key: cord-1025289-solp6ho1 authors: Peiris, J. S. Malik; Poon, Leo L. M. title: Detection of SARS Coronavirus date: 2010-08-16 journal: Diagnostic Virology Protocols DOI: 10.1007/978-1-60761-817-1_20 sha: 4d48998bcab611793b7b42177a47cf470c611385 doc_id: 1025289 cord_uid: solp6ho1 The emergence of severe acute respiratory syndrome (SARS) and its subsequent worldwide spread challenged the global public health community to confront a novel infectious disease. The infection is caused by a coronavirus of animal origin. In this epidemic, molecular detections of SARS coronavirus RNA were shown to be useful for the early diagnosis of SARS. Although this pathogen was eradicated in humans, SARS or SARS-like viruses might reemerge from animals or from laboratory incidents. In this chapter, we describe several polymerase chain reaction (PCR) protocols for detecting SARS coronaviruses. These assays were routinely used for clinical diagnosis during the SARS outbreak. associated with mild respiratory and gastrointestinal diseases. Interestingly, virus surveillance studies over the last few years have identified many novel coronaviruses from different animals. These suggest that there might be a wealth of "unknown" coronaviruses which are yet to be identified. SARS is the first novel infectious respiratory disease in this century. The disease is caused by a coronavirus originating from animals and the clinical presentations of the disease have been extensively reviewed (3) . Further studies also indicated that the virus is a distant relative of bat coronaviruses (4), suggesting bats might be natural carriers of the precursor of SARS coronavirus. However, the nature reservoir of SARS coronavirus is still not confirmed. As the majority of SARS patients seroconverted in the second week of disease onset, serological tests might not be a practical approach for early SARS diagnosis (3) . Because of these reasons, the focus of early diagnosis was mainly concentrated on the development of conventional and quantitative reverse transcriptase (RT) polymerase chain reaction (PCR) assays (3) . Besides, several molecular tests which employ non-PCR-based methods, such as loop mediated isothermal amplification (5) , rolling circle amplification (6) and nucleic acid sequence-based amplification (7) , were also developed for the detection of SARS coronavirus RNA. Here, we share our experiences on the molecular diagnosis of SARS and other coronaviruses. The protocols described in this chapter are directly adopted from our previous publications (8, 9) . The first and second assays are manual RT-PCR (Subheadings 3.2, 3.3, and 3.4) and real-time quantitative RT-PCR (Subheading 3.5) assays, respectively, for SARS coronavirus detections (see Notes 1-3). As there is a possibility that other SARS-like coronaviruses found in bats or other mammals might have zoonotic potential, we also present another PCR assay which is able to detect groups 1 and 2 viruses (Subheading 3.6). This assay might be useful to screen the SARS-like patients whom are negative in the first two assays. It should be noted that the primer set used in the third assay can cross react with a wide range of coronaviruses. Therefore, the identities of all the positive PCR products from the third assay should be formally confirmed by DNA sequencing. In our experiences, this assay is able to detect other common human coronaviruses (e.g. HKU1, NL63, OC43, and 229E). 2. Equilibrate all reagents to room temperature before use. 3. Transfer 140 mL of the sample into a 1.5 mL microcentrifuge tube (see Note 8). 4. Add 560 mL of prepared buffered AVL with carrier RNA to the microcentrifuge tube. 5. Briefly vortex the tubes for 15 s and incubate at room temperature for 10 min. 6. Briefly centrifuge the microcentrifuge tube. Add 560 mL ethanol (96-100%) and mix by pulse-vortexing for 15 s. 7. Briefly centrifuge the microcentrifuge tube. 8. Transfer 630 mL of the solution from the tube to a QIAamp spin column placed in a provided 2 mL collection tube. Centrifuge at 6,000 × g (8,000 RPM) for 1 min at room temperature/4°C. Place the spin column in a clean 2 mL collection tube. Discard the tube containing the filtrate. 9. Open the spin column and repeat step 8. 10. Add 500 mL buffer AW1. Centrifuge at 6,000 × g (8,000 RPM) for 1 min. Place the spin column in a clean 2 mL collection tube. Discard the tube containing the filtrate. 11. Add 500 mL buffer AW2. Centrifuge at 20,000 × g (14,000 RPM) for 3 min. Place the spin column in a clean 2 mL collection tube and centrifuge at 20,000 × g for another 1 min. Place the spin column in a clean 1.5 mL microcentrifuge tube. Discard the tube containing the filtrate. 12. Apply 50 mL buffer AVE equilibrated to room temperature directly on the membrane of the column. Close the cap and incubate at room temperature for 1 min. 13. Centrifuge at 6,000 × g (8,000 RPM) for 1 min. Collect the filtrate for cDNA synthesis. Store the RNA at −20 or −70°C. 2. Vortex and centrifuge the tube briefly. Keep the tube on ice. 3. Add 10 mL of master mix solution into separate 0.5 microcentrifuge tubes. Label the tube accordingly and keep these tubes on ice. 4. Add 10 mL of purified RNA samples into these tubes accordingly. 5. Vortex and centrifuge the tubes briefly. 6. Stand the tubes at room temperature for 10 min and then incubate at 42°C for 50 min. 7. Inactivate the transcription reaction by incubating the tubes at 95°C for 5 min and then chill the samples on ice. Store the cDNA samples at −20°C (see Note 9). 3. Aliquot 48 mL of the master mix into separate 0.5 mL microcentrifuge tubes and label the tube accordingly. 4. Add 2 mL of cDNA generated from the reverse transcription reactions to these tubes accordingly. For the positive control, add 2 mL of SARS coronavirus cDNA into the reaction. For the negative control, add 2 mL of autoclaved water. 5. Vortex and centrifuge the tubes briefly. 6. Run the PCR in the following condition: Step Temperature Time 8. Mix 0.5 mL of the DNA markers with 2 mL of 6× gel loading dye and 9.5 mL of water on a parafilm sheet by repeated pepitting. 9. Mix 10 mL of the PCR products with 2 mL of 6× gel loading dye on a parafilm sheet by pepitting up and down several times. 10. Apply the mixture to the corresponding well of the gel. 11. Close the lid of the electrophoresis apparatus and connect the electrical leads, anode to anode (red to red) and cathode to cathode (black to black). 12. Run the gel at 100 V for 30 min. 13. Turn off the power, remove the cover and retrieve the gel. 14. Soak the gel in 1× TAE with 0.5 mg/mL ethidium bromide for 15 min. Wash the gel with water briefly (see Note 11). 15. Place the gel on top of the transilluminator. Switch on the power of the gel documentary machine (see Note 12) . 16. Adjust the position of the gel and record the results. The size of the expected product for the virus is 182 bp (see Note 13). 1. Turn on the quantitative RT-PCR machine. Activate the Detection Manager from the supplied software and confirm the reporter, quencher, passive reference dyes are FAM, NFQ, and ROX, respectively. Set the cycle conditions as follows: Step 2. In the reaction plate template, input the necessary information for the corresponding samples (e.g. positive standard, negative control, or name of the clinical specimen). Include at least one set of tenfold serially diluted positive controls with known copy numbers of the target sequence (e.g. 10 6 to 10 copies/reaction) and three negative controls (water) in each run. For the positive controls, key in the copy numbers of the target sequence used in the corresponding reactions. 3. Prepare a PCR master mix sufficient for the designated number of samples in a sterile 2.5 mL screw cap tube according to following table. Add additional controls (e.g. purified RNA from the studied samples) as necessary. 10. After the reaction, examine the threshold cycles (Ct) and the amplification curves of the reactions. For a good experiment, the Ct values deduced from the standards should correlate with the log 10 copy numbers of the target sequence used in these reactions (Fig. 1a) . Positive clinical samples will generate amplification signals above the threshold (Fig. 1b) . By contrast, signals from the water controls and negative samples will below the threshold line. Based on the Ct values from the reference standards, the amounts of input target in the positive reactions will be calculated by the software automatically (see Notes 15 and 16). 1. Prepare a PCR master mix sufficient for the designated number of samples in a sterile 0.5 mL microcentrifuge tube according to following table. Include at least one positive control and one negative control (water) for each run. Add additional controls (e.g. purified RNA from the studied samples) as necessary. 3. Aliquot 48 mL of the master mix into separate 0.5 mL microcentrifuge tubes and label them accordingly. 4. Add 2 mL of cDNA generated from the reverse transcription reactions (Subheading 3.2) to these tubes accordingly. For the positive control, add 2 mL of coronavirus cDNA into the reaction. For the negative control, add 2 mL of autoclaved water. 5. Vortex and centrifuge the tubes briefly. 6. Run the PCR using the following conditions: Step Temperature (°C) Time 17) . Alternatively, the products can be kept at −20°C for short-term storage. 1. In our evaluation, the performance of the quantitative RT-PCR assay is better than the manual RT-PCR assays (3) . In addition, the quantitative results generated from the real-time RT-PCR might provide additional data from prognosis (3). 2. In our patient cohort, respiratory samples (e.g. nasopharyngeal aspirate, throat swab) collected from patients within the first week of disease onset have the highest positive rates for SARS coronavirus. By contrast, fecal samples have the highest positive rate after the first week of onset. However, to increase the chance of identifying SARS patients in a nonepidemic period, we recommend testing multiple specimens available from suspected patients. 3. For respiratory samples isolated from early disease onset, the detection rates could be enhanced by increasing the initial extraction volume of the NPA sample from 140 to 560 mL (10). 4. Viral transport medium contains a high concentration of antibiotic to inhibit bacterial growth. 5. The primers and probe used in these assays are perfectly matched to those sequences deduced from SARS coronaviruses in human and civets, including those isolated in 2004. 6. Personal protection equipment should be worn by the healthcare worker taking specimens from suspect or probable SARS patients (http://www.who.int/csr/sars/infectioncontrol/en/). 7. AVL Buffer containing carrier RNA might form white precipitates when stored at 4°C. The precipitate can be dissolved in the buffer by heating the bottle in a water bath. Cool the buffer to room temperature before use. 8. For extracting RNA from suspected infectious samples, the procedure must be handled in a biosafety level (BSL) 2 containment using BSL 3 working practices. (http://www.who. int/csr/sars/biosafety2003_12_18/en/). 9 . General procedures to prevent PCR cross contaminations should be strictly followed. Aerosol-resistant filtered pipette tips could minimize possible carryovers of amplicons. Separate pipettes and areas are used for sampling processing, PCR and post-PCR analysis. It is essential to include multiple positive and negative controls in the PCR reactions when a large number of samples are tested at the same time. 10. Agarose solutions can be superheated in microwave oven. Do not handle bottle immediately after microwaving. Always wear heat-resistant gloves when handling melted agarose. 11. Ethidium bromide is a known mutagen and may be carcinogenic. Handle solutions of ethidium bromide with gloves. 12. UV light can cause severe skin and eye damage. Wear safety glasses and close the photography hood before turning on the UV transilluminator. 13. The conventional RT-PCR protocol is highly specific to SARS coronavirus isolated from respiratory samples. However, we observed a small number of false-positive results from RNA isolated from stools. To overcome this problem, all of our positive fecal samples were retested by the quantitative RT-PCR as described in Subheading 3.5 or using a SYBR green-based RT-PCR assay (11) for confirmation. 14. When performing step 6 in Subheading 3.5, the RNA samples, including those positive standards, must be handled with extreme care. Cross contamination might lead to false-positive or unreliable quantitative results. 15. The amplification curves of all positive samples in the quantitative RT-PCR assays must be examined individually. We occasionally find some clinical specimens might yield high backgrounds and the analytical program might misclassify these samples as positive samples. 16. To exclude negative results due to the poor recovery of RNA, poor performance of the RT-PCR reaction, the presence of PCR inhibitors or human errors, we subsequently modified our quantitative RT-PCR assays to use a duplex assay. The revised test allows simultaneous detection of SARS coronavirus and endogenous 18 S rRNA derived from host cells (12) . The primers and probe for 18 S rRNA are commercially available (TaqMan Ribosomal RNA Control Reagents, Applied Biosystems). 17. The identities of the positive products should be formally confirmed by DNA sequencing. SuperScript II reverse transcriptase, 200 U/mL (Invitrogen) 5× First strand buffer [250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl Random hexamers, 150 ng/mL (Invitrogen) RNaseOUT recombinant ribonuclease inhibitor, 40 U/mL (Invitrogen) Deoxynucleotide triphosphates (dNTP) mix, 10 mM each Autoclaved RNase-free water or equivalents Heating block or equivalents AmpliTaq Gold DNA polymerase, 5 U/mL (Applied Biosystems) 10× Gold PCR buffer (Applied Biosystems) dNTP mix, 10 mM each 25 mM MgCl 2 solution (Applied Biosystems) 10 mM PCR forward primer, 5′-TACACACCT CAGCGTTG-3′. 6. 10 mM PCR reverse primer Applied Biosystems) (see Note 5) 6× Gel loading buffer [10 mM Tris-HCl 1 kb plus DNA ladder markers (Invitrogen) Ethidium bromide, 10 mg/mL Agarose gel electrophoresis apparatus Power supply Gel documentary machine or equivalents 50 mM PCR forward primer A comparative sequence analysis to revise the current taxonomy of the family Coronaviridae Coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus The aetiology, origins, and diagnosis of severe acute respiratory syndrome Emerging nidovirus infections Rapid detection of the severe acute respiratory syndrome (SARS) coronavirus by a loop-mediated isothermal amplification assay Rapid and sensitive detection of severe acute respiratory syndrome coronavirus by rolling circle amplification Real-time NASBA detection of SARS-associated coronavirus and comparison with real-time reverse transcription-PCR Detection of SARS coronavirus in humans and animals by coventional and quantitative (real time) reverse transcription polymerase chain reactions Detection of Group 1 coronaviruses in bats using universial coronavirus reverse transcription polymerase chain reactions Early diagnosis of SARS coronavirus infection by real time RT-PCR Rapid diagnosis of a coronavirus associated with severe acute respiratory syndrome (SARS) A one step quantitative RT-PCR for detection of SARS coronavirus with an internal control for PCR inhibitors