Comparison of the Anyplex T M II RV16 and Seeplex RV12 ACE assays for the detection of respiratory viruses Hee Jae Huh, Kyung Sun Park, Ji-Youn Kim, Hyeon Jeong Kwon, Jong-Won Kim, Chang-Seok Ki, Nam Yong Lee PII: DOI: Reference:
S0732-8893(14)00058-3 doi: 10.1016/j.diagmicrobio.2014.01.025 DMB 13537
To appear in:
Diagnostic Microbiology and Infectious Disease
Received date: Accepted date:
8 October 2013 24 January 2014
Please cite this article as: Huh Hee Jae, Park Kyung Sun, Kim Ji-Youn, Kwon Hyeon Jeong, Kim Jong-Won, Ki Chang-Seok, Lee Nam Yong, Comparison of the AnyplexT M II RV16 and Seeplex RV12 ACE assays for the detection of respiratory viruses, Diagnostic Microbiology and Infectious Disease (2014), doi: 10.1016/j.diagmicrobio.2014.01.025
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Comparison of the AnyplexTM II RV16 and Seeplex® RV12 ACE Assays for the
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Detection of Respiratory Viruses
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Hee Jae Huh1, Kyung Sun Park1, Ji-Youn Kim2, Hyeon Jeong Kwon 2, Jong-Won Kim1,
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Chang-Seok Ki1*, Nam Yong Lee1
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Sungkyunkwan University School of Medicine, Seoul, Republic of Korea; 2Center for
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Clinical Medicine, Samsung Biomedical Research Institute, Samsung Medical Center,
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Running title: Comparison of RV16 and RV12 detection kits
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Seoul, Korea
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Department of Laboratory Medicine and Genetics, Samsung Medical Center,
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Department of Laboratory Medicine and Genetics, Samsung Medical Center,
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Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, Seoul 135-
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710, Korea. Tel: +82-2-3410-2709, fax: +82-2-3410-2719, e-mail: [email protected]
Corresponding Author: Dr. Chang-Seok Ki
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Abstract
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The AnyplexTM II RV16 detection kit (RV16; Seegene, South Korea) is a multiplex
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real-time PCR assay based on tagging oligonucleotide cleavage extension. In this
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prospective study, we evaluated the RV16 assay by comparing with the Seeplex® RV12
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ACE detection kit (RV12; Seegene), a multiplex end-point PCR kit. A total of 365
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consecutive respiratory specimens were tested with both RV16 and RV12 assays in
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parallel and detected 140 (38.4%) and 89 (24.4%) positive cases, respectively. The
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positive percent agreement, negative percent agreement, and kappa values for the two
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assays were 95.6% (95% CI, 89.4 to 98.3%), 80.4% (95% CI, 75.3 to 84.6%), and 0.64
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(95% CI, 0.56 to 0.72), respectively. The monoplex PCR and sequencing for the
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samples with discrepant results revealed that majority of the results were concordant
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with the results from RV16 assays. In conclusion, the RV16 assay produces results
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comparable to the RV12 assay.
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Key Words: Respiratory virus; Multiplex real-time PCR; Validation; Korea
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Introduction
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Respiratory viral infections are responsible for substantial morbidity in both
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pediatric and adult populations. Timely and accurate diagnosis of these infections is
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essential to patient care in settings as diverse as susceptible infants or children, older
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adults, patients with compromised immune systems, or individuals with underlying
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cardiopulmonary diseases (Glezen et al., 2000; Weinberg et al., 2004). Early diagnosis
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of respiratory viruses plays an important role in clinical management, reducing
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complications, antibiotic use, and unnecessary laboratory testing (Jernigan et al., 2011;
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Mahony et al., 2009; Renaud et al., 2012). The Seeplex RV12 ACE detection kit (RV12;
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Seegene, South Korea) enables simultaneous detection of 12 respiratory viruses in two
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reactions per sample using Dual Priming Oligonucleotides (DPOs) as PCR primers. The
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performance of RV12 has been demonstrated in previous studies, showing time and
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resource savings (Bibby et al., 2011; Drews et al., 2008; Weinberg et al., 2004; Yoo et
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al., 2007). Recently, Anyplex II RV16 detection (V1.1) kit (RV16; Seegene) with
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Tagging Oligonucleotide Cleavage and Extension (TOCE) technology has been
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developed. TOCE technology is a novel approach to real-time PCR, using the two
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components, the Pitcher and Catcher, to accomplish a unique signal generation.
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Through target bound Pitcher, TOCE assay moves the detection point from the target
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sequence to the Catcher. By designing unique Catchers, the resulting Duplex Catcher
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will have a predictable melting temperature profile (Cho et al., 2013; Chun, 2012; Lee,
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2012). Both RV16 and RV12 achieved approval from CE, Health Canada, and KFDA,
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being sold in more than 15 countries in Europe, Canada, and Asia. RV16
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simultaneously detects 16 respiratory viruses: human bocavirus (HBoV), human
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enterovirus (HEV), influenza virus (INF) types A and B, parainfluenza virus (PIV)
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types 1, 2, 3, and 4, respiratory syncytial virus (RSV) types A and B, adenovirus (ADV),
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metapneumovirus (MPV), coronavirus (CoV) OC43, 229E, NL63, and human
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rhinovirus (HRV). This profile is similar to the profile of RV12 but further includes
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HBoV, HEV, PIV 4, CoV 229E and CoV NL63.
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The aim of the present study was to compare the performance of RV16 and
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RV12. In addition, we evaluated the analytical performance of RV16. These studies
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were carried out in a routine diagnostic laboratory setting and used nonselective clinical
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specimens from Korean patients.
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Materials and Methods
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Clinical specimens
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This prospective study was approved by the Institutional Review Board of Samsung
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Medical Center. Three hundred sixty-five nonselective consecutive clinical respiratory
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specimens from 302 patients were obtained. The patients included 55 adults and 247
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pediatric patients whose median age was 3 years and ranged from 1day to 93 years.
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There were 320 nasopharyngeal (NPAs) and 45 bronchoalveolar lavage (BAL) fluid
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samples submitted for RV12 testing from January to February 2013. These samples
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were simultaneously analyzed by RV16, and the aliquots of each specimen were
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immediately stored frozen at -70°C. The volume of NPA and BAL fluid was
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approximately 10 ml.
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Nucleic acid extraction
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Nucleic acids were extracted from 100 μl of each specimens by MagNa Pure LC Total
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Nucleic Acid Isolation Kit (Roche, Mannheim, Germany) for RV12 and by
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MICROLAB STARlet (Hamilton, Reno, NV, USA) with STARMag 96 Virus Kit
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(Seegene, Seoul, Korea) for RV16. The final elution volume of each sample was 50 μl 5
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in both kits. In RV16, bacteriophage MS2 was added as an internal control to each
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specimen, according to the manufacturer’s instructions.
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RV12 testing
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Random hexamer-primed cDNA synthesis products were generated using the Revertaid
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First Strand cDNA synthesis kit (Fermentas, Ontario, Canada), according to the
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manufacturer's instructions. Each cDNA preparation was subjected to the RV12 PCR
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procedure according to the manufacturer's instructions (Seegene, Seoul, South Korea).
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Briefly, parallel 20 μl reactions were set up, each containing RV12 mastermix, 8-MOPS
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contamination control reagent, and 3 μl cDNA. One of each pair was supplemented with
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4 ml primer mix A, and the other with 4 ml primer mix B. Thermal cycling conditions
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were as follows: 15 min at 95°C, followed by 30 cycles of 95°C for 30 sec, 60°C for 90
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sec, and 72°C for 90 sec, followed by a single incubation of 10 min at 72°C.
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Amplification products were detected using capillary electrophoresis technology
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(Lab901 Screen Tape System; Lab901 Ltd, Loanhead, UK).
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RV16 testing 6
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cDNA synthesis was performed with cDNA Synthesis Premix (Seegene, Korea) from
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extracted RNAs. Respiratory virus detection kits-A and B (AnyplexTM II RV16
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detection; Seegene, South Korea) were used, according to the manufacturer’s
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instructions. Briefly, the assay was conducted in a final volume of 20 μl containing 8 μl
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cDNA, 5 μl 4×RV primer, and 5 μl 4× master mix with the CFX96 real-time PCR
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detection system (Bio-Rad, Hercules, CA, USA) under the following conditions: 4 min
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at 50°C and 15 min at 95°C, followed by 50 cycles of 95°C for 30 sec, 60°C for 1 min,
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and 72°C for 30 sec. After the reaction, Catcher Melting Temperature Analysis (CMTA)
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was performed by cooling the reaction mixture to 55°C, maintaining the mixture at
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55°C for 30 s, and heating the mixture from 55°C to 85°C. The fluorescence was
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measured continuously during the temperature increase. The melting peaks were
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derived from the initial fluorescence (F) versus temperature (T) curves.
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Comparison of the RV12 and RV16 assays
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Specimens that showed a discrepancy between RV12 and RV16 assay were further
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verified using monoplex PCR and sequencing in a blind manner. The primers for
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monoplex PCR in the single or nested PCR format were identical to the primers of the 7
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RV12 or RV16 assay. The PCR products were purified with a power gel extraction kit
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(TaKaRa Bio Inc., Shiga, Japan). Purified templates were sequenced with a BigDye
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Terminator v3.1 cycle sequencing kit (Life Technologies, Foster City, CA, USA) and
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analyzed on an ABI 3730xl DNA analyzer (Life Technologies). Since RV12 cannot
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detect BoV, HEV, ADV F, PIV4, or HRV C, we excluded those results from
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comparison of the two assays.
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Analytical sensitivity and specificity of the RV16 assay
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Serially diluted plasmids containing the target gene were used for determination of
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analytical sensitivity. pUC19 vector was used for plasmid DNA preparation. Serial
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dilutions of the prepared plasmid DNA were made from 10 6 to 100 copies per reaction
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to determine the analytical sensitivity of the assay. MPV, BoV, and CoV-NL63 samples
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were isolated from patients, and their sequences were confirmed by direct sequencing.
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All other standard strains were obtained from American Type Culture Collection
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(ATCC). Ten replicates of each dilution step were performed. The lower detection limit
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was defined as the lowest concentration detected by 10 replicas of each assay.
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The cross-reactivity of the RV16 assay was assessed using ten different bacteria. 8
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Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus epidermidis,
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Moraxella catarrhalis, Pseudomonas aeruginosa, Escherichia coli, Klebsiella
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pneumoniae, Staphylococcus aureus, Neisseria meningitides, and Haemophilus
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influenzae were obtained from the American Type Culture Collection (ATCC,
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Manassas, VA). The DNA of supplied samples was extracted and assayed with the
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RV16 assay adhering to the same procedures used for sample processing.
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Statistical analysis
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Statistical analyses were performed using SPSS software, version 20.0 (SPSS Inc.,
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Chicago, IL) and the VassarStats website (http://vassarstats.net/). We used inter-rater
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agreement statistics (Kappa calculation) to compare the detection of respiratory viruses
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between the RV12 and RV16 assays. P-values less than 0.05 were considered
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statistically significant.
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Results
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Comparison of the RV16 assay with the RV12 assay 9
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A total of 140 (38.4%) and 89 (24.4%) samples were RV16- and RV12-positive,
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respectively. Among viruses tested using both methods, the positive percent agreement
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between the RV16 and the RV12 assays was 95.5% (95% CI, 88.3 to 98.5), and the
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negative percent agreement was 80.0% (95% CI, 74.8 to 84.2). The kappa value for the
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two methods was 0.64 (95% CI, 0.55 to 0.71). Results by virus are presented in Table 1.
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Sixty-five samples that were identified as positive samples by the RV16 assay
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were identified as negative by the RV12 assay: Eight samples had more than one
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discrepant viruses, and 71 discrepancies by each virus were detected. On the other hand,
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four clinical samples that were negative in the RV16 assay were identified as positive
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by the RV12 assay. For further analysis of the samples that yielded discrepant results
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with the RV16 assay and the RV12 assay, monoplex real-time RT-PCR and gene
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sequencing were performed. In the discrepant RV16-positive and the RV12-negative
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results, the results of monoplex PCR and sequencing revealed that 81.7% (58/71) were
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concordant with the result by the RV16 assay. On the other hand, in discrepant results
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exhibited positivity only in the RV12 assay, none was concordant with the result by the
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RV12 assay. The overall results of monoplex PCR and sequencing revealed that
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majority of the samples that were identified as positive from RV16 assays also exhibited
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positive results, except INF A (Table 2).
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Analytical sensitivity and specificity of the RV16 assay
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The detection limits of the RV16 detection kit for all 16 respiratory viruses were
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approximately 6 copies/μL (50 copies/reaction).
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To evaluate the cross-reactivity and detection specificity, ten different bacterial
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reference strains were tested using the same assay procedure used for the clinical
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samples for RV detection with the RV16 assay. All assay results were negative, and no
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non-specific positive reaction was observed.
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Discussion
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We compared the performance of two multiplex PCR kits, RV12 and RV16, for
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detection of respiratory viruses using clinical respiratory samples. We performed
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monoplex PCR and direct sequencing in a blind manner for the samples that yielded
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discrepant results with the two assays.
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In the present study, 38.4% and 24.4% of samples were RV16- and RV12-
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positive, respectively. The volume of sample added to the RV16 assay was almost 3 11
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times more than the volume added to the RV12 assay and this could have contributed to
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the higher number of positive samples by RV16 compared to RV12 assay.
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Majority of the samples that were identified as positive from RV16 assays were
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confirmed as positive by monoplex real-time RT-PCR and gene sequencing. However,
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in the discrepant RV16-positive and the RV12-negative results for INF A, monoplex
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PCR and sequencing revealed that 50% were negative. This suggests that the false
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positivity for INF A in RV16 assay by nucleic acid contamination or cross-reactivity
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from spurious primer interactions. However, the possibility that the RV16 may detect
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INF A with superior sensitivity compared to the monoplex PCR and sequencing cannot
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be excluded. Recent studies reported on the performance of RV16, which showed
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increased sensitivities compared to the Seeplex® RV15 ACE detection kit (RV15;
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Seegene), although we did not determine the diagnostic accuracy against a reference
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method (Cho et al., 2013; Kim et al., 2013).
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The RV12 system had a limitation regarding its internal control facility. The
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artificial targets included in each PCR mastermix only allow validation of the PCR step,
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without control for the RNA extraction or reverse transcription steps (Bibby et al.,
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2011). As for RV16, the addition of bacteriophage MS2 to each extraction allows the 12
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RT-PCR system to monitor both the RNA extraction and the reverse transcription steps
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(Cho et al., 2013; Dreier et al., 2005). Nevertheless, the internal control in RV16 cannot
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give information for specimen quality or prevent misjudgment from sampling error,
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since it is an exogenous control.
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RV16 has an advantage in workload compare to RV12. Given that the RV12
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assay requires agarose gel detection after PCR, RV16 requires less time and labor than
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RV12 (less than seven hours with reduced hands-on time) (Bibby et al., 2011; Kim et al.,
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2013). In addition, RV16 is a closed PCR system with a reduced chance of amplicon
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contamination.
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The present study has some limitations. First, we could not compare a large
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number of some respiratory viruses between assays due to the low number of virus-
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positive specimens and respiratory virus seasonality, since this was consecutive
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prospective study performed over two months. Second, we did not compare RV16 and
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RV12 with viral cultures or direct fluorescent-antibody assays. Third, we used the same
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primers for RV12 and RV16 in the monoplex PCR and sequencing for discrepant
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analysis. However, we performed monoplex PCR and direct sequencing in a blind
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manner to overcome the limitation of the study design. Fourth, since the analytical
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sensitivities were determined using plasmid DNA, the reverse transcription step could
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not be included in the estimate.
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In conclusion, the RV16 assay produces results comparable to the RV12 assay.
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Further analysis of the samples that yielded discrepant results demonstrated that the
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RV16 assay exhibited a higher concordance with the results of monoplex PCR and
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sequencing, compared to the RV12 assay.
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Acknowledgements
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This study was supported by Seegene Korea. The sponsor had no involvement in the
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study design, data interpretation, or preparation of the manuscript.
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References
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Cho CH, Chulten B, Lee CK, Nam MH, Yoon SY, Lim CS, et al. Evaluation of a novel
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RV15 for simultaneous detection of respiratory viruses. J Clin Virol
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Drews SJ, Blair J, Lombos E, DeLima C, Burton L, Mazzulli T, et al. Use of the
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Seeplex RV Detection kit for surveillance of respiratory viral outbreaks in
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Toronto, Ontario, Canada. Ann Clin Lab Sci 2008;38:376-9.
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Glezen WP, Greenberg SB, Atmar RL, Piedra PA, Couch RB. Impact of respiratory
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Jernigan DB, Lindstrom SL, Johnson JR, Miller JD, Hoelscher M, Humes R, et al.
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Kim HK, Oh SH, Yun KA, Sung H, Kim MN. Comparison of Anyplex II RV16 with
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the xTAG respiratory viral panel and Seeplex RV15 for detection of respiratory
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viruses. J Clin Microbiol 2013;51:1137-41.
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Lee D. TOCE: innovative technology for high multiplex real-time PCR. Seegene Bull
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Renaud C, Crowley J, Jerome KR, Kuypers J. Comparison of FilmArray Respiratory
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2012;74:379-83. Weinberg GA, Erdman DD, Edwards KM, Hall CB, Walker FJ, Griffin MR, et al.
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Table 1. Detection of respiratory viruses by AnyplexTM II RV16 and Seeplex® RV12 ACE assays.
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95% CI
95.5
88.3 – 98.5
80.0
74.8 – 84.2
0.64
0.55 – 0.71
ADV
100
39.6 – 100
95.6
92.8 – 97.4
0.32
0.08 – 0.56
INF A
95.6
76.0 – 99.8
97.1
94.5 – 98.5
0.78
0.66 – 0.91
INF B
NA
NA
100
98.7 – 100
NA
NA
PIV 1
0
0 – 94.5
100
98.7 – 100
0
0–0
PIV 2
100
54.6 - 100
100
98.7 – 100
1.00
1.00 – 1.00
PIV 3
NA
NA
100
98.7 – 100
NA
NA
HRV
90.0
59.6 – 98.2
93.6
93.4 – 97.8
0.53
RSV (A or B)
97.1
83.4 – 99.8
98.2
95.9 – 99.3
0.90
0.82 – 0.97
HMPV
100
46.3 – 100
98.3
96.2 – 99.3
0.62
0.34 – 0.90
CoV
100
69.9 – 100
94.6
91.6 – 96.6
0.54
0.36 – 0.72
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EP
95% CI
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Observed
%
AC C
Virus
Negative
TE
Positive
Kappa value
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Agreement
kappa
95% CI
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ADV, adenovirus; INF A, influenza A virus; INF B, influenza B virus; PIV 1- 3, parainfluenza virus type 1 to 3; HRV, human rhinovirus; RSV, respiratory
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syncytial virus; HMPV, human metapneumovirus; CoV, coronavirus.
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Table 2. Analysis for positive results in AnyplexTM II RV16 and Seeplex® RV12 ACE assays. Virus
RV16+/RV12+
RV16+/RV12No. of positive results in
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No. of samples
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RV16-/RV12+ No. of samples
SC
monoplex PCR and sequencing
5
1
0
0
1
0
0
0
0
13
1
0
4
1
ND*
6
0
0
17
0
0
13
INF A
22
10
PIV 1
0
0
PIV 2
1
0
HRV
9
14
RSV (A or B)
34
6
HMPV
5
6
CoV
12
19
NU
16
MA
0
4
D
monoplex PCR and sequencing 0
ADV
EP
TE
No. of positive results in
ADV, adenovirus; INF A, influenza A virus; PIV 1, parainfluenza virus type 1; HRV, human rhinovirus; RSV, respiratory syncytial virus; HMPV, human
288
metapneumovirus; CoV, coronavirus.
289
* ND, not performed due to lack of sample
290
AC C
287
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S0732-8893(14)00058-3 doi: 10.1016/j.diagmicrobio.2014.01.025 DMB 13537
To appear in:
Diagnostic Microbiology and Infectious Disease
Received date: Accepted date:
8 October 2013 24 January 2014
Please cite this article as: Huh Hee Jae, Park Kyung Sun, Kim Ji-Youn, Kwon Hyeon Jeong, Kim Jong-Won, Ki Chang-Seok, Lee Nam Yong, Comparison of the AnyplexT M II RV16 and Seeplex RV12 ACE assays for the detection of respiratory viruses, Diagnostic Microbiology and Infectious Disease (2014), doi: 10.1016/j.diagmicrobio.2014.01.025
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Comparison of the AnyplexTM II RV16 and Seeplex® RV12 ACE Assays for the
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Detection of Respiratory Viruses
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Hee Jae Huh1, Kyung Sun Park1, Ji-Youn Kim2, Hyeon Jeong Kwon 2, Jong-Won Kim1,
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Chang-Seok Ki1*, Nam Yong Lee1
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Sungkyunkwan University School of Medicine, Seoul, Republic of Korea; 2Center for
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Clinical Medicine, Samsung Biomedical Research Institute, Samsung Medical Center,
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Running title: Comparison of RV16 and RV12 detection kits
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Seoul, Korea
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Department of Laboratory Medicine and Genetics, Samsung Medical Center,
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Department of Laboratory Medicine and Genetics, Samsung Medical Center,
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Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, Seoul 135-
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710, Korea. Tel: +82-2-3410-2709, fax: +82-2-3410-2719, e-mail: [email protected]
Corresponding Author: Dr. Chang-Seok Ki
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Abstract
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The AnyplexTM II RV16 detection kit (RV16; Seegene, South Korea) is a multiplex
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real-time PCR assay based on tagging oligonucleotide cleavage extension. In this
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prospective study, we evaluated the RV16 assay by comparing with the Seeplex® RV12
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ACE detection kit (RV12; Seegene), a multiplex end-point PCR kit. A total of 365
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consecutive respiratory specimens were tested with both RV16 and RV12 assays in
26
parallel and detected 140 (38.4%) and 89 (24.4%) positive cases, respectively. The
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positive percent agreement, negative percent agreement, and kappa values for the two
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assays were 95.6% (95% CI, 89.4 to 98.3%), 80.4% (95% CI, 75.3 to 84.6%), and 0.64
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(95% CI, 0.56 to 0.72), respectively. The monoplex PCR and sequencing for the
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samples with discrepant results revealed that majority of the results were concordant
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with the results from RV16 assays. In conclusion, the RV16 assay produces results
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comparable to the RV12 assay.
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Key Words: Respiratory virus; Multiplex real-time PCR; Validation; Korea
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Introduction
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Respiratory viral infections are responsible for substantial morbidity in both
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pediatric and adult populations. Timely and accurate diagnosis of these infections is
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essential to patient care in settings as diverse as susceptible infants or children, older
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adults, patients with compromised immune systems, or individuals with underlying
43
cardiopulmonary diseases (Glezen et al., 2000; Weinberg et al., 2004). Early diagnosis
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of respiratory viruses plays an important role in clinical management, reducing
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complications, antibiotic use, and unnecessary laboratory testing (Jernigan et al., 2011;
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Mahony et al., 2009; Renaud et al., 2012). The Seeplex RV12 ACE detection kit (RV12;
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Seegene, South Korea) enables simultaneous detection of 12 respiratory viruses in two
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reactions per sample using Dual Priming Oligonucleotides (DPOs) as PCR primers. The
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performance of RV12 has been demonstrated in previous studies, showing time and
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resource savings (Bibby et al., 2011; Drews et al., 2008; Weinberg et al., 2004; Yoo et
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al., 2007). Recently, Anyplex II RV16 detection (V1.1) kit (RV16; Seegene) with
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Tagging Oligonucleotide Cleavage and Extension (TOCE) technology has been
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developed. TOCE technology is a novel approach to real-time PCR, using the two
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components, the Pitcher and Catcher, to accomplish a unique signal generation.
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Through target bound Pitcher, TOCE assay moves the detection point from the target
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sequence to the Catcher. By designing unique Catchers, the resulting Duplex Catcher
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will have a predictable melting temperature profile (Cho et al., 2013; Chun, 2012; Lee,
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2012). Both RV16 and RV12 achieved approval from CE, Health Canada, and KFDA,
59
being sold in more than 15 countries in Europe, Canada, and Asia. RV16
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simultaneously detects 16 respiratory viruses: human bocavirus (HBoV), human
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enterovirus (HEV), influenza virus (INF) types A and B, parainfluenza virus (PIV)
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types 1, 2, 3, and 4, respiratory syncytial virus (RSV) types A and B, adenovirus (ADV),
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metapneumovirus (MPV), coronavirus (CoV) OC43, 229E, NL63, and human
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rhinovirus (HRV). This profile is similar to the profile of RV12 but further includes
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HBoV, HEV, PIV 4, CoV 229E and CoV NL63.
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The aim of the present study was to compare the performance of RV16 and
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RV12. In addition, we evaluated the analytical performance of RV16. These studies
68
were carried out in a routine diagnostic laboratory setting and used nonselective clinical
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specimens from Korean patients.
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Materials and Methods
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Clinical specimens
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This prospective study was approved by the Institutional Review Board of Samsung
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Medical Center. Three hundred sixty-five nonselective consecutive clinical respiratory
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specimens from 302 patients were obtained. The patients included 55 adults and 247
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pediatric patients whose median age was 3 years and ranged from 1day to 93 years.
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There were 320 nasopharyngeal (NPAs) and 45 bronchoalveolar lavage (BAL) fluid
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samples submitted for RV12 testing from January to February 2013. These samples
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were simultaneously analyzed by RV16, and the aliquots of each specimen were
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immediately stored frozen at -70°C. The volume of NPA and BAL fluid was
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approximately 10 ml.
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Nucleic acid extraction
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Nucleic acids were extracted from 100 μl of each specimens by MagNa Pure LC Total
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Nucleic Acid Isolation Kit (Roche, Mannheim, Germany) for RV12 and by
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MICROLAB STARlet (Hamilton, Reno, NV, USA) with STARMag 96 Virus Kit
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(Seegene, Seoul, Korea) for RV16. The final elution volume of each sample was 50 μl 5
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in both kits. In RV16, bacteriophage MS2 was added as an internal control to each
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specimen, according to the manufacturer’s instructions.
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RV12 testing
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Random hexamer-primed cDNA synthesis products were generated using the Revertaid
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First Strand cDNA synthesis kit (Fermentas, Ontario, Canada), according to the
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manufacturer's instructions. Each cDNA preparation was subjected to the RV12 PCR
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procedure according to the manufacturer's instructions (Seegene, Seoul, South Korea).
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Briefly, parallel 20 μl reactions were set up, each containing RV12 mastermix, 8-MOPS
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contamination control reagent, and 3 μl cDNA. One of each pair was supplemented with
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4 ml primer mix A, and the other with 4 ml primer mix B. Thermal cycling conditions
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were as follows: 15 min at 95°C, followed by 30 cycles of 95°C for 30 sec, 60°C for 90
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sec, and 72°C for 90 sec, followed by a single incubation of 10 min at 72°C.
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Amplification products were detected using capillary electrophoresis technology
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(Lab901 Screen Tape System; Lab901 Ltd, Loanhead, UK).
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RV16 testing 6
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cDNA synthesis was performed with cDNA Synthesis Premix (Seegene, Korea) from
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extracted RNAs. Respiratory virus detection kits-A and B (AnyplexTM II RV16
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detection; Seegene, South Korea) were used, according to the manufacturer’s
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instructions. Briefly, the assay was conducted in a final volume of 20 μl containing 8 μl
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cDNA, 5 μl 4×RV primer, and 5 μl 4× master mix with the CFX96 real-time PCR
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detection system (Bio-Rad, Hercules, CA, USA) under the following conditions: 4 min
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at 50°C and 15 min at 95°C, followed by 50 cycles of 95°C for 30 sec, 60°C for 1 min,
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and 72°C for 30 sec. After the reaction, Catcher Melting Temperature Analysis (CMTA)
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was performed by cooling the reaction mixture to 55°C, maintaining the mixture at
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55°C for 30 s, and heating the mixture from 55°C to 85°C. The fluorescence was
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measured continuously during the temperature increase. The melting peaks were
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derived from the initial fluorescence (F) versus temperature (T) curves.
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Comparison of the RV12 and RV16 assays
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Specimens that showed a discrepancy between RV12 and RV16 assay were further
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verified using monoplex PCR and sequencing in a blind manner. The primers for
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monoplex PCR in the single or nested PCR format were identical to the primers of the 7
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RV12 or RV16 assay. The PCR products were purified with a power gel extraction kit
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(TaKaRa Bio Inc., Shiga, Japan). Purified templates were sequenced with a BigDye
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Terminator v3.1 cycle sequencing kit (Life Technologies, Foster City, CA, USA) and
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analyzed on an ABI 3730xl DNA analyzer (Life Technologies). Since RV12 cannot
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detect BoV, HEV, ADV F, PIV4, or HRV C, we excluded those results from
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comparison of the two assays.
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Analytical sensitivity and specificity of the RV16 assay
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Serially diluted plasmids containing the target gene were used for determination of
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analytical sensitivity. pUC19 vector was used for plasmid DNA preparation. Serial
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dilutions of the prepared plasmid DNA were made from 10 6 to 100 copies per reaction
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to determine the analytical sensitivity of the assay. MPV, BoV, and CoV-NL63 samples
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were isolated from patients, and their sequences were confirmed by direct sequencing.
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All other standard strains were obtained from American Type Culture Collection
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(ATCC). Ten replicates of each dilution step were performed. The lower detection limit
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was defined as the lowest concentration detected by 10 replicas of each assay.
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The cross-reactivity of the RV16 assay was assessed using ten different bacteria. 8
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Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus epidermidis,
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Moraxella catarrhalis, Pseudomonas aeruginosa, Escherichia coli, Klebsiella
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pneumoniae, Staphylococcus aureus, Neisseria meningitides, and Haemophilus
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influenzae were obtained from the American Type Culture Collection (ATCC,
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Manassas, VA). The DNA of supplied samples was extracted and assayed with the
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RV16 assay adhering to the same procedures used for sample processing.
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Statistical analysis
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Statistical analyses were performed using SPSS software, version 20.0 (SPSS Inc.,
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Chicago, IL) and the VassarStats website (http://vassarstats.net/). We used inter-rater
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agreement statistics (Kappa calculation) to compare the detection of respiratory viruses
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between the RV12 and RV16 assays. P-values less than 0.05 were considered
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statistically significant.
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Results
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Comparison of the RV16 assay with the RV12 assay 9
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A total of 140 (38.4%) and 89 (24.4%) samples were RV16- and RV12-positive,
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respectively. Among viruses tested using both methods, the positive percent agreement
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between the RV16 and the RV12 assays was 95.5% (95% CI, 88.3 to 98.5), and the
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negative percent agreement was 80.0% (95% CI, 74.8 to 84.2). The kappa value for the
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two methods was 0.64 (95% CI, 0.55 to 0.71). Results by virus are presented in Table 1.
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Sixty-five samples that were identified as positive samples by the RV16 assay
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were identified as negative by the RV12 assay: Eight samples had more than one
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discrepant viruses, and 71 discrepancies by each virus were detected. On the other hand,
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four clinical samples that were negative in the RV16 assay were identified as positive
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by the RV12 assay. For further analysis of the samples that yielded discrepant results
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with the RV16 assay and the RV12 assay, monoplex real-time RT-PCR and gene
167
sequencing were performed. In the discrepant RV16-positive and the RV12-negative
168
results, the results of monoplex PCR and sequencing revealed that 81.7% (58/71) were
169
concordant with the result by the RV16 assay. On the other hand, in discrepant results
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exhibited positivity only in the RV12 assay, none was concordant with the result by the
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RV12 assay. The overall results of monoplex PCR and sequencing revealed that
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majority of the samples that were identified as positive from RV16 assays also exhibited
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positive results, except INF A (Table 2).
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Analytical sensitivity and specificity of the RV16 assay
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The detection limits of the RV16 detection kit for all 16 respiratory viruses were
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approximately 6 copies/μL (50 copies/reaction).
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To evaluate the cross-reactivity and detection specificity, ten different bacterial
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reference strains were tested using the same assay procedure used for the clinical
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samples for RV detection with the RV16 assay. All assay results were negative, and no
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non-specific positive reaction was observed.
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Discussion
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We compared the performance of two multiplex PCR kits, RV12 and RV16, for
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detection of respiratory viruses using clinical respiratory samples. We performed
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monoplex PCR and direct sequencing in a blind manner for the samples that yielded
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discrepant results with the two assays.
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In the present study, 38.4% and 24.4% of samples were RV16- and RV12-
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positive, respectively. The volume of sample added to the RV16 assay was almost 3 11
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times more than the volume added to the RV12 assay and this could have contributed to
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the higher number of positive samples by RV16 compared to RV12 assay.
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Majority of the samples that were identified as positive from RV16 assays were
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confirmed as positive by monoplex real-time RT-PCR and gene sequencing. However,
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in the discrepant RV16-positive and the RV12-negative results for INF A, monoplex
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PCR and sequencing revealed that 50% were negative. This suggests that the false
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positivity for INF A in RV16 assay by nucleic acid contamination or cross-reactivity
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from spurious primer interactions. However, the possibility that the RV16 may detect
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INF A with superior sensitivity compared to the monoplex PCR and sequencing cannot
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be excluded. Recent studies reported on the performance of RV16, which showed
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increased sensitivities compared to the Seeplex® RV15 ACE detection kit (RV15;
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Seegene), although we did not determine the diagnostic accuracy against a reference
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method (Cho et al., 2013; Kim et al., 2013).
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The RV12 system had a limitation regarding its internal control facility. The
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artificial targets included in each PCR mastermix only allow validation of the PCR step,
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without control for the RNA extraction or reverse transcription steps (Bibby et al.,
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2011). As for RV16, the addition of bacteriophage MS2 to each extraction allows the 12
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RT-PCR system to monitor both the RNA extraction and the reverse transcription steps
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(Cho et al., 2013; Dreier et al., 2005). Nevertheless, the internal control in RV16 cannot
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give information for specimen quality or prevent misjudgment from sampling error,
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since it is an exogenous control.
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RV16 has an advantage in workload compare to RV12. Given that the RV12
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assay requires agarose gel detection after PCR, RV16 requires less time and labor than
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RV12 (less than seven hours with reduced hands-on time) (Bibby et al., 2011; Kim et al.,
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2013). In addition, RV16 is a closed PCR system with a reduced chance of amplicon
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contamination.
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The present study has some limitations. First, we could not compare a large
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number of some respiratory viruses between assays due to the low number of virus-
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positive specimens and respiratory virus seasonality, since this was consecutive
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prospective study performed over two months. Second, we did not compare RV16 and
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RV12 with viral cultures or direct fluorescent-antibody assays. Third, we used the same
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primers for RV12 and RV16 in the monoplex PCR and sequencing for discrepant
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analysis. However, we performed monoplex PCR and direct sequencing in a blind
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manner to overcome the limitation of the study design. Fourth, since the analytical
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sensitivities were determined using plasmid DNA, the reverse transcription step could
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not be included in the estimate.
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In conclusion, the RV16 assay produces results comparable to the RV12 assay.
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Further analysis of the samples that yielded discrepant results demonstrated that the
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RV16 assay exhibited a higher concordance with the results of monoplex PCR and
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sequencing, compared to the RV12 assay.
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Acknowledgements
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This study was supported by Seegene Korea. The sponsor had no involvement in the
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study design, data interpretation, or preparation of the manuscript.
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References
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Bibby DF, McElarney I, Breuer J, Clark DA. Comparative evaluation of the Seegene
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Seeplex RV15 and real-time PCR for respiratory virus detection. J Med Virol
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Cho CH, Chulten B, Lee CK, Nam MH, Yoon SY, Lim CS, et al. Evaluation of a novel
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real-time RT-PCR using TOCE technology compared with culture and Seeplex
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RV15 for simultaneous detection of respiratory viruses. J Clin Virol
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Drews SJ, Blair J, Lombos E, DeLima C, Burton L, Mazzulli T, et al. Use of the
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Seeplex RV Detection kit for surveillance of respiratory viral outbreaks in
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Table 1. Detection of respiratory viruses by AnyplexTM II RV16 and Seeplex® RV12 ACE assays.
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95% CI
95.5
88.3 – 98.5
80.0
74.8 – 84.2
0.64
0.55 – 0.71
ADV
100
39.6 – 100
95.6
92.8 – 97.4
0.32
0.08 – 0.56
INF A
95.6
76.0 – 99.8
97.1
94.5 – 98.5
0.78
0.66 – 0.91
INF B
NA
NA
100
98.7 – 100
NA
NA
PIV 1
0
0 – 94.5
100
98.7 – 100
0
0–0
PIV 2
100
54.6 - 100
100
98.7 – 100
1.00
1.00 – 1.00
PIV 3
NA
NA
100
98.7 – 100
NA
NA
HRV
90.0
59.6 – 98.2
93.6
93.4 – 97.8
0.53
RSV (A or B)
97.1
83.4 – 99.8
98.2
95.9 – 99.3
0.90
0.82 – 0.97
HMPV
100
46.3 – 100
98.3
96.2 – 99.3
0.62
0.34 – 0.90
CoV
100
69.9 – 100
94.6
91.6 – 96.6
0.54
0.36 – 0.72
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Total
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%
EP
95% CI
D
SC
Observed
%
AC C
Virus
Negative
TE
Positive
Kappa value
RI
Agreement
kappa
95% CI
283
ADV, adenovirus; INF A, influenza A virus; INF B, influenza B virus; PIV 1- 3, parainfluenza virus type 1 to 3; HRV, human rhinovirus; RSV, respiratory
284
syncytial virus; HMPV, human metapneumovirus; CoV, coronavirus.
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Table 2. Analysis for positive results in AnyplexTM II RV16 and Seeplex® RV12 ACE assays. Virus
RV16+/RV12+
RV16+/RV12No. of positive results in
RI
No. of samples
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RV16-/RV12+ No. of samples
SC
monoplex PCR and sequencing
5
1
0
0
1
0
0
0
0
13
1
0
4
1
ND*
6
0
0
17
0
0
13
INF A
22
10
PIV 1
0
0
PIV 2
1
0
HRV
9
14
RSV (A or B)
34
6
HMPV
5
6
CoV
12
19
NU
16
MA
0
4
D
monoplex PCR and sequencing 0
ADV
EP
TE
No. of positive results in
ADV, adenovirus; INF A, influenza A virus; PIV 1, parainfluenza virus type 1; HRV, human rhinovirus; RSV, respiratory syncytial virus; HMPV, human
288
metapneumovirus; CoV, coronavirus.
289
* ND, not performed due to lack of sample
290
AC C
287
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