Coronaviruses are enveloped positive-str and RNA viruses with 27-33 kb genomes. These viruses are classified into four genera,namely Alphacoronavirus,Betacor-onavirus,Gammacoronavirus, and Deltacoronavirus(Adams and Carstens, 2012). The Middle East respiratory syndrome coronavirus(MERS-CoV),which was first and only recently identified in the Middle East,belongs to the genus Betacoronavirus(Zaki et al., 2012). The human coronaviruses HCoV-NL63,HCoV-229E,SARS-CoV,HCoV-OC43,MERS-CoV, and HCoV-HKU1 are associated with high-morbidity respiratory distress,including acute respiratory tract infection,pneumonia, and bronchiolitis(Gaunt et al., 2010; Zaki et al., 2012; Lu et al., 2014). Of these,HCoV-NL63,HCoV-229E,HCoV-OC43, and HCoV-HKU1 are frequently isolated from patients, and are globally distributed,although prevalence varies with time and geographical region(Geng and Tan, 2013). On the other h and ,an outbreak of SARS between 2002 and 2003 afflicted approximately 8,000 people,with 774 deaths(Stadler et al., 2003).
Timely diagnosis is critical in managing coronavirus infections, and in tracing possible sources. Many early diagnostic technologies relied on cumbersome and insensitive methods such as serology,virus cultures, and antigen detection. Molecular diagnostic tests have since confirmed that coronaviruses are causative agents of respiratory distress, and have allowed identification to species. Today,reverse-transcription polymerase chain reaction(RT-PCR),real-time PCR with melt curve analysis, and probe-based real-time RT-PCR are routinely used to detect human coronaviruses in nasopharyngeal swabs(Theamboonlers et al., 2007; Gaunt et al., 2010). However,these techniques have limited sensitivity or are low throughput,precluding rapid screening of large numbers of samples.
In this study,we developed a two-tube multiplex real-time RT-PCR assay for sensitive and specific detection of all known human coronaviruses. Total nucleic acids were extracted from 100 μL samples using QIAamp Viral RNA Mini Kit. Primer sets(Table 1)quoted from the references were modified based on the Nucleotide Collection Database(National Center for Biotechnology Information,Bethesda,MD,USA)for our study using Primer Premier 5.0. Different primer and probe combinations were evaluated in preliminary experiments. Based on these experiments,primers for NL63,229E, and SARS were grouped into one triplex reaction,while those for MERS,OC43, and HKU1 were grouped into another(Table 1). Viral targets were amplified on a CFX96 real-time PCR system(Bio-Rad,USA)using One Step RT-PCR Enzyme Mix(TaKaRa,Japan)in 25 μL reactions containing 12.5 µL 2 × PCR buffer,0.5 µL RT enzyme mix,0.5 µL Taq enzyme mix,2 µL template DNA,as well as primers and probes added from 10 × mixtures. Final concentrations are listed Table 1. Reactions consisted of 5 min reverse transcription at 42 °C,10 s denaturation at 95 °C, and 40 cycles at 95 °C for 10 s and 62 °C for 45 s. Human RNase P gene was amplified as internal control. Data were analyzed by univariate statistics and binary logistic regression. P values < 0.05 were considered statistically significant.
Virus (target) Target gene Primer Sequence ConcentrationA(nmol/L) Reference Multiplex PCR #1 HCoV-NL63 Nucleoprotein Forward AGGACCTTAAATTCAGACAACGTTCT 100 Theamboon-Alers et al., 2007 Reverse GATTACGTTTGCGATTACCAAGACT 50 Probe FAM-TAACAGTTTTAGCACCTTCCTTAAGCAACCCAAACA-TAMRA 25 HCoV-229E Nucleoprotein Forward CGCAAGAATTCAGAACCAGAG 50 Adapted from Theamboon-Alers et al., 2007 Reverse GGCAGTCAGGTTCTTCAACAA 75 Probe HEX-CCACACTTCAATCAAAAGCTCCCAAAATG-TAMRA 25 SARS-CoV Nucleoprotein Forward TGGACCCACAGATTCAACTGA 50 Adapted from Theamboon-Alers et al., 2007 Reverse GCTGTGAACCAAGACGCAGTAT 50 Probe CY5-TAACCAGAATGGAGGACGCAATGAG-BHQ2 25 Multiplex PCR #2 HCoV-OC43 Nucleoprotein Forward GCTCAGGAAGGTCTGCTCC 50 Theamboon-Alers et al., 2007 Reverse TCCTGCACTAGAGGCTCTGC 25 Probe FAM-TTCCAGATCTACTTCGCGCACATACC-TAMRA 25 MERS-CoV Nucleoprotein Forward GGCACTGAGGACCCACGTT 50 Adapted from Lu et al., 2014 Reverse TTGCGACATACCCATAAAAGCA 50 Probe CY5-CCCCAAATTGCTGAGCTTGCTCCATACA-BHQ2 25 HCoV-HKU1 Replicase 1b Forward CCTTGCGAATGAATGTGCT 50 Adapted from Dare et al., 2007 Reverse TTGCATCACCACTGCTAGTACCAC 375 Probe HEX-TGTGTGGCGGTTGCTATTATGTTAAAGCCTG-TAMRA 25
Table 1. Primers for two-tube multiplex real-time RT-PCR.
Preliminary experiments indicate that the two-tube multiplex assay was internally specific for each coronavi-rus. Importantly,cross-reactivity was not observed with influenza A virus,influenza B virus,parainfluenza virus 1-4,respiratory syncytial virus,metapneumovirus,adenovirus,bocavirus,rhinovirus,echovirus,mumps virus,measles virus, and Staphylococcus aureus.
Sensitivity was assessed using in vitro transcripts of all six coronaviruses,which were obtained using a T7 large-scale RNA production system(Promega,WI,USA). These transcripts were serially diluted 10-fold, and amplified in triplicate by two-tube multiplex RT-PCR and by previously established monoplex RT-PCR. We found that Ct values did not differ significantly between monoplex and multiplex reactions(data not shown). PCR products were cloned into pGEM-T Easy and confirmed by sequencing. Detection limits were determined using samples containing one virus,or all six viruses in equal proportion. St and ard curves were generated from samples containing one virus by plotting Ct values against the log of copies/μL(Supplementary Figure S1). The high sensitivity of the assay was confirmed using synthesized RNA st and ards at 10 copies/reaction.
To obtain additional performance data and explore possible applications,we tested the ability of two-tube multiplex RT-PCR to detect viral RNA in monkeys experimentally infected with known titers of MERS-CoV(Yao et al., 2014). The MERS-CoV strain hCoV-EMC was generously provided by Drs. Fouchier and Haagmans at Erasmus Medical Centre,The Netherl and s, and was propagated and titered in Vero cells. Swabs from infected monkeys were collected according to published methods(Yao et al., 2014)by Professor Qin Chuan at Institute of Laboratory Animal Sciences,Chinese Academy of Medical Sciences. Viral RNA was detected in nasal,throat, and anal swabs within two days of infection,after which point viral RNA was most abundant in throat swabs(Supplementary Table S1). Notably,multiplex PCR was able to distinguish virus from Vero cell cultures at different titers(Supplementary Figure S2).
In addition,we tested the performance of two-tube real-time RT-PCR against whole blood and pharyngeal swabs collected in 2015 in Guangdong Province,China,from a Korean patient with suspected MERS(Lu et al., 2015). The virus was detected in all specimens(Supplementary Table S1). Results were comparable to in-house monoplex RT-PCR reactions(Lu et al., 2014).
Moreover,we assessed the ability of two-tube multiplex RT-PCR to monitor HCoV-HKU1 propagation in human airway epithelial cells(Zhu et al., 2015). The HCoV-HKU1 stock was provided by the National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention. Copies of HCoV-HKU1 RNA increased with time,peaking at 96 h post-inoculation(Supplementary Table S2),in line with in-house monoplex reactions(Dare et al., 2007).
Finally,clinical performance was evaluated using 346 nasopharyngeal swabs obtained in 2014 from children under 14 years who were hospitalized with acute respiratory infection. This study was approved by the Institutional Review Boards of the Chinese Center for Disease Control and Prevention, and written informed consent was obtained from parents or guardians of all patients. As shown in Table 2,two-tube multiplex real-time RT-PCR detected viruses in 24(6.94%)samples,of which five(1.46%)were infected with NL63, and 10(2.89%)were infected with 229E. Six samples(1.73%)tested positive for OC43, and HKU1 was detected in three(0.87%)samples. There were no differences between two-tube multiplex real-time RT-PCR and a previously established,one-tube multiplex RT-PCR assay with inline electrophoresis(QIAxcel,Qiagen)(Niu et al., 2014). In addition,co-infection was detected by both assays in three patients(0.87%),of whom one was co-infected with NL63 and 229E,while the other two were co-infected with OC43 and 229E(Table 2). Infection was confirmed(data not shown)by in-house monoplex real-time PCR(Lu et al., 2012). Notably,one case of OC43 and two cases of HKU1 were detected by two-tube multiplex RT-PCR,but not by one-tube multiplex RT-PCR with inline electrophoresis. Nested RT-PCR and gene sequencing confirmed results from two-tube multiplex RT-PCR(data not shown),highlighting its potentially higher sensitivity for these viruses.
Target Two-tube multiplex real-time RT-PCR One-tube multiplex real-time RT-PCR Awith inline electrophoresis Range of Ct values Positive samples (%) Positive samples (%) HCoV-NL63 25-33.5 5 (1.46) 5 (1.46) HCoV-229E 26-34 10 (2.89) 10 (2.89) SARS-CoV 0 0 HCoV-OC43 26-35 6 (1.73) 5 (1.46) MERS-CoV 0 0 HCoV-HKU1 28-34.5 3 (0.87) 1 (0.29) Co-infectiona 3 (0.87) 3 (0.87) Total 25-35 24 (6.94) 21 (6.1) Note: a HCoV-NL63 and HCoV-229E (n = 1), HCoV-OC43 and HCoV-229E (n = 2).
Table 2. Detection of coronavirus in 346 clinical samples.
Real-time RT-PCR is an established,rapid and effective method to detect multiple viral pathogens of the respiratory tract(Dare et al., 2007; Gaunt et al., 2010; Lu et al., 2012). We have developed a sensitive and specific real-time RT-PCR assay to detect all six human coronav-iruses. Its ability to monitor HKU1 replication in cultures of human airway epithelial cells,to quantitatively measure viral RNA in monkeys experimentally infected with MERS, and to detect MERS in a human patient was demonstrated . In addition,the assay was used to assess disease burden and epidemiology of coronaviruses among hospitalized patients with acute respiratory infection, and able to detect co-infection. Finally,the assay requires significantly less sample than monoplex real-time RT-PCR. Thus,the assay will be widely used in coronav-irus research.
The authors thank Dr. Bart Haagmans and Ron Fouchier at Erasmus Medical Center,Rotterdam,the Netherl and s for providing MERS-CoV isolate hCoV-EMC/2012. We also thank Dr. Qin Chuan at Institute of Laboratory Animal Sciences,Chinese Academy of Medical Sciences for providing swabs from rhesus monkeys challenged with MERS-CoV. This work was supported by grants from the State Megaproject for Infectious Disease Research of China(2014ZX10004001-002,2013ZX10004101,2013ZX10004805-002). The funding agency did not participate in study design,data collection and analysis,decision to publish,or preparation of the manuscript. The authors declare that they have no conflict of interest. All the animal tests comply with Chinese Center for Disease Control and Prevention laboratory animal management approach and the requirement of animal welfare. Written informed consent was obtained from parents or guardians of all patients.
Supplementary figures/tables are available on the website of Virologica Sinica: www.virosin.org;link.springer.com/journal/12250.
Figure S1. Two-tube multiplex real-time RT-PCR to simultaneously detect six human coronaviruses. (A-C) Amplification plots for 101 -107 copies/μL each of NL63 (A), 229E (B), and SARS (C), which were amplified simultaneously in one tube. (D) Standard curves generated from (A-C) by plotting Ct values against the log of copies/μL. (E-G) Amplification plots for 101 -107 copies/μL of OC43 (E), MERS (F), and HKU1 (G), which were amplified at the same time in a single reaction. (H) Standard curves generated from (E-G) by plotting Ct values against the log of copies/μL.
Figure S2. Multiplex real-time RT-PCR of specimens from vero cell cultures with 2,000 pfu (a), 200 pfu (b), 20 pfu (c), and 2 pfu (d) MERS-CoV.
Samples Ct values Monoplex real-time RT-PCRa Two-tube multiplex real-time RT-PCR Rhesus monkeys challenged with MERS-CoV Nasal swab 1 day post infection 33.86 34.00 Nasal swab 2 days post infection 32.46 32.50 Nasal swab 1 day pre infection Throat swab 2 days post infection 30.13 30.00 Throat swab 1 day pre infection Anal swab 1 day post infection 34.05 34.20 Anal swab 2 days pre infection Human patient with suspected MERS Pharyngeal swab 1 31.21 Pharyngeal swab 2 31.27 Pharyngeal swab 3 30.76 Whole blood 36.50 Note: a In-house monoplex real-time PCR for MERS-CoV N genes. The linear relationship can be described by y = –2.78x + 34.2.
Table S1. Two-tube multiplex real-time RT-PCR of clinical specimens.
Sample(Culture hours) Ct values Monoplex real-time RT-PCRa Two-tube multiplex real-time RT-PCR 0 31.74 30.91 2 27.22 27.14 24 25.59 25.78 48 25.03 25.32 96 23.86 24.34 120 25 25.29 144 25.02 25.31 168 25.56 25.76 Uninfected cells 2 × TCID50 of HCoV-HKU1 25 25.29 Note: a In-house monoplex real-time PCR for HCoV-HKU1 replicase 1b. The linear relationship can be described by y = –3.2x + 33.4.
Table S2. Detection of HCoV-HKU1 in the supernatant of infected cultures of human airway epithelial cells.