Real-Time Reverse Transcription–Polymerase Chain Reaction Assay for SARS-associated Coronavirus

A real-time reverse transcription–polymerase chain reaction (RT-PCR) assay was developed to rapidly detect the severe acute respiratory syndrome–associated coronavirus (SARS-CoV). The assay, based on multiple primer and probe sets located in different regions of the SARS-CoV genome, could discriminate SARS-CoV from other human and animal coronaviruses with a potential detection limit of <10 genomic copies per reaction. The real-time RT-PCR assay was more sensitive than a conventional RT-PCR assay or culture isolation and proved suitable to detect SARS-CoV in clinical specimens. Application of this assay will aid in diagnosing SARS-CoV infection.

I n late 2002, a life-threatening febrile respiratory illness appeared in Guangdong Province, China, and quickly spread throughout Asia and to other parts of the world (1)(2)(3)(4). Designated "severe acute respiratory syndrome" (SARS), the etiologic agent was later identified as a hitherto unrecognized coronavirus (SARS-CoV) (5,6). A diagnosis of SARS is based primarily on clinical and epidemiologic criteria, but many respiratory viruses can cause similar symptoms, and therefore rapid, reliable diagnostic tests for SARS-CoV infection were needed. In response to this need, three types of diagnostic tests for SARS-CoV were quickly developed: tissue culture isolation, antibody detection, and reverse transcription-polymerase chain reaction (RT-PCR) assays.
A variety of RT-PCR assays were developed during the epidemic for SARS-CoV (1,(5)(6)(7)(8), including a commercial ready-to-use RT-PCR kit (Artus Biotech, Hamburg, Germany). Early RT-PCR assays based on conventional designs required postamplification product processing (e.g., gel electrophoresis), were time-consuming, and were prone to false-positive results from amplicon contamination. Conversely, real-time RT-PCR assays based on detecting and quantifying a fluorescent signal generated during amplification do not require postamplification processing and therefore eliminate one potential avenue for template contamination.
A variant of the real-time format, based on TaqMan probe hydrolysis technology (Applied Biosystems, Foster City, CA), has been shown to provide sensitive, specific, and quantifiable results in viral diagnostic assays (9) and has been used successfully to study emerging virus infections (10,11), including SARS (6,12). In response to the SARS public health emergency, we developed and evaluated a TaqMan real-time RT-PCR assay based on three distinct targets in the SARS-CoV genome for rapid deployment to the National Laboratory Response Network for Bioterrorism (LRN) (http://www.cdc.gov/programs/bio.htm).

Clinical Specimens
A total of 340 clinical specimens collected from 246 persons with confirmed or suspected SARS-CoV infection (13) were used in this study. Specimens included oroand nasopharyngeal swabs (dry and in viral transport media), sputa, nasal aspirates and washes, bronchoalveolar lavage, and lung tissue specimens collected at autopsy. Specimen processing was performed in a class II biological safety cabinet using biosafety level three (BSL3) work practices. Three 100-µL aliquots of each specimen were distributed to vials each containing 900 µL of NucliSens lysis buffer (bioMérieux, Durham, NC) and stored at -70°C until testing.

Real-Time Reverse Transcription-Polymerase Chain
Reaction Assay for SARS-associated Coronavirus

Virus Culture
Vero E6 cells were inoculated with clinical specimens and observed for cytopathic effect, consisting of cell rounding with a refractive appearance followed by detachment from the flask surface (5). Plaque titrations were conducted by standard methods (14).

Nucleic Acid Extraction
Nucleic acids were recovered from clinical specimens using the automated NucliSens extraction system (bioMérieux). Following manufacturer's instructions, specimens received in NucliSens lysis buffer were incubated at 37°C for 30 min with intermittent mixing, and 50 µL of silica suspension, provided in the extraction kit, was added and mixed. The contents of the tube were then transferred to a nucleic acid extraction cartridge and processed on an extractor workstation. Approximately 40-50 µL of total nucleic acid eluate was recovered into nuclease-free vials and either tested immediately or stored at -70°C.

Primers and Probes
Multiple primer and probe sets were designed from the Urbani strain of SARS-CoV polymerase 1b and nucleocapsid gene sequences (15) by using Primer Express software version 1.5 or 2.0.0 (Applied Biosystems) with the following default settings: primer melting temperature (T M ) set at 60°C; probe T M set at 10°C greater than the primers at approximately 70°C; and no guanidine residues permitted at the 5′ probe termini. All primers and probes were synthesized by standard phosphoramidite chemistry techniques at the Biotechnology Core Facility at the Centers for Disease Control and Prevention (CDC).
TaqMan probes were labeled at the 5′-end with the reporter molecule 6-carboxy-fluorescein (FAM) and at the 3′-end with the quencher Blackhole Quencher 1 (Biosearch Technologies, Inc., Novato, CA). Optimal primer and probe concentrations were determined by cross-titration of serial twofold dilutions of each primer against a constant amount of purified SARS-CoV RNA. Primer and probe concentrations that gave the highest amplification efficiencies in this study were selected for further study (Table 1).

Real-Time RT-PCR Assay
The real-time RT-PCR assay was performed by using the Real-Time One-Step RT-PCR Master Mix (Applied Biosystems). Each 25-µL reaction mixture contained 12.5 µL of 2X Master Mix, 0.625 µL of the 40X MultiScribe and RNase Inhibitor mix, 0.25 µL of 10 µM probe, 0.25 µL each of 50 µM forward and reverse primers, 6.125 µL of nuclease-free water, and 5 µL of nucleic acid extract. Amplification was carried out in 96well plates on an iCycler iQ Real-Time Detection System (Bio-Rad, Hercules, CA). Thermocycling conditions consisted of 30 min at 48°C for reverse transcription, 10 min at 95°C for activation of the AmpliTaq Gold DNA polymerase, and 45 cycles of 15 s at 95°C and 1 min at 60°C. Each run included one SARS-CoV genomic template control and at least two no-template controls for the extraction (to check for contamination during sample processing) and one no-template control for the PCR-amplification step. As a control for PCR inhibitors, and to monitor nucleic acid extraction efficiency, each sample was tested by real-time RT-PCR for the presence of the human ribonuclease (RNase) P gene (GenBank accesssion number NM_ 006413) by using the following primers and probe: forward primer 5′-AGATTTGGACCTGCGAGCG-3′; reverse primer 5′-GAGCGGCTGTCTCCACAAGT-3′; probe 5′-TTCTGACCTGAAGGCTCTGCGCG-3′. The assay reaction was performed identically to that described above except that primer concentrations used were 30 µM each. Fluorescence measurements were taken and the threshold cycle (C T ) value for each sample was calculated by determining the point at which fluorescence exceeded a threshold limit set at the mean plus 10 standard deviations above the baseline. A test result was considered positive if two or more of the SARS genomic targets showed positive results (C T <45 cycles) and all positive and negative control reactions gave expected values. Clinical specimens submitted to CDC for SARS-CoV testing that gave positive results were confirmed with a TaqMan real-time RT-PCR assay based on three different primer and probe sets (Table 1). This assay was performed independently in a separate laboratory using newly extracted nucleic acid from a second specimen aliquot. The confirmatory assay used the SuperScript One-Step RT-PCR (Invitrogen Corp., Carlsbad, CA) and the Mx4000 Multiplex Quantitative PCR system (Stratagene, La Jolla, CA).

Synthesis of RNA Transcripts
Template for the nucleocapsid gene RNA was plasmid DNA (pCRII, Invitrogen Corp.) containing a full-length copy of the open reading frame for the SARS-CoV nucleocapsid gene oriented behind a T7 promoter. The plasmid was linearized by digestion with SpeI. The template for the polymerase RNA was a RT-PCR product generated by using the following primers: Cor-p-F2-T7, 5′-GTAATA CGACTCACTATAGGGCTAACATGCTTAGGATAA TGG-3′ and Cor-p-R2, 5′-CCTATTTCTATAGAGACA CTC-3′. Approximately 1 µg of RNA from Vero cells infected with SARS-CoV was used in RT-PCR reactions performed by using the SuperScript RT-PCR kit (Invitrogen Corp.) according to the manufacturer's instructions; both templates were purified by phenol-chloroform extraction and ethanol precipitation before being used for in vitro transcription. RNA was synthesized in vitro by using the MegaScript kit (Ambion Inc., Austin, TX) according to the standard protocol. Synthetic RNA was treated with RNase-free DNase before being purified by phenol-chloroform extraction and ethanol precipitation. The concentration of RNA was determined by use of UV spectroscopy. Synthetic RNA was positive sense and 1,369 nt in length for N and 325 nt in length for polymerase.

Real-Time RT-PCR Sensitivity and Reproducibility
Tenfold serial dilutions of the polymerase and nucleo-capsid RNA transcripts were tested to assess the copy detection limits and dynamic range of our optimized realtime RT-PCR assays. The lower potential limit of detection was approximately 2 transcript copies per reaction for SARS2 and SARS3, and 7.5 copies per reaction for SARS1 (Figure). The confirmatory assays, which employ three different primer and probe sets (N3, 3′NTR, and M), showed potential limits of detection similar to the SARS2 and SARS3 assays. Strong linear correlations (r 2 >0.99) were obtained between C T values and transcript quantity over at least a 6-log range from approximately 10 2 to 10 7 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10 copies per reaction for the three primer/probe sets. Linearity was markedly reduced for copy numbers exceeding 10 6 (data not shown). Assay reproducibility was tested by using replicate 10fold serial dilutions of the RNA transcripts and intra-and interassay variability evaluated for each dilution point in triplicate on three different days. At the lower copy detection limit for SARS2 and SARS3 (2 copies per reaction), assay reproducibility exceeded 90%. In contrast, the lower copy detection limit for SARS1 (7.5 copies per reaction) was positive in <50% of replicate reactions. One hundred percent reproducibility with SARS1 was achieved at the dilution that contained 75 transcript copies per reaction. Over the linear range of the assay, the coefficient of variation of the mean C T values within and between runs was 0.46%-2.54% and 0.64%-2.39%, respectively ( Table 2).
To assess the efficiency of amplification of the RNA transcripts in the presence of exogenous nucleic acid and potential RT-PCR inhibitors, 10-fold serial dilutions of the RNA transcripts were prepared in water and pooled total nucleic acid extract from 20 SARS-CoV-negative human respiratory specimens (nasopharyngeal aspirates, bronchial washes, sputum, naso-and oropharyngeal swabs, and lung tissue). Exogenous nucleic acid had no discernible effect on amplification efficiency of the SARS1 and SARS3 primer/probe sets, as demonstrated by the similarity in linear regression slopes and endpoint detection limits in the presence and absence of specimen extract (Table 3). In contrast, the standard curve for SARS2 had a more efficient slope (-3.21) in water than in the presence of spiked extract (-3.48) and with greater variation in the C T values at 20 target copies or lower, suggesting that the amplification reaction was less efficient in the presence of the specimen extract. This observation was confirmed on two additional repetitions of the same experiment.
The real-time RT-PCR assay was compared with a previously described conventional RT-PCR for SARS-CoV by using fluorescent dye-labeled primers and GeneScan amplicon analysis (5). Tenfold serial dilutions of a pretitrated SARS-CoV stock adjusted to 1 x 10 7 PFUs/mL were prepared in triplicate and tested by all assays ( Table  4). The real-time RT-PCR assays were positive with 100% frequency at a 10 -8 dilution. Accordingly, the lowest virus quantity detected was 0.01 PFU/100 µL of specimen extract. The conventional RT-PCR assay was at least 10fold less sensitive in repeat comparisons.

Specificity
We compared our primer and probe sets with sequences for 14 SARS-CoV field isolates that became available during the course of this study (16)  as human and nonhuman primate cell lines were tested. No positive reactions were obtained with any of the primer and probe sets.

Evaluation with Clinical Specimens
The real-time RT-PCR assay was used to test 14 clinical specimens (including throat swab [2 specimens], sputum [1 specimen], throat wash [5 specimens], and lung autopsy tissues [6 specimens]) from 10 patients with laboratory confirmed SARS-CoV infection (Table 5). Assay results were positive with all specimens for all three primer/probe sets. An additional, 326 respiratory specimens collected during the course of the outbreak from 236 suspected U.S. SARS patients who were serologically negative for SARS-CoV infection were also negative by the real-time RT-PCR.

Discussion
In response to the SARS outbreak, we developed a realtime RT-PCR assay based on multiple primer and probe sets designed to different genomic targets to facilitate sensitive and specific detection of SARS-CoV in clinical specimens. A potential detection limit of <10 transcript copies per reaction was achieved with greater relative sen-sitivity than cell culture isolation or conventional RT-PCR. The potential for quantitation over a wide dynamic range (at least 6 logs) was demonstrated with low intra-and interassay variability and limited inhibition from exogenous nucleic acid extract from respiratory secretions. The increased sensitivity of the real-time RT-PCR assay over cell culture and conventional RT-PCR methods may aid detection of the virus at earlier stages of infection, when the virus is present at low titer in respiratory secretions (8). In addition, by eliminating the need for postamplification product processing, the real-time RT-PCR format permitted shortened turnaround time for reporting results, which proved critical during the SARS outbreak.
Although real-time RT-PCR offers clear advantages over more conventional RT-PCR formats, assay results must still be interpreted with caution. For example, the effectiveness of RT-PCR for detection of SARS-CoV in clinical specimens has been shown to be greatly influenced by the quantity, type, and timing of specimen collection (8,17). False-negative results due to poor quality nucleic acid or presence of RT-PCR inhibitors can also be a concern. We addressed this by simultaneously testing for the human RNase P gene, which should be present in all adequately collected samples. False-negative results could also potentially arise from mutations occurring in the primer and probe target regions in the SARS-CoV genome. We addressed this by including multiple genetic targets in our assay and by carefully comparing our primer and probe sequences against published sequences of SARS-CoV as they became available. To avoid falsepositive results, meticulous care was taken to prevent introduction of contaminating viral RNA or previously amplified DNA during preparation of the nucleic acid extracts and amplification reactions. In addition, all RT-PCR-positive specimens were retested from a second, unopened sample aliquot and confirmed in a second laboratory by using a real-time assay based on different genetic targets.  Slope determined from the formula: Y = Y intercept -slope log10. Slopes calculated for SARS1 (7.5 x 10 6 to 7.5 x 10 1 ); SARS2 (2 x 10 6 to 2 x 10 1 ); SARS3 (2 x 10 6 to 2 x 10 0 ).  In conclusion, our real-time RT-PCR assay permitted rapid, sensitive, and specific detection of SARS-CoV in clinical specimens and provided needed diagnostic support during the recent SARS outbreak. Widely deploying this assay through the LRN will enhance our ability to provide a rapid response in the event of the possible return of SARS. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10