An improved RT-qPCR method for direct quantification of enveloped RNA viruses

Reverse transcription quantitative PCR (RT-qPCR) has emerged as the gold standard for virus detection and quantification, being utilized in numerous diagnostic and research applications. However, the direct detection of viruses has so far posed a challenge as the viral genome is often encapsidated by a proteinaceous layer surrounded by a lipid envelope. This necessitates an additional and undesired RNA extraction step prior to RT-qPCR amplification. To circumvent this limitation, we have developed a direct RT-qPCR method for the detection of RNA viruses. In our method, we provide a proof-of-concept using phage phi6, a safe-to-use proxy for pathogenic enveloped RNA viruses that is commonly utilized in e.g. aerosolization studies. First, the phage phi6 envelope is removed by 1% chloroform treatment and the virus is then directly quantified by RT-qPCR. To identify false negative results, firefly luciferase is included as a synthetic external control. Thanks to the duplex format, our direct RT-qPCR method reduces the reagents needed and provides an easy to implement and broadly applicable, fast, and cost-effective tool for the quantitative analysis of enveloped RNA viruses.• One-step direct RT-qPCR quantification of phage phi6 virus without prior RNA isolation.• Reduced reaction volume for sustainable and cost-effective analysis.


Description of protocol
The COVID-19 pandemic has called to attention the need for rapid yet sensitive and costeffective methods for direct detection and quantification of airborne viruses. The most commonly used methods rely on isolating and purifying the viral RNA genome followed by reverse transcription (RT) and quantitative polymerase chain reaction (qPCR). However, the applicability of these methods has been hampered by the high demand for viral RNA purification reagents. A handful of direct (i.e. RNA extraction-free) RT-qPCR methods have been recently developed for SARS-CoV-2 virus detection [ 1 , 2 ]. As SARS-CoV-2 is a single-stranded positive-sense RNA ( + ssRNA) enveloped virus [3] , these direct detection approaches rely on removing the lipid envelope, either by heat-inactivation or by use of detergents, such as Igepal [ 1 , 2 ], prior to amplification.
Since there are inherent risks in working with pathogenic viruses, bacteriophages are often utilized as safe-to-use alternative models [4][5][6] . Due to its structural resemblance to numerous pathogenic viruses, phage phi6 has become a popular analog for airborne transmission studies. Phage phi6 has a tripartite double-stranded (ds)RNA genome enclosed within a capsid ( Fig. 1 A). Stripping the lipid envelope weakens the capsid structure. During the RT step, the weakened capsid is prone to degrade due to the increased internal pressure caused by the elevated reaction temperature, thus exposing the genomic RNA and enabling direct RT-qPCR detection. Similarly, synthetically produced capsids (viruslike particles) of SARS-CoV-2 have been shown to be unstable and readily degrade as the temperature exceeds 34 °C [7] . The lipid envelope is frequently removed using various organic solvents [5] , which often inhibit the enzymatic activity in downstream steps. Consequently, we tested the inhibitory effects of organic solvents (0.01% and 1% 1-bromo-3-chloropropane, 0.01% and 1% chloroform) and a detergent (0.005% and 0.5% sodium deoxycholate) using end-point RT-PCR. The most efficient amplification and lowest inhibitory effect was observed when using 1% chloroform treatment, which was chosen as the standard treatment condition ( Fig. 1 B, Supplementary Fig. 1). It is worth noting that this approach is equally applicable for analyzing other enveloped viruses, although the inhibitory effects and efficacy of lipid envelope removal need to be tested separately for each virus. Importantly, quantification of phage phi6 is facilitated by the tightly regulated genome packaging mechanism, whereby the mature viral particle contains only one copy of each genome segment [8] . Therefore, the detection of any of the viral genes directly reflects the number of viral particles present in the sample. Moreover, we included a synthetic control template (Firefly Luciferase) to detect possible inhibitory effects of com pounds present in the sam ple. In conclusion, this method provides a comprehensive protocol for the absolute quantification of phage phi6 and other enveloped RNA viruses by direct RT-qPCR using hydrolysis probes, as well as a protocol to enzymatically synthesize all necessary RNA standards.
Required reagents and equipment (round points). The 10 0 dilution was not used for construction of calibration curve due to a non-linear response. The method was validated by quantifying three dilutions of 1x purified phage phi6 (rectangles). n = 3, primer effciency = eff%.

Procedure Preparation of RNA standard and firefly luciferase control RNA
The single-stranded RNA standards used for generating the calibration curve can be prepared by in vitro transcription (IVT) using T7 polymerase. The template plasmids can be easily propagated in Escherichia coli and linearized with a single restriction enzyme ( Eco RI). For RNA preparation, all buffers, reagents, and disposable material need to be RNase-free grade and good laboratory practices for work with RNA should be followed.
Template preparation for in vitro RNA synthesis 1. Propagate template vectors and isolate the plasmid. For plasmid isolation, we use E.Z.N.A.® Plasmid DNA Mini Kit I (Omega Bio-tek), but any other plasmid purification kit can be used. 2. Linearize the plasmid by Eco RI cleavage. An example for the reaction set-up is given in Table 3 .
Carry out the cleavage reaction for 1 h at 37 °C. 3. Separate the linearized plasmid on a 0.8% agarose gel in 1xTAE buffer, extract the plasmid from gel and purify it using a gel extraction kit (recommended e.g. E.Z.N.A.® Gel Extraction Kit, Omega Bio-tek). Alternatively, the linearized plasmid can be purified directly, without  The typical yield from this reaction is between 40 and 70 μg of purified RNA.

Virus sample preparation before RT-qPCR
Note: Samples containing virus should be stored at −80 °C to preserve the RNA quality and obtain unbiased data. Place the samples on ice and proceed with quantification by RT-qPCR.

Quantification of virus by RT-qPCR
The RT-qPCR enzyme mix used in this assay is designed for use with hybridization probes such as TaqMan®. The enzyme mix utilizes ROX passive reference and therefore, the instrument compatibility needs to be checked prior to designing the assay. The RT reaction can be carried out in a wide range of temperatures (42 °C to 60 °C), however 50 °C proved to be the most optimal (data not shown).
The assay design includes the Firefly Luciferase (FFluc) internal control template to detect possible inhibitory contaminants from the sample. The primer and probe designs were done using IDT PrimerQuest TM and primers and probes were checked for cross-reactivity with the IDT OligoAnalyzer TM tool. The primers and probes for the duplex reaction were designed according to the recommendations for multiplex qPCR design with minimal cross-reactivity between primers and probes ( Fig. 1 C). The selection of reporter dyes was limited by the availability of filters on the qPCR system. Consequently, we selected the reporter dyes with the smallest overlap in their emission spectra.
Furthermore, a wide range of primer/probe concentrations were tested in the RT-qPCR set-up to find the most optimal efficiency of the reaction. The final reaction mix constitutes the most efficient amplification (primer efficiency ∼95%) with the widest detection range (10 1 -10 9 genome copies) ( Fig. 1 D).
1. Prepare the reaction mastermix as described in Table 5 . 2. Place the qPCR plate on ice and pipette 5 μL of the reaction mastermix into each well used of the qPCR plate. 3. Add 5 μL of template/well and seal the plate. As a negative control, add 5 μL of double-distilled H 2 O (ddH 2 O) instead of the template. All samples should be analyzed in technical triplicates.
Note: At the same time, construct the calibration curve using the P2 RNA standard (see section below).
4. Centrifuge the plate for 1 min at 10 0 0 g to collect the reaction mix to the bottom of the wells. 5. Run the RT-qPCR reaction in a cycler using the program described in Table 6 . Set the correct filters, the reaction volume to 10 μL, and the lid temperature to 105 °C. Table 6 qRT-PCR cycling program.  Table 7 Dilution series for generating the calibration curve.
To validate our method, we quantified three different concentrations of 1x purified virus diluted in Tris-HCl, pH 7.2 buffer ( Fig. 1 D, rectangles) using viral infectivity (PFU/reaction) as a measure for the virus amount. The analyzed samples contained 10 1 , 10 3 and 10 5 PFU/reaction. As expected, the amount of genome copies detected was approx. 10-fold higher than the infective counts. This difference can be attributed to the presence of non-infective virus particles. Despite the tightly regulated packaging mechanism of phage phi6, which ensures the addition of one copy of each genome fragment into the viral capsid [7] , infectivity of the particle is also dependent on additional virus and host factors. Indeed, loss of infectivity of the mature virion may be ascribed to incomplete outer shell assembly, loss of the viral envelope, or loss of the spike protein, as well as to host factors, such as a reduced pili expression (entry path for phage phi6) by the bacterium [9] .
Establishing the calibration curves 1. Make a stock solution of the P2 RNA standard containing 10 10 molecules/μL. For a calculation guide, please refer to the Supplementary Information. 2. Prepare a dilution series containing P2 RNA standard at concentrations from 10 0 to 10 9 molecules per 5 μL (as described in Table 7 ). Vortex thoroughly each dilution prior to making the next one. 3. Next, to ascertain the possible inhibitory effect of chloroform on the activity of the enzymes in the RT-qPCR mix, add chloroform to the dilutions at an amount equivalent to that used for the samples. 4. Vortex all dilutions and centrifuge the tubes at 10 0 0 0 g for 1 min. 5. Store the tubes on ice until assembling the reaction. 6. Prepare the RT-qPCR reactions as described above (section Quantification of virus by RT-qPCR, Table 5 ) and use the dilution series as a template. Each standard dilution should be analyzed in technical triplicates. 7. Create the calibration curve. A representative calibration curve is show in Fig. 1 D, and a template for generating the calibration curve is provided in the Supplementary Information.

Data analysis
The data analysis was performed using the analysis module of QuantStudio TM Design and Analysis software, v1.5.1 (Thermo Fisher Scientific) using the standard curve generated as described above. Alternatively, the analysis can be performed using the formula below, where Cq is the quantitation cycle and Yintercept as well as the slope are derived from the calibration curve ( Fig. 1 D). An example of such a calculation is provided in the Supplementary Information.

Quant it y per wel l =
Cq − Y intercept sl ope

Conclusions
Here we describe a simple RNA-extraction free method for direct RT-qPCR quantification of phage phi6. Additionally, we provide a detailed protocol for in-house synthesis of RNA standards, making the method cost-effective and easy to implement. Similarly to other direct RT-qPCR methods, the limitations of this method are primarily dictated by the sample volume [2] . For example, some aerosol collection experiments may generate very large sample volumes, which introduces a dilution factor that could prevent quantification due to the limited amount of viral RNA genome copies that are transferred to the RT-qPCR reaction. To circumvent this limitation and ensure a reliable and quantitative outcome, the amount of chloroform added to the virus containing sample is kept to a minimum, whereas the maximal possible volume of the final processed sample is added to the RT-qPCR reaction.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.