Real-time reverse transcription-polymerase chain reaction assay for SARS-associated coronavirus.A real-time reverse transcription-polymerase chain reaction (RT-PCR RT-PCR
reverse transcriptase-polymerase chain reaction. See PCR1. ) assay was developed to rapidly detect the severe acute respiratory syndrome-associated coronavirus coronavirus /co·ro·na·vi·rus/ (ko-ro´nah-vi?rus) any virus belonging to the family Coronaviridae.
Coronavirus /Co·ro·na·vi·rus/ (ko-ro´nah-vi?rus (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.
In late 2002, a life-threatening febrile febrile /feb·rile/ (feb´ril) pertaining to or characterized by fever.
Of, relating to, or characterized by fever; feverish. respiratory illness Noun 1. respiratory illness - a disease affecting the respiratory system
respiratory disease, respiratory disorder
adult respiratory distress syndrome, ARDS, wet lung, white lung - acute lung injury characterized by coughing and rales; inflammation of the appeared in Guangdong Province Noun 1. Guangdong province - a province in southern China
Guangdong, Kwangtung , China, and quickly spread throughout Asia and to other parts of the world (1-4). Designated "severe acute respiratory syndrome Severe Acute Respiratory Syndrome (SARS) Definition
Severe acute respiratory syndrome (SARS) is the first emergent and highly transmissible viral disease to appear during the twenty-first century. " (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 (1,5-8), 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 gel electrophoresis
Electrophoresis performed in a gel composed of agarose, polyacrylamide, or starch. ), 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 hydrolysis (hīdrŏl`ĭsĭs), chemical reaction of a compound with water, usually resulting in the formation of one or more new compounds. technology (Applied Biosystems Applied Biosystems, Inc. (formerly NASDAQ: ABIO) is the original name of a pioneer biotechnology company founded in 1981 in Foster City, California, among the Silicon Valley cities of the southern San Francisco Bay Area. , 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 LRN Linux.ru.net (website)
LRN Laboratory Response Network
LRN Location Routing Number
LRN Local Routing Number
LRN Learning Resource iNterchange (Microsoft)
LRN Lead Round Nose ) (http://www.cdc.gov/programs/bio.htm).
Materials and Methods
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 oro- and nasopharyngeal nasopharyngeal
pertaining to the nasal and pharyngeal cavities.
see nasopharyngeal meatus.
see reverse sneeze. swabs (dry and in viral transport media), sputa, nasal aspirates and washes, bronchoalveolar lavage Bronchoalveolar lavage
A way of obtaining a sample of fluid from the airways by inserting a flexible tube through the windpipe. Used to diagnose the type of lung disease. , and lung tissue specimens collected at autopsy. Specimen processing was performed in a class II biological safety cabinet using biosafety level biosafety level Epidemiology A classification for the degree of caution required when working with specific groups of pathogens. See Maximum containment facility. three (BSL (language) BSL - A variant of IBM's PL/S systems language. Versions: BSL1, BSL2. 3) work practices. Three 100-[micro]L aliquots of each specimen were distributed to vials each containing 900 [micro]L of NucliSens lysis buffer A lysis buffer is used for the purpose of lysing cells for use in experiments that analyze the compounds of the cells (e.g. western blot). There are many different kind of lysis buffers that one can apply, depending on what analysis the cell lysate will be used for. (bioMerieux, Durham, NC) and stored at -70[degrees]C until testing.
Vero E6 cells were inoculated with clinical specimens and observed for cytopathic effect Cytopathic effect (CPE) refers to degenerative changes in cells (especially in tissue culture) associated with the multiplication of certain viruses. When in tissue culture, the spread of virus is restricted by an overlay of agar (or other suitable substance) and thus the , consisting of cell rounding with a refractive refractive
capacity to refract light.
a difference between the focal length of the cornea and lens, and the length of the eye, resulting in myopia or hyperopia. appearance followed by detachment from the flask surface (5). Plaque titrations were conducted by standard methods (14).
Nucleic Acid nucleic acid, any of a group of organic substances found in the chromosomes of living cells and viruses that play a central role in the storage and replication of hereditary information and in the expression of this information through protein synthesis. Extraction
Nucleic acids Nucleic acids
The cellular molecules DNA and RNA that act as coded instructions for the production of proteins and are copied for transmission of inherited traits. were recovered from clinical specimens using the automated NucliSens extraction system (bioMerieux). Following manufacturer's instructions, specimens received in NucliSens lysis buffer were incubated at 37[degrees]C for 30 min with intermittent mixing, and 50 [micro]L 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 [micro]L of total nucleic acid eluate eluate /el·u·ate/ (el´u-at) the substance separated out by, or the product of, elution or elutriation.
The solution of solvent and dissolved matter resulting from elution. was recovered into nuclease-free vials and either tested immediately or stored at -70[degrees]C.
Primers and Probes
Multiple primer and probe sets were designed from the Urbani strain of SARS-CoV polymerase 1b and nucleocapsid nucleocapsid /nu·cleo·cap·sid/ (noo?kle-o-kap´sid) a unit of viral structure, consisting of a capsid with the enclosed nucleic acid.
n. 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 Melting temperature may refer to:
tr.v. quenched, quench·ing, quench·es
1. To put out (a fire, for example); extinguish.
2. To suppress; squelch: 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 RNA: see nucleic acid.
in full ribonucleic acid
One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic . 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-[micro]L reaction mixture contained 12.5 [micro]L of 2X Master Mix, 0.625 [micro]L of the 40X MultiScribe and RNase Inhibitor mix, 0.25 [micro]L of 10 [micro]M probe, 0.25 [micro]L each of 50 [micro]M forward and reverse primers, 6.125 [micro]L of nuclease-free water, and 5 [micro]L of nucleic acid extract. Amplification was carried out in 96-well plates on an iCycler iQ Real-Time Detection System (Bio-Rad, Hercules, CA). Thermocycling conditions consisted of 30 min at 48[degrees]C for reverse transcription reverse transcription
The process by which DNA is synthesized from an RNA template. , 10 min at 95[degrees]C for activation of the AmpliTaq Gold DNA polymerase DNA polymerase /DNA po·lym·er·ase/ (pah-lim´er-as) any of various enzymes catalyzing the template-directed incorporation of deoxyribonucleotides into a DNA chain, particularly one using a DNA template. , and 45 cycles of 15 s at 95[degrees]C and 1 min at 60[degrees]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 PCR polymerase chain reaction.
polymerase chain reaction
Polymerase chain reaction (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 Ribonuclease
A group of enzymes, widely distributed in nature, which catalyze hydrolysis of the internucleotide phosphodiester bonds in ribonucleic acid (RNA). (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 [micro]M each. Fluorescence measurements were taken and the threshold cycle ([C.sub.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 positivo if two or more of the SARS genomic targets showed positive results ([C.sub.T] [less than or equal to] 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 aliquot (al-ee-kwoh) adj. a definite fractional share, usually applied when dividing and distributing a dead person's estate or trust assets. (See: share) . The confirmatory assay used the SuperScript Any letter, digit or symbol that appears above the line. For example, 10 to the 9th power is written with the 9 in superscript (109). Contrast with subscript. One-Step RT-PCR (Invitrogen Corp., Carlsbad, CA) and the Mx4000 Multiplex Quantitative PCR system (Stratagene, La Jolla La Jolla (lə hoi`yə), on the Pacific Ocean, S Calif., an uninc. district within the confines of San Diego; founded 1869. The beautiful ocean beaches, in particular La Jolla shores and Black's Beach, and sea-washed caves attract visitors and , CA).
Synthesis of RNA Transcripts
Template for the nucleocapsid gene RNA was plasmid DNA Noun 1. plasmid DNA - a small cellular inclusion consisting of a ring of DNA that is not in a chromosome but is capable of autonomous replication
plasmid (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 Spel. 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 [micro]g 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 in vitro /in vi·tro/ (in ve´tro) [L.] within a glass; observable in a test tube; in an artificial environment.
In an artificial environment outside a living organism. 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 nucleocapsid RNA transcripts were tested to assess the copy detection limits and dynamic range of our optimized real-time 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.sup.2] [greater than or equal to] 0.99) were obtained between [C.sub.T] values and transcript quantity over at least a 6-log range from approximately [10.sup.2] to [10.sup.7] copies per reaction for the three primer/probe sets. Linearity was markedly reduced for copy numbers exceeding [l0.sup.6] (data not shown).
Assay reproducibility was tested by using replicate 10-fold 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 Coefficient of Variation
A measure of investment risk that defines risk as the standard deviation per unit of expected return. of the mean [C.sub.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 bronchial /bron·chi·al/ (brong´ke-al) pertaining to or affecting one or more bronchi.
Relating to the bronchi, the bronchial tubes, or the bronchioles. washes, sputum sputum /spu·tum/ (spu´tum) [L.] expectoration; matter ejected from the trachea, bronchi, and lungs through the mouth.
sputum cruen´tum bloody sputum. , naso- and oropharyngeal oropharyngeal /oro·pha·ryn·ge·al/ (-fah-rin´je-al)
1. pertaining to the mouth and pharynx.
2. pertaining to the oropharynx. 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 Linear regression
A statistical technique for fitting a straight line to a set of data points. 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.sub.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 pre-titrated SARS-CoV stock adjusted to 1 x [10.sup.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.sup.-8] dilution. Accordingly, the lowest virus quantity detected was 0.01 PFU/100 [micro]L of specimen extract. The conventional RT-PCR assay was at least 10-fold less sensitive in repeat comparisons.
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) and found no nucleotide mismatches. In contrast, alignments with other published human and animal coronaviruses (GenBank accession no.: human coronaviruses X69721 and AF124989; bovine coronaviruses NC003045 and AF 124985; murine murine /mu·rine/ (mur´en) pertaining to, derived from, or characteristic of mice or rats.
adj. hepatitis viruses NC001846 and M55148; sialodacryoadenitis virus AF124990; canine coronavirus AF124986; feline infectious peritonitis Feline infectious peritonitis (FIP) is a fatal, incurable disease that affects cats. It is caused by Feline Infectious Peritonitis Virus (FIPV), which is a mutation of Feline Enteric Coronavirus (FECV/FeCoV). Experts do not always agree on the specifics of FIP. virus AF124987; porcine porcine /por·cine/ (por´sin) pertaining to swine.
pertaining to pig. See also hog (1), swine.
porcine circovirus 1
a nonpathogenic virus. hemagglutinating encephalomyelitis encephalomyelitis /en·ceph·a·lo·my·eli·tis/ (en-sef?ah-lo-mi?e-li´tis) inflammation of the brain and spinal cord.
acute disseminated encephalomyelitis virus AF124988, Z34093, and AF124992; turkey coronavirus AF124991; and avian infectious bronchitis virus Avian infectious bronchitis virus is a virus that infects poultry. It and its associated disease, avian infectious bronchitis, are usually abbreviated to IBV, which stands for "Infectious bronchitis virus". References
1. NC_001451) showed little sequence identity with our primer and probe sets. To further assess the potential for crossreactions with other members of the Coronaviridae family, the RT-PCR assays were tested against nucleic acid extracts of human respiratory coronaviruses OC43 (VR-759) and 229E (VR-740), feline infectious peritonitis virus (VR-3004), mouse hepatitis virus Mouse hepatitis virus is a virus of the family Coronaviridae, genus coronavirus. References
Capable of being conveyed from one person to another. gastroenteritis gastroenteritis: see enteritis.
Acute infectious syndrome of the stomach lining and intestines. Symptoms include diarrhea, vomiting, and abdominal cramps. virus (VR-743), and avian infectious bronchitis virus (VR-841), obtained from the American Type Culture Collection American Type Culture Collection (ATCC) is a private, not-for-profit biological resource center whose mission focuses on the acquisition, authentication, production, preservation, development and distribution of standard reference microorganisms, cell lines and other materials for (Manassas, VA), and human enteric enteric /en·ter·ic/ (en-ter´ik) within or pertaining to the small intestine.
1. Of, relating to, or within the intestine.
2. coronavirus (VR-1475). In addition, nucleic acid extracts of field isolates of influenza A influenza A
Influenza caused by infection with a strain of influenza virus type A.
influenza A Infectious disease An avian virus, especially of ducks–which in China live near the pig reservoir and 'vector'; and B; parainfluenza parainfluenza Infectious disease A virus that causes URIs–up to 50% of croup and 10–15% of bronchiolitis, bronchitis, pneumonias in toddlers Clinical Rhinorrhea, cold-like Sx Risk factors Preschool children; by school age most children have been exposed 1, 2, and 3; rhinovirus rhinovirus
Any of a group of picornaviruses capable of causing common colds in humans. The virus is thought to be transmitted to the upper respiratory tract by airborne droplets. ; adenovirus adenovirus
Any of a group of spheroidal viruses, made up of DNA wrapped in a protein coat, that cause sore throat and fever in humans, hepatitis in dogs, and several diseases in fowl, mice, cattle, pigs, and monkeys. ; human metapnuemovirus; and respiratory syncytial virus respiratory syncytial virus (sĭnsĭsh`əl): see cold, common. , as well as human and nonhuman primate nonhuman primate
see 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.
In response to the SARS outbreak, we developed a real-time 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 sensitivity 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 titer /ti·ter/ (ti´ter) the quantity of a substance required to react with or to correspond to a given amount of another substance. in respiratory secretions (8). In addition, by eliminating the need for postamplification product processing, the real-time RT-PCR format permitted shortened turnaround time (1) In batch processing, the time it takes to receive finished reports after submission of documents or files for processing. In an online environment, turnaround time is the same as response 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 false-positive results, meticulous care was taken to prevent introduction of contaminating viral RNA or previously amplified DNA DNA: see nucleic acid.
or deoxyribonucleic acid
One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. 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.
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.
Table 1. Primers and probes used for real-time RT-PCR assays (a) Primer/ Assay ID probe Sequence (5'>3') Primary diagnostic assay SARS1 F CAT GTG TGG CGG CTC ACT ATA T R CAC ACT ATT AGC ATA AGC ACT TGT AGC A P TTA AAC CAG GTG GAA CAT CAT CCG GTG SARS2 F GGA GCC TTG AAT ACA CCC AAA G R GCA CGG TGG CAG CAT TG P CCA CAT TGG CAC CCG CAA TCC SARS3 F CAA ACA TTG GCC GCA AAT T R CAA TGC GTG ACA TTC CAA AGA P CAC AAT TTG CTC CAA GTG CCT CTG CA To confirm positive results N3 F GAA GAA CCA TCT GGG GCT GAG R CCG AAG AGC TAC CCG ACG P CTC TTT CAT TTT GCC GTC ACC ACC AC 3'NTR F AGC TCT CCC TAG CAT TAT TCA CTG R CAC CAC ATT TTC ATC GAG GC P TAC CCT CGA TCG TAC TCC GCG T M F TGT AGG CAC TGA TTC AGG TTT TG R CGG CGT GGT CTG TAT TTA ATT TA P CTG CAT ACA ACC GCT ACC GTA TTG GAA Primer/ Assay ID probe Genomic region Location (b) Primary diagnostic assay SARS1 F RNA polymerase 15370-15392 R 15422-15449 P 15395-15420 SARS2 F Nucleocapsid 28531-28552 R 28581-28597 P 28559-28574 SARS3 F Nucleocapsid 29016-29034 R 29063-29083 P 29036-29061 To confirm positive results N3 F Nucleocapsid 28432-28452 R 28383-28400 P 28406-28431 3'NTR F 3' nontranslated region 29619-29642 R 29576-29595 P 29597-29618 M F Membrane protein 26951-26973 R 27005-27027 P 26974-27000 (a) RT-PCR, reverse transcription-polymerase chain reaction; F, forward primer; R, reverse primer; P, probe. (b) Location based on the severe acute respiratory syndrome-associated coronavirus, Urbani strain (GenBank accession no. AY278741). Table 2 Reproducibility of real-time RT-PCR assays (a) RNA transcript copy number (b) 7.5 x [10.sup.1] 7.5 x [10.sup.2] SARS1 CV within assay (%) (c) 2.53 0.96 CV between assays (%) (d) 2.39 1.09 2.0 x [10.sup.1] 2.0 x [10.sup.2] SARS2 CV within assay (%) 1.27 0.57 CV between assays (%) 1.54 1.18 SARS3 CV within assay (%) 0.8 0.55 CV between assays (%) 0.94 0.64 RNA transcript copy number (b) 7.5 x [10.sup.3] 7.5 x [10.sup.4] SARS1 CV within assay (%) (c) 0.49 0.69 CV between assays (%) (d) 0.82 0.64 2.0 x [10.sup.3] 2.0 x [10.sup.4] SARS2 CV within assay (%) 0.46 0.72 CV between assays (%) 0.97 1.47 SARS3 CV within assay (%) 0.65 0.5 CV between assays (%) 1.07 1.13 RNA transcript copy number (b) 7.5 x [10.sup.5] 7.5 x [10.sup.6] SARS1 CV within assay (%) (c) 1.66 0.7 CV between assays (%) (d) 2.1 0.79 2.0 x [10.sup.5] 2.0 x [10.sup.6] SARS2 CV within assay (%) 0.84 0.67 CV between assays (%) 1.54 1.32 SARS3 CV within assay (%) 0.27 1.25 CV between assays (%) 1.24 1.65 (a) RT-PCR, reverse transcription-polymerase chain reaction; CV, coefficient of variation. (b) Ten-fold dilutions of the polymerase and nucleocapsid RNA transcripts; copies per reaction; dilution series thawed on 3 different days and assays performed in triplicate for each dilution. (c) Determined from three replicates within each assay. (d) Determined from three independent assays performed on different days. Table 3. Efficiency of real-time PCR assays Mean [C.sub.T.sup.b] values at estimated RNA transcript copy number 7.5 x [10.sup.0] 7.5 x [10.sup.1] SARS1 RNA transcript alone Neg 38.65 [+ or -] 1.48 RNA transcript + extract (e) Neg 38.05 [+ or -] 0.92 2 x [10.sup.0] 2 x [10.sup.1] SARS2 RNA transcript alone 35.4 [+ or -] 0.57 32.1 [+ or -] 0.14 RNA transcript + extract Neg 34.55 [+ or -] 1.91 SARS3 RNA transcript alone 39.3 36.2 [+ or -] 0.42 RNA transcript + extract 40.3 36.2 [+ or -] 0.28 Mean [C.sub.T.sup.b] values at estimated RNA transcript copy number 7.5 x [10.sup.2] 7.5 x [10.sup.3] SARS1 RNA transcript alone 34.25 [+ or -] 0.57 31.1 [+ or -] 0.14 RNA transcript + extract (e) 34.85 [+ or -] 0.21 31.55 [+ or -] 0.07 2 x [10.sup.2] 2 x [10.sup.3] SARS2 RNA transcript alone 29.45 [+ or -] 0.64 26.15 [+ or -] 0.07 RNA transcript + extract 29.2 [+ or -] 0.28 26.2 SARS3 RNA transcript alone 32.8 29.1 [+ or -] 0.14 RNA transcript + extract 33.4 [+ or -] 0.28 29.9 [+ or -] 0.21 Mean [C.sub.T.sup.b] values at estimated RNA transcript copy number 7.5 x [10.sup.4] 7.5 x [10.sup.5] SARS1 RNA transcript alone 27.5 24.2 RNA transcript + extract (e) 27.75 [+ or -] 0.07 24.4 2 x [10.sup.4] 2 x [10.sup.5] SARS2 RNA transcript alone 22.9 [+ or -] 0.14 19.4 RNA transcript + extract 23.1 19.6 [+ or -] O.14 SARS3 RNA transcript alone 25.9 22.15 [+ or -] 0.07 RNA transcript + extract 26.05 [+ or -] 0.07 22.55 [+ or -] 0.21 Mean [C.sub.T.sup.b] values at estimated RNA transcript copy number 7.5 x [10.sup.6] SARS1 RNA transcript alone 20.55 [+ or -] 0.07 RNA transcript + extract (e) 20.6 2 x [10.sup.6] SARS2 RNA transcript alone 16.35 [+ or -] 0.07 RNA transcript + extract 16.6 SARS3 RNA transcript alone 19.2 RNA transcript + extract 19.65 [+ or -] 0.21 Efficiency Slope (c) (%) (d) SARS1 RNA transcript alone -3.55 91.1 RNA transcript + extract (e) -3.49 93.3 SARS2 RNA transcript alone -3.21 104.9 RNA transcript + extract -3.48 93.9 SARS3 RNA transcript alone -3.39 97.1 RNA transcript + extract -3.42 96.1 (a) PCR, polymerase chain reaction; CT, threshold cycle number.; neg, negative. (b) Values shown are mean of triplicate samples [+ or -] standard deviations. (c) Slope determined from the formula: Y = Y intercept - slope [log.sub.10]. Slopes calculated for SARS1 (7.5 x [10.sup.6] to 7.5 x [l0.sup.1]); SARS2 (2 x [10.sup.6] to 2 x [10.sup.1]); SARS3 (2 x [10.sup.6] to 2 x [10.sup.0]). (d) Efficiency = [[10.sup.(-1/slope)]] - 1. (e) Reactions performed in presence of pooled total nucleic acid extract from 20 human respiratory specimens. Table 4. Comparison of real-time RT-PCR assay with culture and conventional RT-PCR (a) Real-time RT-PCR SARS-CoV Conventional dilution (b) RT-PCR SARS1 SARS2 SARS3 [10.sup.-4] 3/3 (c) 3/3 3/3 3/3 [10.sup.-5] 3/3 3/3 3/3 3/3 [10.sup.-6] 3/3 3/3 3/3 3/3 [10.sup.-7] 3/3 3/3 3/3 3/3 [10.sup.-8] 0/3 3/3 3/3 3/3 [10.sup.-9] 0/3 0/3 1/3 0/3 [10.sup.-10] 0/3 0/3 0/3 0/3 (a) RT-PCR, reverse transcription-polymerase chain reaction; SARS-CoV, severe acute respiratory syndrome-associated coronavirus. (b) Serial 10-fold dilution of SARS-CoV stock culture containing 1 x [10.sup.7] PFUs/mL. (c) Number of positive results divided by the number of replicates tested. Table 5. Results of real-time RT-PCR assay with specimens from patients with laboratory-confirmed SARS-CoV infection Vero E6 Case ID Location Specimen ID Specimen Serology culture 05078 Toronto 2003756523 Lung, RM N/A -- 2003756525 Lung, RU -- 05077 Thailand 2003756502 Throat swab + + 05000 Hong Kong 2003757035 Lung, RU + -- 2003757036 Lung, LU -- 2003757037 Lung, RM -- 2003757038 Lung, LL -- 00220 Utah, USA 2003757508 Sputum + + 05001 Vietnam 2003757190 Throat wash + + 05008 Vietnam 2003757229 Throat wash + -- 05010 Vietnam 2003757239 Throat wash + -- 05013 Vietnam 2003757251 Throat wash + -- 05017 Vietnam 2003757268 Throat wash + + 05316 Vietnam 2003759760 Throat swab N/A + Real-time RT-PCR [C.sub.T.sup.c] values Conventional Case ID Specimen ID RT-PCR (b) SARS1 SARS2 SARS3 RNase P 05078 2003756523 + 24.2 21.6 23 23.9 2003756525 + 24.9 21.5 23 23.7 05077 2003756502 + 37.5 36.2 39.8 29.3 05000 2003757035 + 26.7 22.6 24.1 24.7 2003757036 + 27.2 24.9 26.5 26 2003757037 + 34.9 37.5 31.9 27.4 2003757038 + 29.6 27 28.6 24.5 00220 2003757508 + 24.7 23 24.8 30.6 05001 2003757190 + 23.7 22.4 24.1 30.1 05008 2003757229 + 35.5 35.5 36.7 30 05010 2003757239 + 31.1 29.3 31.5 34.2 05013 2003757251 + 29.5 28.4 30.3 28.8 05017 2003757268 + 26 24.7 26.4 27.9 05316 2003759760 N/A 25 25.3 28.2 28 (a) RT-PCR, reverse transcription polymerase chain reaction; SARS-CoV, severe acute respiratory syndrome-associated coronavirus; CT, threshold cycle number; RM, right middle; RU, right upper; LU, left upper; LL, left lower; N/A, not applicable. (b) Ref. 5. (c) Values shown mean of duplicate values.
We are indebted to Le Thi Quynh Mai, Nghiem Kim Ha, Nguyen Le Khang Hang, Nguyen Thi Hong Hang, Hoang Thuy Long, and the staff of the National Institute of Hygiene and Epidemiology, Hanoi, Vietnam, who provided some of the serum and respiratory specimens used in this study. We also thank James Luby for providing the human enteric coronavirus used in our study.
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Shannon Emery is currently earning her Master of Science in Epidemiology from Emory University and working as a guest researcher at the Centers of Disease Control and Prevention in Atlanta, GA. Her research interests include developing real-time PCR platforms for other human respiratory viruses including respiratory syncytial virus and human metapnuemovirus.
Shannon L. Emery, * Dean D. Erdman, * Michael D. Bowen, * Bruce R. Newton, * Jonas M. Winchell, * Richard F. Meyer, * Suxiang Tong, * Byron T. Cook, * Brian P. Holloway, * Karen A. McCaustland, * Paul A. Rota, * Bettina Bankamp, * Luis E. Lowe, * Tom G. Ksiazek, * William J. Bellini, * and Larry J. Anderson *
* Centers for Disease Control and Prevention, Atlanta, Georgia, USA