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Development of an efficient qRT-PCR assay for quality control and cellular quantification of respiratory samples Cécile Resa a,b,c,∗ , Stéphane Magro c , Patricia Marechal c , Côme Barranger c , Martine Joannes c , Fabien Miszczak a,b,d , Astrid Vabret a,b,d a
Normandie University, France UNICAEN, EA4655 – U2RM, F-14032 Caen, France c bioMérieux, Parc Technologique Delta Sud, 09340 Verniolle, France d Department of Virology, University Hospital, F-14033 Caen, France b
a r t i c l e
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Article history: Received 5 September 2013 Received in revised form 25 March 2014 Accepted 31 March 2014 Keywords: Respiratory samples Respiratory viral infections Normalization Cellular control Cellular quantification
a b s t r a c t Background: Sample quality is a fundamental parameter for the successful diagnosis of respiratory viruses. This parameter depends upon the concentration of epithelial cells. Respiratory samples are usually heterogeneous, which makes relative quantification of the viral load, against the quantity of cells, the most suitable measurement. The quantification of viral load in the field of respiratory viruses is a vital piece of information. Quantification is required from RNA or DNA viral genomes extracted. Objectives: To design (RT-)PCR assays for reference genes, which show stable expression during viral infection, to be used as cellular controls and cellular quantification tools. Study design: Assays were designed for two reference genes: hypoxanthine phosphoribosyltransferase 1 (HPRT1) and ubiquitin C (UBC). The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was used as a reference for this study. The transcriptional activity of the three genes was studied during infection with respiratory syncytial virus and adenovirus. The HPRT1 q(RT-)PCR assay was used on clinical samples. Results: All the analysis methods concluded that the three reference genes were stably expressed during viral infection. The HPRT1 q(RT-)PCR assay indicated that the majority of clinical samples (n = 301, 69%) had a cellular load of between 100 and 10,000 cells/PCR. The data showed that the concentration decreased as the age of patient increased. Conclusions: A new tool has been developed and commercialized for quality control and evaluation of cellular concentration in respiratory samples. © 2014 Elsevier B.V. All rights reserved.
1. Background Acute respiratory diseases are estimated to account for the majority of all acute morbidities in developed countries [1–3]. Although the polymerase chain reaction (PCR) is a rapid and highly sensitive tool for the detection of a large number of respiratory viruses, there are still samples in which the causative agent is not found [4–7]. The sensitivity and specificity of viral detection assays
Abbreviations: PCR, real-time polymerase chain reaction; RT-PCR, real-time reverse transcriptase-polymerase chain reaction; qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction; (RT-)PCR, simultaneous RT-PCR and PCR; RSVA, respiratory syncytial virus subgroup A; Ad5, adenovirus serotype 5; NAATs, Nucleic-Acid Amplification Tests; hpi, hour(s) post infection; BAL, bronchoalveolar lavages; CT , threshold cycle; I, infected; NI, non-infected. ∗ Corresponding author at: bioMérieux, Parc Technologique Delta Sud, 09340 Verniolle, France. Tel.: +33 (0)5 81 54 60 43; fax: +33 (0)5 61 69 61 01. E-mail address: [email protected] (C. Resa).
are largely influenced by the type and quality of the clinical samples. Furthermore, the detection of viral genomes and total RNA are predominantly intracellular, particularly for respiratory viruses that cause the formation of syncytia, i.e. the respiratory syncytial virus. Thus the specimen quality depends on the concentration of cells. For a good diagnosis, sufficient epithelial cells must be collected [8]. Detection of respiratory viruses can be performed on various sample types, which are dependent upon the symptoms. The nasopharyngeal wash is the most common sampling method used in children. For non-cooperative patients, or those in respiratory distress, nasal swabs provide an alternative [9]. Invasive specimens, such as bronchoalveolar lavages (BAL) or bronchial secretions, are recommended for lower respiratory tract infections but are more difficult to obtain [10,11]. The quantification of viral load can be used to improve our understanding of the pathophysiology of respiratory viruses, to predict the evolution of emergent respiratory viruses, as a
http://dx.doi.org/10.1016/j.jcv.2014.03.019 1386-6532/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Resa C, et al. Development of an efficient qRT-PCR assay for quality control and cellular quantification of respiratory samples. J Clin Virol (2014), http://dx.doi.org/10.1016/j.jcv.2014.03.019
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2 Table 1 Evaluated housekeeping genes. Acronyms
Gene
Function
Localization
Amplified fragment size (bp)
UBC HPRT1 GAPDH
Ubiquitin C Hypoxanthine phosphoribosyltransferase 1 Glyceraldehyde-3-phosphate dehydrogenase
Protein degradation Purine synthesis in salvage pathway Oxidoreductase in glycolysis and gluconeogenesis
12q24.3-1 Exon Xq26.1-1 Exon 12p13-1 Exon
163 108 226
prognostic factor for disease progression and to prevent nosocomial infections. Quantification is also useful for monitoring a patient after treatment, in particular the monitoring of the influenza virus in a patient undergoing anti-viral treatment [12]. In patients for whom many viruses are detected by quantitative real-time RTPCR (qRT-PCR), quantitative data may help to distinguish between the virus that is causing the acute respiratory disease and those that have no causal link with the actual clinical symptoms [13]. However, respiratory samples are heterogeneous because the cells are collected in a variable volume of viral buffer. Therefore, normalization of the viral load, against the cellular concentration, is required [14]. Reliable estimation of viral load and disease management also require sample quality control assessment. This can be achieved through cellular gene quantification especially when repeated specimens are collected from an infected individual [15]. Respiratory viruses include both RNA and DNA viruses. The extraction process, before the diagnosis by the Nucleic-Acid Amplification Tests (NAATs), is dependent on the type of genome under investigation and the available laboratory equipment. Quantification of viral nucleic acids can be performed with either qPCR or qRT-PCR. Numerous studies have shown that hypoxanthine phosphoribosyltransferase 1 (HPRT1), ubiquitin C (UBC) and glyceraldehyde-3phosphate dehydrogenase (GAPDH) have stable expression levels and can be used as reference genes for normalization studies [16–18]. However, during a viral infection the virus hijacks and manipulates the cellular machinery, which can affect the transcription levels of genes.
2. Objectives The objective was to develop a useful tool for the quality control of respiratory samples and the normalization of viral load. This was achieved by the development of a q(RT-)PCR assay that amplified a stably expressed reference gene.
3. Study design 3.1. Expression of the HPRT1, UBC and GAPDH genes in infected and non-infected cells The respiratory syncytial virus subgroup A (RSVA) (ATCC, ref. VR-26) and adenovirus serotype 5 (Ad5) (ATCC, ref.VR-5) strains, titrated at 2.4e5 ID50 /mL and 2.2e4 ID50 /mL, respectively, were cultivated on MRC-5 cells (human lung fibroblast cells). Culture medium for the infection step, composed of D-MEM supplemented with fetal calf serum, 1% penicillin/streptomycin, 2% l-glutamine (200 mM) and 1% of trypsin solution from porcine pancreas [150 g/ml]), was added to the cells after a one hour incubation of the virus at 37 ◦ C. The MRC-5 cells were cultivated at 3e5 cells/mL in two 24well-plates (one control and one infected plate), in a final volume of 600 L per well. Culture medium was aspirated and 100 L of viral supernatant from RSVA or Ad5 cultures(as described
previously) were placed on cells from the infected plate. Culture medium (600 L, as described previously) was placed on cells from the control plate. At zero hour post infection (hpi), the viral supernatant was removed and cells were collected after a PBS-wash and a trypsinization. At other time points, 500 L of culture medium were added after 30 min of incubation at 37 ◦ C with the viral supernatant, and cells were collected as described above. Nucleic acids extractions were performed on the MagNa Pure Compact system (Roche Diagnostics, Meylan, France). Total RNA was extracted with the RNA Isolation Kit I, using the “RNA cells” protocol. Total nucleic acids were extracted with the Nucleic Acid Isolation Kit I, using the “Total NA plasma 100 400” protocol. The (RT-)PCR assays for HPRT1 and UBC were designed within exons to amplify both mRNA and DNA. The GAPDH RT-PCR assay has been described previously [19] (Table 1). All TaqMan® probes were labeled with 5 6-Hexachlorofluorescein and 3 Eclipse® Dark Quencher (Eurogentec France SA, Angers, France). Amplification mixes were composed of a PCR buffer (1× final), 0.2 mM dNTPs, 0.1 M specific probe, 0.2 M sense primer, 0.4 M antisense primer, three units of HotStarTaq DNA Polymerase (QIAGEN, Courtaboeuf, France), 0.05 units Omniscript RT (QIAGEN) and molecular-grade water, in a total volume of 15.1 L per reaction. The (RT-)PCR reactions were performed, using 10 L of eluate from nucleic acid extraction mixed with 15 L of amplification mix, on the Rotor-gene 6000 (Corbett, Archamps, France) for RSVA and on the LightCycler480 (Roche) for Ad5. The amplification protocol consisted of 30 min of reverse transcription at 50 ◦ C, followed by 15 min of Taq activation at 95 ◦ C and 45 cycles of denaturation at 95 ◦ C for 10 s, annealing at 54 ◦ C for 30 s, with optical reading in the yellow channel, and an extension for 10 s at 72 ◦ C. Detection of the viral genomes was performed with commercialized kits, in accordance with the manufacturer’s instructions. The RSVA/B r-gene® primers/probes (ref. 71-007, bioMerieux, Marcy l’Etoile, France) were used for RSVA detection on the Rotorgene 6000 (Corbett). The Adenovirus r-gene® primers/probes (ref. 71-010, bioMerieux) were used for Ad5 detection on a Smart Cycler 2 (Cepheid, Maurens-Scopont, France). The GeNorm software determines the most stable reference genes from a set of tested genes in a given sample panel. It calculates the gene expression stability measure, M, for a reference gene as the average pairwise variation, V, for that gene against all other tested reference genes. Genes with the lowest M value have the most stable expression [16]. The default limit for M is 1.5 [20]. The CT value was calculated by subtracting the CT value of the GAPDH RT-PCR from the CT value of the HPRT1 or UBC RT-PCR. The CT value was calculated by subtracting the CT of the infected condition from the CT of the non-infected condition [19].
3.2. HPRT1 q(RT-)PCR on clinical specimens Four hundred and thirty-six nasal swabs were collected between September 17th, 2009, and January 4th, 2010, atthe
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Fig. 1. RNA transcription levels of GAPDH, HPRT1 and UBC reference genes in RSVA and Ad5 infection kinetics of MRC-5 cells. CT values of the RT-PCRs of GAPDH, HPRT1 and UBC in RSVA-infected cells (I) and non-infected cells (NI) (a) and in Ad5-infected cells (I) and non-infected cells (NI) (b) are shown. Each point shows the CT values obtained for each gene and each point of measure, 0, 24, 30, 48 and 72 h post infection in Ad5 kinetics and the mean of the three CT values obtained for each gene and each point of measure, 0, 24, 30, 48 and 72 h post infection in RSVA kinetics. The CT values of GAPDH RT-PCRs are presented in the top figure, of HPRT1 RT-PCRs in the central figures and of UBC RT-PCRs in the bottom figures. On each figure, CT values obtained by RSVA RT-PCRs or Ad5 PCR amplification in RSVA-infected cells and Ad5-infected cells at 0, 24, 30, 48 and 72 hpi are shown to follow the viral infection.
University hospital of Caen. They were stored at −80 ◦ C. The age distribution of the tested patients was: 25% less than two years old, 44% between three years old and 15 years old, 28% between 16 years old and 65 years old and 3% more than 66 years old. Total nucleic acids were extracted on the QIAsymphony® SP platform (QIAGEN), using the QIAsymphony Virus/bacteria mini kit with the Pathogen complex 200 protocol (QIAGEN). Sample volume was 300 L and elution volume was 85 L. For quantification, the HPRT1 PCR product was cloned into the pCR® 2.1-TOPO vector using the TOPO TA Cloning kit (Invitrogen, Cergy Pontoise, France). The concentration of the plasmid solution was estimated by spectrophotometry. Three quantification standards, composed of plasmid solutions that were diluted to 105 , 104 and 103 HPRT1 plasmid copies/PCR, were tested. As the HPRT1 gene is a single copy gene (http://www.ncbi.nlm.nih.gov/) and we estimate that there are in mean two copies in each cell, the three quantification standards were expressed as 50,000, 5000 and 500 cells/PCR. The amplification was performed on
Applied Biosystems® 7500 Real-Time PCR Systems (Life Technologies, Saint-Aubin, France). 4. Results 4.1. Expression of the HPRT1, UBC and GAPDH genes in infected and non-infected cells The kinetics of RSVA and Ad5 infections was assessed, in vitro, in MRC-5 cells, to follow the transcriptional activity of the reference genes HPRT1, UBC and GAPDH in infected and non-infected cells. The CT values that represented the RSVA or Ad5 genome detection were measured at 0, 24, 30, 48 and 72 hpi and are shown in Fig. 1. No virus was detected in non-infected cells. In infected cells, the CT values obtained for RSVA and Ad5 genome detection showed an exponential decrease (from 44.0 to 20.9 cycles for RSVA and from 31.3 to 14.9 for Ad5, at 0 and 72 hpi, respectively), which demonstrated increasing infection of the cells.
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Fig. 2. Ranking of the three candidate reference genes, UBC, HPRT1 and GAPDH, using the geNorm software. The software ranks the candidate reference genes by their stability parameter, M. Lower M values represent higher stability of expression.
The mean CT values for each gene, in infected and non-infected conditions, were very close for both infections, which indicated the absence of regulation of their transcriptional activity in the presence of these viruses. In the RSVA assay, the average CT values were 27.1 vs. 27.4, 22.6 vs. 22.5 and 20.1 vs. 20.1, for HPRT1, UBC and GAPDH, in infected and non-infected cells, respectively. For Ad5, the CT values, for HPRT1, UBC and GAPDH, in infected and non-infected cells were 28 vs. 28.5, 26.5 vs. 26.2 and 23.4 vs. 23.8, respectively. No significant difference was observed in obtained CT values between infected and non-infected cells for any of the three genes (ANOVA p-values for the three genes > 0.05), at any of the time points, for both RSVA and Ad5. The GeNorm software has also been used to analyze the stability of expression of the three genes. The M values are shown in Fig. 2. The GAPDH gene gave the lowest M value of the three genes for both viruses (0.634 in RSVA and 0.572 in Ad5). This indicated that GAPDH is the most stable of the three genes. The M values obtained for the UBC and HPRT1 genes were similar for RSVA. The UBC gene had a higher M value (0.719) than HPRT1 (0.612), for Ad5, which signified that HPRT1 is more stable than UBC in cells infected with this virus. The stability of expression of HPRT1 and UBC was analyzed by the CT method with GAPDH as a reference. The CT value reflects the changes in RNA transcription caused by the infection. A positive CT indicates down-regulation of gene transcription, whereas a negative CT indicates up-regulation. [19] The calculated CT values for the three reference genes are given in Fig. 3. The CT values for HPRT1 and UBC showed low levels of regulation of their expression for both viruses (maximum CT values
Fig. 4. Ranking of the 436 samples by cellular concentration and patient age. Cells from respiratory specimens were quantified after a total nucleic acid extraction and an HPRT1 q(RT-)PCR. Samples were quantified by three quantification standards and were ranked in accordance with the concentration ranges of cells/PCR and the age of the patient. Median values (Med) are shown.
obtained for HPRT1 of −(1.2) at 0 hpi with RSVA and −(1.1) at 30 hpi with Ad5, and for UBC of −(1.1) at 0 hpi with RSVA and 1.65 with Ad5 at 72 hpi). All the measured variations were close to the values that correspond to three times the inter-assay variability of these RT-PCRs (data not shown). 4.2. Study on clinical samples The data of the three genes indicated that they could all be developed and used as cellular controls. We selected the HPRT1 assay because the design was original, unlike the GAPDH design that was taken from a publication, and this gene showed more stable expression than UBC (see Section 4.1). Cellular concentrations of the clinical samples were quantified by HPRT1 q(RT-)PCR and are shown in Fig. 4. The cellular concentrations fell into the following categories: between 100 and 10,000 cells/PCR (69.11%, n = 301, median age = 9), between 10,000 and 50,000 cells/PCR (24.94%, n = 92, median age = 6), more than 50,000 cells/PCR (3.89%, n = 26, median age = 2) and less than 100 cells/PCR (2.06%, n = 17, median age = 30). 5. Discussion Comparisons of the stability of reference genes are frequently performed between tumor and non-tumor tissues of the same type, in order to identify new biomarkers [21–24]. The stability
Fig. 3. Normalized expression of HPRT1 and UBC genes in RSVA and Ad5 infected cells at each point of measure. The CT value is indicated for each gene (HPRT1 and UBC) at each point of measure, 0, 24, 30, 48 and 72 hpi. The CT was calculated from changes in GAPDH RNA transcription, induced by Ad5 (a) and RSVA (b) infection, and was normalized to RNA transcription changes in non-infected cells.
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of reference genes during viral infection in humans, animals or plant cells has also been studied [25–30]. Our study is the first to examine the expression of GAPDH, HPRT1 and UBC genes, during in vitro viral infection with RSVA and Ad5 viruses, in order to develop a cellular control. These are common respiratory viruses, with either an enveloped RNA genome (RSVA) or a non-enveloped DNA genome (Ad5). The GeNorm software showed that all three genes were stable with both viruses, as all M values were below the default limit [16]. The transcription of the genes in infected cells, in comparison with non-infected cells, was not affected, as shown by CT values and CT analysis. The software defined GAPDH as the most stable gene, which is concordant with published data [26,27]. The three (RT-)PCR assays could be used as cellular controls in potentially infected respiratory samples. Nevertheless, these statements may require confirmation by further studies with other pathogens. The HPRT1 q(RT-)PCR assay was tested on clinical samples that contained both DNA and mRNA, which could be amplified simultaneously by the assay. In an experiment on 20 clinical samples, which may or may not have been infected by various respiratory viruses, we obtained similar cellular concentrations by HPRT1 qPCR (without reverse transcriptase) and q(RT-)PCR (with reverse transcriptase) (data not shown). The HPRT1 q(RT-)PCR assay allowed us to make an accurate estimation of the cellular concentration. The study on 436 nasal swabs highlighted the heterogeneity of the cellular concentrations in this type of sample and also showed that the concentration decreased as the age of patient increased. Further studies are ongoing to confirm this data. Nevertheless, sampling in the elderly is known to be more difficult than in other age groups [31]. The choice of sampling tool and site can improve the quantity of cells collected and aid the viral diagnostic. Indeed, it has been shown that the viral load was 4.8 times higher in flocked swabs than in rayon swabs, and 19 times higher in the nasopharynx than in the oropharynx [32]. There is no definition of a “rich” or “poor” sample, in terms of cells. In our study, all 436 samples were Influenza A positive. The usefulness of the cellular controls has been demonstrated on upper respiratory samples [15]. However, further studies are required for lower respiratory samples. Indeed, BAL, tracheal aspirates or bronchial secretions are the samples of choice for the diagnosis of acute lower respiratory infections. Across different laboratories and samplers, samples are heterogeneous and there is no standardized consensus protocol. However, a published clinical study on BAL, for the normalization of HSV1 viral load, in which our assay was used, showed homogeneity of cellular loads [33]. In light of these findings, there is a growing interest in the accurate quantification of respiratory viruses [34,35]. The developed cell control assay allows accurate respiratory virus quantification and may contribute to our knowledge of the physiopathology of respiratory viruses in two ways: firstly, for follow-up after the administration of therapeutics; secondly, to analyze and prevent transmissibility and nosocomial infections. This tool has been commercialized in the Cell control r-gene® and Rhino&EV/CC r-gene® kits (bioMerieux). Funding None. Competing interests The authors RESA Cécile, MAGRO Stéphane, MARECHAL Patricia, BARRANGER Côme and JOANNES Martine are in an employment relationship with company Biomerieux SA.
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Development of an efficient qRT-PCR assay for quality control and cellular quantification of respiratory samples Cécile Resa a,b,c,∗ , Stéphane Magro c , Patricia Marechal c , Côme Barranger c , Martine Joannes c , Fabien Miszczak a,b,d , Astrid Vabret a,b,d a
Normandie University, France UNICAEN, EA4655 – U2RM, F-14032 Caen, France c bioMérieux, Parc Technologique Delta Sud, 09340 Verniolle, France d Department of Virology, University Hospital, F-14033 Caen, France b
a r t i c l e
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Article history: Received 5 September 2013 Received in revised form 25 March 2014 Accepted 31 March 2014 Keywords: Respiratory samples Respiratory viral infections Normalization Cellular control Cellular quantification
a b s t r a c t Background: Sample quality is a fundamental parameter for the successful diagnosis of respiratory viruses. This parameter depends upon the concentration of epithelial cells. Respiratory samples are usually heterogeneous, which makes relative quantification of the viral load, against the quantity of cells, the most suitable measurement. The quantification of viral load in the field of respiratory viruses is a vital piece of information. Quantification is required from RNA or DNA viral genomes extracted. Objectives: To design (RT-)PCR assays for reference genes, which show stable expression during viral infection, to be used as cellular controls and cellular quantification tools. Study design: Assays were designed for two reference genes: hypoxanthine phosphoribosyltransferase 1 (HPRT1) and ubiquitin C (UBC). The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was used as a reference for this study. The transcriptional activity of the three genes was studied during infection with respiratory syncytial virus and adenovirus. The HPRT1 q(RT-)PCR assay was used on clinical samples. Results: All the analysis methods concluded that the three reference genes were stably expressed during viral infection. The HPRT1 q(RT-)PCR assay indicated that the majority of clinical samples (n = 301, 69%) had a cellular load of between 100 and 10,000 cells/PCR. The data showed that the concentration decreased as the age of patient increased. Conclusions: A new tool has been developed and commercialized for quality control and evaluation of cellular concentration in respiratory samples. © 2014 Elsevier B.V. All rights reserved.
1. Background Acute respiratory diseases are estimated to account for the majority of all acute morbidities in developed countries [1–3]. Although the polymerase chain reaction (PCR) is a rapid and highly sensitive tool for the detection of a large number of respiratory viruses, there are still samples in which the causative agent is not found [4–7]. The sensitivity and specificity of viral detection assays
Abbreviations: PCR, real-time polymerase chain reaction; RT-PCR, real-time reverse transcriptase-polymerase chain reaction; qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction; (RT-)PCR, simultaneous RT-PCR and PCR; RSVA, respiratory syncytial virus subgroup A; Ad5, adenovirus serotype 5; NAATs, Nucleic-Acid Amplification Tests; hpi, hour(s) post infection; BAL, bronchoalveolar lavages; CT , threshold cycle; I, infected; NI, non-infected. ∗ Corresponding author at: bioMérieux, Parc Technologique Delta Sud, 09340 Verniolle, France. Tel.: +33 (0)5 81 54 60 43; fax: +33 (0)5 61 69 61 01. E-mail address: [email protected] (C. Resa).
are largely influenced by the type and quality of the clinical samples. Furthermore, the detection of viral genomes and total RNA are predominantly intracellular, particularly for respiratory viruses that cause the formation of syncytia, i.e. the respiratory syncytial virus. Thus the specimen quality depends on the concentration of cells. For a good diagnosis, sufficient epithelial cells must be collected [8]. Detection of respiratory viruses can be performed on various sample types, which are dependent upon the symptoms. The nasopharyngeal wash is the most common sampling method used in children. For non-cooperative patients, or those in respiratory distress, nasal swabs provide an alternative [9]. Invasive specimens, such as bronchoalveolar lavages (BAL) or bronchial secretions, are recommended for lower respiratory tract infections but are more difficult to obtain [10,11]. The quantification of viral load can be used to improve our understanding of the pathophysiology of respiratory viruses, to predict the evolution of emergent respiratory viruses, as a
http://dx.doi.org/10.1016/j.jcv.2014.03.019 1386-6532/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Resa C, et al. Development of an efficient qRT-PCR assay for quality control and cellular quantification of respiratory samples. J Clin Virol (2014), http://dx.doi.org/10.1016/j.jcv.2014.03.019
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2 Table 1 Evaluated housekeeping genes. Acronyms
Gene
Function
Localization
Amplified fragment size (bp)
UBC HPRT1 GAPDH
Ubiquitin C Hypoxanthine phosphoribosyltransferase 1 Glyceraldehyde-3-phosphate dehydrogenase
Protein degradation Purine synthesis in salvage pathway Oxidoreductase in glycolysis and gluconeogenesis
12q24.3-1 Exon Xq26.1-1 Exon 12p13-1 Exon
163 108 226
prognostic factor for disease progression and to prevent nosocomial infections. Quantification is also useful for monitoring a patient after treatment, in particular the monitoring of the influenza virus in a patient undergoing anti-viral treatment [12]. In patients for whom many viruses are detected by quantitative real-time RTPCR (qRT-PCR), quantitative data may help to distinguish between the virus that is causing the acute respiratory disease and those that have no causal link with the actual clinical symptoms [13]. However, respiratory samples are heterogeneous because the cells are collected in a variable volume of viral buffer. Therefore, normalization of the viral load, against the cellular concentration, is required [14]. Reliable estimation of viral load and disease management also require sample quality control assessment. This can be achieved through cellular gene quantification especially when repeated specimens are collected from an infected individual [15]. Respiratory viruses include both RNA and DNA viruses. The extraction process, before the diagnosis by the Nucleic-Acid Amplification Tests (NAATs), is dependent on the type of genome under investigation and the available laboratory equipment. Quantification of viral nucleic acids can be performed with either qPCR or qRT-PCR. Numerous studies have shown that hypoxanthine phosphoribosyltransferase 1 (HPRT1), ubiquitin C (UBC) and glyceraldehyde-3phosphate dehydrogenase (GAPDH) have stable expression levels and can be used as reference genes for normalization studies [16–18]. However, during a viral infection the virus hijacks and manipulates the cellular machinery, which can affect the transcription levels of genes.
2. Objectives The objective was to develop a useful tool for the quality control of respiratory samples and the normalization of viral load. This was achieved by the development of a q(RT-)PCR assay that amplified a stably expressed reference gene.
3. Study design 3.1. Expression of the HPRT1, UBC and GAPDH genes in infected and non-infected cells The respiratory syncytial virus subgroup A (RSVA) (ATCC, ref. VR-26) and adenovirus serotype 5 (Ad5) (ATCC, ref.VR-5) strains, titrated at 2.4e5 ID50 /mL and 2.2e4 ID50 /mL, respectively, were cultivated on MRC-5 cells (human lung fibroblast cells). Culture medium for the infection step, composed of D-MEM supplemented with fetal calf serum, 1% penicillin/streptomycin, 2% l-glutamine (200 mM) and 1% of trypsin solution from porcine pancreas [150 g/ml]), was added to the cells after a one hour incubation of the virus at 37 ◦ C. The MRC-5 cells were cultivated at 3e5 cells/mL in two 24well-plates (one control and one infected plate), in a final volume of 600 L per well. Culture medium was aspirated and 100 L of viral supernatant from RSVA or Ad5 cultures(as described
previously) were placed on cells from the infected plate. Culture medium (600 L, as described previously) was placed on cells from the control plate. At zero hour post infection (hpi), the viral supernatant was removed and cells were collected after a PBS-wash and a trypsinization. At other time points, 500 L of culture medium were added after 30 min of incubation at 37 ◦ C with the viral supernatant, and cells were collected as described above. Nucleic acids extractions were performed on the MagNa Pure Compact system (Roche Diagnostics, Meylan, France). Total RNA was extracted with the RNA Isolation Kit I, using the “RNA cells” protocol. Total nucleic acids were extracted with the Nucleic Acid Isolation Kit I, using the “Total NA plasma 100 400” protocol. The (RT-)PCR assays for HPRT1 and UBC were designed within exons to amplify both mRNA and DNA. The GAPDH RT-PCR assay has been described previously [19] (Table 1). All TaqMan® probes were labeled with 5 6-Hexachlorofluorescein and 3 Eclipse® Dark Quencher (Eurogentec France SA, Angers, France). Amplification mixes were composed of a PCR buffer (1× final), 0.2 mM dNTPs, 0.1 M specific probe, 0.2 M sense primer, 0.4 M antisense primer, three units of HotStarTaq DNA Polymerase (QIAGEN, Courtaboeuf, France), 0.05 units Omniscript RT (QIAGEN) and molecular-grade water, in a total volume of 15.1 L per reaction. The (RT-)PCR reactions were performed, using 10 L of eluate from nucleic acid extraction mixed with 15 L of amplification mix, on the Rotor-gene 6000 (Corbett, Archamps, France) for RSVA and on the LightCycler480 (Roche) for Ad5. The amplification protocol consisted of 30 min of reverse transcription at 50 ◦ C, followed by 15 min of Taq activation at 95 ◦ C and 45 cycles of denaturation at 95 ◦ C for 10 s, annealing at 54 ◦ C for 30 s, with optical reading in the yellow channel, and an extension for 10 s at 72 ◦ C. Detection of the viral genomes was performed with commercialized kits, in accordance with the manufacturer’s instructions. The RSVA/B r-gene® primers/probes (ref. 71-007, bioMerieux, Marcy l’Etoile, France) were used for RSVA detection on the Rotorgene 6000 (Corbett). The Adenovirus r-gene® primers/probes (ref. 71-010, bioMerieux) were used for Ad5 detection on a Smart Cycler 2 (Cepheid, Maurens-Scopont, France). The GeNorm software determines the most stable reference genes from a set of tested genes in a given sample panel. It calculates the gene expression stability measure, M, for a reference gene as the average pairwise variation, V, for that gene against all other tested reference genes. Genes with the lowest M value have the most stable expression [16]. The default limit for M is 1.5 [20]. The CT value was calculated by subtracting the CT value of the GAPDH RT-PCR from the CT value of the HPRT1 or UBC RT-PCR. The CT value was calculated by subtracting the CT of the infected condition from the CT of the non-infected condition [19].
3.2. HPRT1 q(RT-)PCR on clinical specimens Four hundred and thirty-six nasal swabs were collected between September 17th, 2009, and January 4th, 2010, atthe
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Fig. 1. RNA transcription levels of GAPDH, HPRT1 and UBC reference genes in RSVA and Ad5 infection kinetics of MRC-5 cells. CT values of the RT-PCRs of GAPDH, HPRT1 and UBC in RSVA-infected cells (I) and non-infected cells (NI) (a) and in Ad5-infected cells (I) and non-infected cells (NI) (b) are shown. Each point shows the CT values obtained for each gene and each point of measure, 0, 24, 30, 48 and 72 h post infection in Ad5 kinetics and the mean of the three CT values obtained for each gene and each point of measure, 0, 24, 30, 48 and 72 h post infection in RSVA kinetics. The CT values of GAPDH RT-PCRs are presented in the top figure, of HPRT1 RT-PCRs in the central figures and of UBC RT-PCRs in the bottom figures. On each figure, CT values obtained by RSVA RT-PCRs or Ad5 PCR amplification in RSVA-infected cells and Ad5-infected cells at 0, 24, 30, 48 and 72 hpi are shown to follow the viral infection.
University hospital of Caen. They were stored at −80 ◦ C. The age distribution of the tested patients was: 25% less than two years old, 44% between three years old and 15 years old, 28% between 16 years old and 65 years old and 3% more than 66 years old. Total nucleic acids were extracted on the QIAsymphony® SP platform (QIAGEN), using the QIAsymphony Virus/bacteria mini kit with the Pathogen complex 200 protocol (QIAGEN). Sample volume was 300 L and elution volume was 85 L. For quantification, the HPRT1 PCR product was cloned into the pCR® 2.1-TOPO vector using the TOPO TA Cloning kit (Invitrogen, Cergy Pontoise, France). The concentration of the plasmid solution was estimated by spectrophotometry. Three quantification standards, composed of plasmid solutions that were diluted to 105 , 104 and 103 HPRT1 plasmid copies/PCR, were tested. As the HPRT1 gene is a single copy gene (http://www.ncbi.nlm.nih.gov/) and we estimate that there are in mean two copies in each cell, the three quantification standards were expressed as 50,000, 5000 and 500 cells/PCR. The amplification was performed on
Applied Biosystems® 7500 Real-Time PCR Systems (Life Technologies, Saint-Aubin, France). 4. Results 4.1. Expression of the HPRT1, UBC and GAPDH genes in infected and non-infected cells The kinetics of RSVA and Ad5 infections was assessed, in vitro, in MRC-5 cells, to follow the transcriptional activity of the reference genes HPRT1, UBC and GAPDH in infected and non-infected cells. The CT values that represented the RSVA or Ad5 genome detection were measured at 0, 24, 30, 48 and 72 hpi and are shown in Fig. 1. No virus was detected in non-infected cells. In infected cells, the CT values obtained for RSVA and Ad5 genome detection showed an exponential decrease (from 44.0 to 20.9 cycles for RSVA and from 31.3 to 14.9 for Ad5, at 0 and 72 hpi, respectively), which demonstrated increasing infection of the cells.
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Fig. 2. Ranking of the three candidate reference genes, UBC, HPRT1 and GAPDH, using the geNorm software. The software ranks the candidate reference genes by their stability parameter, M. Lower M values represent higher stability of expression.
The mean CT values for each gene, in infected and non-infected conditions, were very close for both infections, which indicated the absence of regulation of their transcriptional activity in the presence of these viruses. In the RSVA assay, the average CT values were 27.1 vs. 27.4, 22.6 vs. 22.5 and 20.1 vs. 20.1, for HPRT1, UBC and GAPDH, in infected and non-infected cells, respectively. For Ad5, the CT values, for HPRT1, UBC and GAPDH, in infected and non-infected cells were 28 vs. 28.5, 26.5 vs. 26.2 and 23.4 vs. 23.8, respectively. No significant difference was observed in obtained CT values between infected and non-infected cells for any of the three genes (ANOVA p-values for the three genes > 0.05), at any of the time points, for both RSVA and Ad5. The GeNorm software has also been used to analyze the stability of expression of the three genes. The M values are shown in Fig. 2. The GAPDH gene gave the lowest M value of the three genes for both viruses (0.634 in RSVA and 0.572 in Ad5). This indicated that GAPDH is the most stable of the three genes. The M values obtained for the UBC and HPRT1 genes were similar for RSVA. The UBC gene had a higher M value (0.719) than HPRT1 (0.612), for Ad5, which signified that HPRT1 is more stable than UBC in cells infected with this virus. The stability of expression of HPRT1 and UBC was analyzed by the CT method with GAPDH as a reference. The CT value reflects the changes in RNA transcription caused by the infection. A positive CT indicates down-regulation of gene transcription, whereas a negative CT indicates up-regulation. [19] The calculated CT values for the three reference genes are given in Fig. 3. The CT values for HPRT1 and UBC showed low levels of regulation of their expression for both viruses (maximum CT values
Fig. 4. Ranking of the 436 samples by cellular concentration and patient age. Cells from respiratory specimens were quantified after a total nucleic acid extraction and an HPRT1 q(RT-)PCR. Samples were quantified by three quantification standards and were ranked in accordance with the concentration ranges of cells/PCR and the age of the patient. Median values (Med) are shown.
obtained for HPRT1 of −(1.2) at 0 hpi with RSVA and −(1.1) at 30 hpi with Ad5, and for UBC of −(1.1) at 0 hpi with RSVA and 1.65 with Ad5 at 72 hpi). All the measured variations were close to the values that correspond to three times the inter-assay variability of these RT-PCRs (data not shown). 4.2. Study on clinical samples The data of the three genes indicated that they could all be developed and used as cellular controls. We selected the HPRT1 assay because the design was original, unlike the GAPDH design that was taken from a publication, and this gene showed more stable expression than UBC (see Section 4.1). Cellular concentrations of the clinical samples were quantified by HPRT1 q(RT-)PCR and are shown in Fig. 4. The cellular concentrations fell into the following categories: between 100 and 10,000 cells/PCR (69.11%, n = 301, median age = 9), between 10,000 and 50,000 cells/PCR (24.94%, n = 92, median age = 6), more than 50,000 cells/PCR (3.89%, n = 26, median age = 2) and less than 100 cells/PCR (2.06%, n = 17, median age = 30). 5. Discussion Comparisons of the stability of reference genes are frequently performed between tumor and non-tumor tissues of the same type, in order to identify new biomarkers [21–24]. The stability
Fig. 3. Normalized expression of HPRT1 and UBC genes in RSVA and Ad5 infected cells at each point of measure. The CT value is indicated for each gene (HPRT1 and UBC) at each point of measure, 0, 24, 30, 48 and 72 hpi. The CT was calculated from changes in GAPDH RNA transcription, induced by Ad5 (a) and RSVA (b) infection, and was normalized to RNA transcription changes in non-infected cells.
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of reference genes during viral infection in humans, animals or plant cells has also been studied [25–30]. Our study is the first to examine the expression of GAPDH, HPRT1 and UBC genes, during in vitro viral infection with RSVA and Ad5 viruses, in order to develop a cellular control. These are common respiratory viruses, with either an enveloped RNA genome (RSVA) or a non-enveloped DNA genome (Ad5). The GeNorm software showed that all three genes were stable with both viruses, as all M values were below the default limit [16]. The transcription of the genes in infected cells, in comparison with non-infected cells, was not affected, as shown by CT values and CT analysis. The software defined GAPDH as the most stable gene, which is concordant with published data [26,27]. The three (RT-)PCR assays could be used as cellular controls in potentially infected respiratory samples. Nevertheless, these statements may require confirmation by further studies with other pathogens. The HPRT1 q(RT-)PCR assay was tested on clinical samples that contained both DNA and mRNA, which could be amplified simultaneously by the assay. In an experiment on 20 clinical samples, which may or may not have been infected by various respiratory viruses, we obtained similar cellular concentrations by HPRT1 qPCR (without reverse transcriptase) and q(RT-)PCR (with reverse transcriptase) (data not shown). The HPRT1 q(RT-)PCR assay allowed us to make an accurate estimation of the cellular concentration. The study on 436 nasal swabs highlighted the heterogeneity of the cellular concentrations in this type of sample and also showed that the concentration decreased as the age of patient increased. Further studies are ongoing to confirm this data. Nevertheless, sampling in the elderly is known to be more difficult than in other age groups [31]. The choice of sampling tool and site can improve the quantity of cells collected and aid the viral diagnostic. Indeed, it has been shown that the viral load was 4.8 times higher in flocked swabs than in rayon swabs, and 19 times higher in the nasopharynx than in the oropharynx [32]. There is no definition of a “rich” or “poor” sample, in terms of cells. In our study, all 436 samples were Influenza A positive. The usefulness of the cellular controls has been demonstrated on upper respiratory samples [15]. However, further studies are required for lower respiratory samples. Indeed, BAL, tracheal aspirates or bronchial secretions are the samples of choice for the diagnosis of acute lower respiratory infections. Across different laboratories and samplers, samples are heterogeneous and there is no standardized consensus protocol. However, a published clinical study on BAL, for the normalization of HSV1 viral load, in which our assay was used, showed homogeneity of cellular loads [33]. In light of these findings, there is a growing interest in the accurate quantification of respiratory viruses [34,35]. The developed cell control assay allows accurate respiratory virus quantification and may contribute to our knowledge of the physiopathology of respiratory viruses in two ways: firstly, for follow-up after the administration of therapeutics; secondly, to analyze and prevent transmissibility and nosocomial infections. This tool has been commercialized in the Cell control r-gene® and Rhino&EV/CC r-gene® kits (bioMerieux). Funding None. Competing interests The authors RESA Cécile, MAGRO Stéphane, MARECHAL Patricia, BARRANGER Côme and JOANNES Martine are in an employment relationship with company Biomerieux SA.
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