key: cord-0005548-0pgcfk6b authors: Sidoti, Francesca; Bergallo, Massimiliano; Terlizzi, Maria Elena; Piasentin Alessio, Elsa; Astegiano, Sara; Gasparini, Giorgio; Cavallo, Rossana title: Development of a Quantitative Real-Time Nucleic Acid Sequence-Based Amplification Assay with an Internal Control Using Molecular Beacon Probes for Selective and Sensitive Detection of Human Rhinovirus Serotypes date: 2011-07-05 journal: Mol Biotechnol DOI: 10.1007/s12033-011-9432-4 sha: c515bc2f154c41d35b7c4fac6686284f9091a49e doc_id: 5548 cord_uid: 0pgcfk6b Evidence demonstrating that human rhinovirus (HRV) disease is not exclusively limited to the upper airways and may cause lower respiratory complications, together with the frequency of HRV infections and the increasing number of immunocompromised patients underline the need for rapid and accurate diagnosis of HRV infections. In this study, we developed the first quantitative real-time nucleic acid sequence-based amplification assay with an internal control using molecular beacon probes for selective and sensitive detection of human rhinovirus serotypes. We described a simple method to accurately quantify RNA target by computing the time to positivity (TTP) values for HRV RNA. Quantification capacity was assessed by plotting these TTP values against the starting number of target molecules. By using this simple method, we have significantly increased the diagnostic accuracy, precision, and trueness of real-time NASBA assay. Specificity of the method was verified in both in silico and experimental studies. Moreover, for assessment of clinical reactivity of the assay, NASBA has been validated on bronchoalveolar lavage (BAL) specimens. Our quantitative NASBA assay was found to be very specific, accurate, and precise with high repeatability and reproducibility. Human rhinoviruses (HRVs) are the most frequent cause of acute upper respiratory tract infections in humans and are usually responsible for 30-50% of cases of common cold [1] [2] [3] . However, they may also be associated with moresevere lower respiratory tract infections. Rhinoviruses have been isolated from cases of cystic fibrosis, otitis media, sinusitis, asthma, exacerbations of chronic obstructive pulmonary disease (COPD), and pneumonia, especially in children, in the elderly, and in immunocompromised patients [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] . Evidence demonstrating that HRV disease is not exclusively limited to the upper airways and may cause lower respiratory complications, together with the frequency of HRV infections and the increasing number of immunocompromised patients underline the need for rapid and accurate diagnosis of HRV infections. Two nucleic acid amplification techniques (NAATs) are actually available for the detection of HRV: reverse transcription-PCR (RT-PCR) [15, 16] , and nucleic acid sequence-based amplification (NASBA) [17, 18] . NASBA has proven to be highly sensitive, specific, and more rapid than RT-PCR technique [19, 20] . Currently, only qualitative NASBA kits for the detection of HRV are commercially available (registered trademarks owned of bioMérieux, Marcy L'Etoile, France), while there are no quantitative NASBA kits. Some quantitative molecular beacon real-time NAS-BA assays have been described in the literature, mainly for the identification of human immunodeficiency virus (HIV), respiratory syncytial virus A and B, influenza A virus (H1N1), Trypanosoma brucei, Aspergillus fumigatus, Plasmodium species, and Listeria monocytogenes [21] [22] [23] [24] [25] [26] [27] [28] [29] . However, these real-time NASBA assays use mathematical models for the analysis of results that requires employing of specific complex software calibrated to each target [30] . Other quantitative NASBA assays, instead, compute the ratio of the time to positivity (TTP) values for both the target RNA and internal control by using standard curves with a correlation coefficient less than 0.99 (R 2 \ 0.99), index of an insensitive assay [19, 24, 26] . Aim of this study was to develop the first quantitative real-time nucleic acid sequence-based amplification assay internally controlled using molecular beacon for selective and sensitive detection of HRV serotypes. Validation and standardization were performed by evaluating diagnostic trueness, precision, and accuracy of real-time NASBA assay. Prototype human rhinovirus serotype 16 (HRV-16) was obtained from the American Type Culture Collection (ATCC, Manassas, Virginia). Rhinovirus serotype 16 (ATCC VR-283), originally isolated from a human clinical specimen, was extracted by using an automatic extractor NucliSENS easyMAG platform (bioMérieux, France), according to the manufacturer's recommendations. Onehundred-microliters of HRV-16 were used for the extraction, RNA was eluted in 25 ll of nuclease-free water and stored at -80°C. To evaluate the specificity of the HRV NASBA assay, purified RNA templates from 12 HRV isolates, and 14 selected respiratory viruses other than HRV were used for inclusivity and exclusivity testing (Table 1) . In Vitro RNA Transcription Viral cDNA was generated, first by incubation of random primers (600 ng/ll) and dNTPs (10 mM) (Invitrogen) with 10 ll of HRV-16 RNA for 5 min at 70°C. Subsequently, a mix containing buffer 59 [250 mM Tris-HCl (pH 8.3 at 25°C), 375 mM KCl, and 50 mM DTT], MgCl 2 (25 mM), ImpromII RT (1 U/ll), and Recombinant RNasin Ò Ribonuclease Inhibitor (40 U/ll) (Promega) was added. The total volume (20 ll) of the reaction mixture was incubated for 5 min at 25°C, 60 min at 42°C, and 15 min at 70°C using 9800 Fast Thermal Cycler (Applied Biosystems, Monza, Italy). cRNA production was carried out using T7-RiboMAX Large Scale RNA Production Systems (Promega, USA) at 37°C for 4 h. One-tenth of cRNA product was treated with RQ1 RNase-Free DNase (Promega, USA) at 37°C for 15 min followed by incubation with EDTA for 15 min at 65°C. HRV-16 cRNA was purified using RNAgent kit (Promega, USA) following manufacturer's instructions. HRV-16 cRNA was quantified using Quant-iT DNA BR assay on Qubit TM fluorometer (Invitrogen, Carlsbad, USA), and the number of molecules per microliter calculated from the molecular weight of HRV-16 amplicon (70,950 MW) and Avogadro number (6.023 9 10 23 ). Ten-fold dilutions of RNA standards were generated in order to amplify from 10 8 to 1 copy per reaction, and frozen at -80°C until use. For the production of internal control (IC) RNA, we used the human U1A housekeeping gene encoding the ''A'' protein present in the human U1 small nuclear ribonucleoprotein (snRNP) particle. To generate the IC RNA, the U1A molecule was extracted from a clinical specimen, precisely from a human bronchoalveolar lavage sample, and subjected to reverse transcription (RT) reaction by using random primers (600 ng/ll) and ImpromII RT (1 U/ll). IC cDNA product was amplified by using adapted NASBA primers containing the T7 RNA polymerase promoter site. Briefly, 2 ll of IC cDNA was added to 28 ll of PCR solution containing Flexi Buffer 59, 50 mM MgCl 2 , 1 unit GoTaq Ò Hot Start Polymerase (Promega), 200 lM of each dNTP, and 25 lM of each U1A primer. After an initial denaturation step of 2 min at 94°C, the first-round PCR amplification was carried out under the following conditions: 95°C for 0 s, 58°C for 15 s, 72°C for 10 s for 35 cycles, then one cycle at 72°C for 7 min using the 9800 Fast Thermal Cycler (Applied Biosystems). PCR product was transcribed in vitro using T7-RiboMAX, as previously described (see ''In vitro transcription'' in the ''Materials and methods'' section). Ten-fold dilutions of IC RNA standards were generated in order to amplify from 10 8 to 1 copy per reaction. Primers and molecular beacon probes were obtained from literature [31, 32] (Table 2) , synthesized by Eurogentec (Seraing, Belgium), and diluted to a final concentration of 100 lmol/l. Moreover, to maximize the oligonucleotides stabilization, we added 60% dimethyl sulfoxide (DMSO) to primers and beacons mixture. The HRV beacon was labeled with FAM at its 5 0 -end and quencher DABCYL at its 3 0 -end, while the IC beacon contained a ROX at its 5 0 -end and a DABCYL quencher at the 3 0 -end. The stability and predicted structure of the beacons were analyzed by using the European MFOLD server (http://frontend. bioinfo.rpi.edu/applications/mfold/cgi-bin/dna-form1.cgi). Real-time NASBA reaction was performed on a NucliSens EasyQ analyzer (BioMérieux) using the NucliSENS EasyQ Basic Kit Version 2 (bioMérieux, Lyon, France) for the amplification according to the manufacturer's manual. To obtain the best amplification efficiency, conditions for the real-time NASBA assay were optimized until the best primers, beacons, and KCl concentrations were determined. Titrations of IC RNA (between 10 and 10 6 copies) and HRV RNA were performed to determine the optimal amount of internal control to generate the greatest dynamic range for the assay without interference with the detection of HRV RNA (data not shown). As a result, each reaction was run with the addition of 10 5 copies of the IC RNA. Briefly, a total volume of 10 ll of reaction mixture containing 80 mM KCl and 0.3 lM of the HRV-and IC-specific primers was incubated with 2.5 ll HRV RNA and 2.5 ll IC RNA in the presence of 0.05 lM of HRVand IC-molecular beacons at 65°C for 2 min to denature secondary structure RNA. The reaction was subsequently cooled to 41°C for 2 min to anneal the primers before adding 5 ll of enzyme mixture containing avian myeloblastosis virus retrotranscriptase, RNase H, and T7 RNA polymerase. After a brief centrifugation and gentle mixing by tapping, the tubes were then incubated at 41°C for 90 min. To estimate the dynamic range of the real-time NASBA assay (range of concentrations over which the method performs in a linear manner with an acceptable level of trueness and precision), we used HRV standard dilutions from 10 8 copies/ll to 1 copy/ll. Sensitivity of NASBA assay was determined by the lowest standard dilution consistently detectable in replicate reactions at frequency of 100%, whereas the limit of detection by the lowest concentration of target quantified. Nuclease-free water was included as the no-template control (NTC) to serve as a control for background fluorescence. The Optimal real-time NASBA assay conditions that allowed efficient amplification of the HRV target were established. Sensitivity and limit of detection of NASBA assay were assessed by repeated testing of serial logarithmic dilutions of the HRV RNA standards ranging from 10 8 to 1 copy/ reaction. In particular, HRV NASBA dynamic range was calculated from TTP value (time point at which emitted fluorescence exceeds the baseline emission) regressed against the standard curve by using an Excel spreadsheet created ''ad hoc'' by us (Table 3) . Results from linear regression show that HRV real-time NASBA assay was able to quantify from 10 8 to 10 copies/reaction. The standard curve of HRV dilutions plotted versus NASBA amplifications (expressed as TPP) is shown in Fig. 1 , whereas plots for the amplification of HRV standard dilutions (from 10 8 to 10 copies/reaction), and the optimal amount of IC RNA (10 5 copies/reaction), are shown in Fig. 2 . The consistency of replicates was measured by the correlation coefficient (R 2 ), which indicates the linearity of TPP values plotted in the standard curve. The R 2 index for HRV was 0.9948. Sensitivity of real-time NASBA assay was determined by the lowest standard dilution consistently detectable in replicate reactions at frequency of 100%. HRV sensitivity was 10 copies/reaction, whereas the limit of detection for reliable quantification was 1 copy/ reaction. Based on the data available at the BLAST alignment software, primers were tested in silico for potential crossreactivity with respiratory viruses other than HRV, and demonstrated no significant homologies to any other sequences. Moreover, HRV primer and probe set, tested on respiratory viruses, was able to detect only HRV isolates, thus being the inclusivity of 100% (Table 1 ). The assay's specificity was further demonstrated by its ability to exclude all respiratory viruses other than HRV listed in Table 1 . No positive results were demonstrated for the other respiratory viruses, indicating that this molecular assay is highly specific for HRV isolates, thus being the exclusivity of 100% (Table 1) . Diagnostic trueness of HRV real-time NASBA method, defined as the degree of agreement between the average value obtained from a large series of test results and an accepted reference value, was evaluated. To establish the level of trueness and concordance with the assigned value, data from 10 replicate measures of each dilution that we performed (10 2 , 10 3 , 10 4 , and 10 5 ) were analyzed using a Student's t test to compare the mean concentrations from each dilution with an accepted reference value. The mean concentrations from each dilution for the method are shown in Table 4 with the t test results, which indicate the significance of the differences between each experimental mean and the assigned value. Analysis of the t statistics showed that the method had t-calc values lower than the t-tab value, demonstrating a significant trueness of HRV assay. Precision of method was expressed as the coefficient of variation (CV) in the log 10 values of the concentration. Repeatability and intermediate reproducibility of HRV assay were evaluated over different concentrations ranging from 10 2 to 10 5 copies/reaction from 10 replicate measures (n = 10) of each reference viral quantification standard within a single run or in 10 different run experiments performed by three different operators. The precision associated with each dilution measurement (10 2 , 10 3 , 10 4 , and 10 5 ) was assessed by calculation of the CV for each. The CVs within a single run (repeatability) ranged from 0.73 to 13.23%; whereas, the CVs in different runs (intermediate reproducibility) ranged from 3.71 to 16.71% (Table 4 ), indicating that the precision of the assay is satisfactory. Diagnostic accuracy includes both trueness and precision. The measure of accuracy is usually expressed numerically in terms of bias (lack of agreement). Accuracy shall be within ±25% of the accepted reference value over the whole dynamic range, according to document ISO 16140 [33] . Data for the percentage of inaccuracy HRV method are reported in Table 4 . The developed real-time NASBA assay was able to detect HRV RNA in 7/33 (21.2%) BAL specimens. The TTP values of these NASBA-positive samples ranged between 39 and 51 min when plotted against the standard curve in Fig. 1 (data not shown) . All the results were validated by the addition of internal control RNA to rule out inhibition of amplification. In all cases, amplification of the control RNA was observed, thus, confirming that all the negative and positive results are valid. Moreover, all negative control reactions were NASBA negative, demonstrating the absence of amplicon contamination. Viral respiratory tract infections have been recognized as a predominant cause of human disease. To improve clinical management of such patients, it is important to obtain an accurate diagnosis and to identify the causative agent early in infection to ensure appropriate treatment. In this study, we developed the first quantitative NASBA assay for the detection of HRV serotypes. By combining NASBA amplification with molecular beacon probes, this assay becomes a real-time analysis tool that offers faster results than conventional RT-PCR technique. Since NASBA amplification involves three separate enzymes each with their own kinetic parameters, variability in every measurement is inevitable [19] . Weusten et al. [30] were the first to describe a mathematical model for RNA amplification of both target and internal calibrator RNA in a molecular beacon-based NASBA reaction to normalize enzyme efficiency differences between reactions. However, the description of this model did not include all of the essential parameters needed to operate the model. Consequently, analysis using this model requires software calibrated to each target and is commercially available for only a few specific targets. Here, we describe an alternative method for normalizing NASBA data by using a simple TPP calculation in the presence of an internal control that reduces the variability between replicates and increases the precision, trueness, and accuracy for predicting unknown concentrations of HRV RNA. It has been shown that tube to tube variation within NASBA can be normalized with the addition of an internal control in each reaction [34, 35] . In particular, the optimal concentration for the internal calibrator should be determined as the concentration providing the greatest dynamic range of amplification of both the target and internal control RNA. In our study, a fixed amount of 10 5 copies of the internal calibrator RNA was optimal for the assay reported here (data not shown). Furthermore, the addition of an internal control is fundamental to identify false negative results because of reaction failure, and to monitor the effects of unknown sample factors that might interfere with the amplification kinetics. As concerns the importance of primers and KCl for NASBA optimization, their role has not been emphasized to date. NASBA is an isothermal nucleic acid amplification method able to specifically amplify target RNA by using specific primers in a KCl background. Initially, the concentration of the primers relative to the total concentration of amplifiable RNA is very high, is not rate limiting, and relatively small amounts of primers are consumed in depletion of the initially present pool of RNA copies (linear phase of NASBA process). At some time point, obviously, the primers' concentrations do become rate limiting, and decline to practically zero. At this time point, the DNA intermediate levels have reached their maximum and RNA production proceeds at high speed. From now on, the only reaction that can proceed is T7 RNA polymerase-mediated formation of RNA from the DNA intermediate templates. This time interval represents the second phase of NASBA process characterized by an exponential kinetics. We observed that high concentrations of primers and KCl elongate the linear phase of NASBA process by shorting the exponential amplification; whereas, low concentrations of primers and KCl promote the exponential phase. In particular, in this study we used relatively low concentrations of primers and KCl (0.3 lM and 80 mM, respectively) to elongate the exponential phase of NASBA process, and accordingly, to minimize the reaction-toreaction variation. By using this simple expedient, we have significantly increased our accuracy, precision, and trueness of prediction over the standard TTP calculations. In summary, we describe a simplified method of calculating unknown concentrations of target RNA using an internal calibrator. This method allowed for greater precision, accuracy, and trueness for predicting HRV RNA over the standard TTP analysis. In conclusion, we described the first real-time NASBA assay capable of accurate quantification of HRV RNA making it a valuable tool in the molecular diagnostics of HRV serotypes. Respiratory virus infections and aeroallergens in acute bronchial asthma Recurrent wheezy bronchitis and viral respiratory infections Viruses and bacteria in the etiology of the common cold Rhinovirus in acute otitis media Improved detection of rhinoviruses in nasal and throat swabs by seminested RT-PCR Use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms Lower airways inflammation during rhinovirus colds in normal and in asthmatic subjects Effect of respiratory virus infections including rhinovirus on clinical status in cystic fibrosis Acute viral infections of upper respiratory tract in elderly people living in the community: Comparative, prospective, population based study of disease burden Detection of rhinovirus in sinus brushings of patients with acute community-acquired sinusitis by reverse transcription-PCR Rhinovirus infections in myelosuppressed adult blood and marrow transplant recipients. Clinical Infectious Diseases Conventional respiratory viruses recovered from immunocompromised patients: clinical considerations Polymerase chain reaction is more sensitive than viral culture and antigen testing for the detection of respiratory viruses in adults with hematological cancer and pneumonia Detection of human rhinoviruses in the lower respiratory tract of lung transplant recipients Amplification of rhinovirus specific nucleic acids from clinical samples using the polymerase chain reaction Development of a RT real-time PCR for the detection and quantification of human rhinoviruses Development and application of a new method for amplification and detection of human rhinovirus RNA Improved detection of rhinoviruses by nucleic acid sequence-based amplification after nucleotide sequence determination of the 5 0 noncoding regions of additional rhinovirus strains Increased precision of microbial RNA quantification using NASBA with an internal control Detection of rhinoviruses by tissue culture and two independent amplification techniques, nucleic acid sequence-based amplification and reverse transcription-PCR, in children with acute respiratory infections during a winter season Real-time nucleic acid sequencebased amplification is more convenient than real-time PCR for quantification of Plasmodium falciparum Evaluation of a real-time nucleic acid sequence-based amplification assay using molecular beacons for detection of human immunodeficiency virus type 1 Detection and identification of human Plasmodium species with real-time quantitative nucleic acid sequence-based amplification Rapid and highly sensitive qualitative realtime assay for detection of respiratory syncytial virus A and B using NASBA and molecular beacon technology A molecular beacon-based real time NASBA assay for detection of Listeria monocytogenes in food products: role of target mRNA secondary structure on NASBA design Detection of Trypanosoma brucei parasites in blood samples using real-time nucleic acid sequence-based amplification Quantitative determination of Plasmodium vivax gametocytes by real-time quantitative nucleic acid sequence-based amplification in clinical samples Detection of novel swine origin influenza A virus (H1N1) by realtime nucleic acid sequence-based amplification Detection of Aspergillus fumigatus in a rat model of invasive pulmonary aspergillosis by real-time nucleic acid sequence-based amplification Principles of quantitation of viral loads using nucleic acid sequence-based amplification in combination with homogeneous detection using molecular beacons The role of respiratory viruses in cystic fibrosis Human cytomegalovirus virions differentially incorporate viral and host cell RNA during the assembly process Microbiology of food and animal feeding stuffs. Protocol for the validation of alternative method. ISO/DIS 16140 Usefulness of quantitative nucleic acid sequence-based amplification for diagnosis of malaria in an academic hospital setting Molecular-based methods for quantifying HIV viral load