPCR were within the range of DENV 3'UTR RNA standard, and the lower detection limit (8 x 10-2 PFU) was calculated to contain 11,400 ± 7,077 RNA copies (Figure 3B). Of note, as reported in several flavivirus studies18,19,20, the larger ratio of viral RNA copies to virus infectious titer (i.e., PFU) in DENV samples was also observed in this study. This overestimation is assumed to be largely due to the presence of defective (i.e., non-infectious) viruses or free viral RNA released from infected and dead cells. Although the ratio of RNA copies:PFU obtained in this study (1.1 x 105 to 1.3 x 105 RNA copies/PFU) was inconsistent with the data shown previously (ratios range from 1 to 3 log)21,20, this may be attributed to the difference in virus strains, cells, or culture conditions employed. Another likely explanation for the discrepancy is that, since no RNA purification process was involved in the direct RT-qPCR assay described herein, more DENV RNA in the culture supernatant could be detected by real-time PCR analysis without the loss of viral genetic materials.
The simplicity of the direct RT-qPCR enhances the ability to handle a large number of samples in multi-well formats, such as a 96-well plate. Therefore, this property will assist the future application of the direct RT-qPCR assay to a high-throughput screening study for the discovery of anti-DENV drugs. Indeed, in this report, application of the direct RT-qPCR assay for the validation of an anti-DENV inhibitor, MPA, was examined, and the result showed that an IC50 value of MPA obtained by the direct RT-qPCR assay (Figure 4) was comparable to that reported in previous studies using plaque assay12,13. This demonstrates that direct RT-qPCR is a useful assay for appropriately evaluating the inhibitory effect of antiviral agents and can be adopted into a drug screening study in future.
In this study, attempts were made to apply the direct RT-qPCR to the detection of other RNA viruses. As shown in Figure 5, when 10-fold dilutions of three other RNA viruses (YFV, CHIKV, and MeV) classified into different genera (Flaviviridae, Togaviridae, and Paramyxoviridae, respectively) were examined using previously reported primers and fluorogenic probes specific to the respective viral RNA sequences. Good correlations between infectious titers and Ct values were obtained by direct RT-qPCR assays, and the correlations were 4-6 linear orders of magnitude. This suggests that the direct RT-qPCR protocol is adaptable to various types of RNA viruses by simply changing the virus-specific primers and fluorogenic probes.
Nevertheless, the sensitivity of detection varied among the viruses tested in this study (Figure 5). One modification of the protocol that can improve the detection of target virus RNA should be to use a new set of primer/probe sequences. Additionally, a key step for the successful direct RT-qPCR assay is thought to be the efficient release of the viral genome during the sample processing step (steps 3.1–3.4). This would be of particular importance for the quantitative detection of stable viruses such as a non-enveloped virus. Although as a preliminary experiment, Coxsackievirus B3 (CVB3), a non-enveloped RNA virus belonging to Picornaviridae, was subjected to the direct RT-qPCR assay using previously described CVB3-specific primers and fluorescent probe22, a positive amplicon signal could not be detected even with a high-titer (1 x 105 PFU/mL) virus stock. This is considered to be largely attributed to the high physical integrity of the non-enveloped virus. So far, some RT-PCR assays that do not require the purification of nucleic acid have been developed to detect several RNA viruses, which employ different protocols in the RNA release step23,24,25,26. Thus, use of the alternative (or additional) treatments, such as an incubation of a virus sample at a high temperature, potentially increases the sensitivity of detection.
In summary, the direct RT-qPCR protocol developed in this study was a simple and rapid but reliable method for quantifying viral RNA in a culture supernatant of infected cells. Because of this simplicity, the direct RT-qPCR assay could process a number of samples in a short time, which holds promise for the high-throughput analysis of virus replication. Furthermore, the application of the direct RT-qPCR protocol to other RNA viruses was also demonstrated. This approach should, therefore, be a useful tool for efficiently monitoring the RNA virus infections in a research laboratory setting and, possibly, in clinical diagnoses.
The authors have nothing to disclose.
The authors thank Subhash G. Vasudevan (Duke-NUS Graduate Medical School, Singapore) for the DENV-2 isolate EDEN2 3295 and Shunro Imura (Kobe Quarantine Station, Japan) for the YFV 17D vaccine strain. The authors are also grateful to the members of the Department of Microbiology and Infection Control for their assistance. This work is supported by MEXT KAKENHI Grant Number JP16737900.
PrimeSTAR Max DNA Polymerase | Takara Bio. Inc | R045A | Comparable PCR reagent kit can be used. |
SimpliAmp Thermal Cycler | Thermo Fisher Scientific | A24811 | Comparable thermal cycler instrument, but PCR cycle condition needs to be optimized. |
6x Gel Loading Dye | New England BioLabs | B7024S | Comparable gel loading dye can be used. |
2-Log DNA Ladder | New England BioLabs | N3200 | 0.1-10.0 kilobase pairs. Comparable DNA molecular ladder can be used. |
Gel Scene Tablet | Astec | GST-33 | Comparable gel imaging system can be used. |
illustra GFX PCR Purification Kit | GE Healthcare Life Sciences | 28-9034-70 | Comparable gel extraction kit can be used. |
BioSpectrometer | Eppendorf | 6135000905 | Comparable spectrophotometer can be used. |
MEGAscrip T7 Transcription Kit | Thermo Fisher Scientific | AM1334 | |
TURBO DNase | Thermo Fisher Scientific | AM2238 | Included in MEGAscrip T7 Transcription Kit. |
Recombinant RNase Inhibitor | Takara Bio. Inc | 2313A | 40 units/μL |
RNeasy Mini Kit | Qiagen | 74104 | Comparable RNA purification kit can be used. |
CellAmp Direct RNA Prep Kit | Takara Bio. Inc | 3733Q | |
Proteinase K | Nacalai Tesque | 15679-06 | > 600 units/mL. Comparable reagent can be used. |
iTaq Universal Probes One-Step Kit | Bio-Rad | 1725141 | |
MicroAmp Fast Optical 96-Well Reaction Plate, 0.1 mL | Thermo Fisher Scientific | 4346907 | Comparable plate or tube can be used dependent on the real-time PCR instrument, but PCR cycle condition needs to be optimized. |
MicroAmp Optical Adhesive Film | Thermo Fisher Scientific | 4311971 | Comparable film or optical cap can be used dependent on the real-time PCR plate or tube. |
StepOnePlus Real-Time PCR System | Thermo Fisher Scientific | StepOnePlus-01 | Comparable real-time PCR instrument, but PCR cycle condition needs to be optimized. |