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Virology

Evaluation of Reverse Transcription-PCR Assays for Rapid Diagnosis of Severe Acute Respiratory Syndrome Associated with a Novel Coronavirus

W. C. Yam, K. H. Chan, L. L. M. Poon, Y. Guan, K. Y. Yuen, W. H. Seto, J. S. M. Peiris
W. C. Yam
Department of Microbiology, Queen Mary Hospital, The University of Hong Kong, Hong Kong, People's Republic of China
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K. H. Chan
Department of Microbiology, Queen Mary Hospital, The University of Hong Kong, Hong Kong, People's Republic of China
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L. L. M. Poon
Department of Microbiology, Queen Mary Hospital, The University of Hong Kong, Hong Kong, People's Republic of China
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Y. Guan
Department of Microbiology, Queen Mary Hospital, The University of Hong Kong, Hong Kong, People's Republic of China
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K. Y. Yuen
Department of Microbiology, Queen Mary Hospital, The University of Hong Kong, Hong Kong, People's Republic of China
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W. H. Seto
Department of Microbiology, Queen Mary Hospital, The University of Hong Kong, Hong Kong, People's Republic of China
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J. S. M. Peiris
Department of Microbiology, Queen Mary Hospital, The University of Hong Kong, Hong Kong, People's Republic of China
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  • For correspondence: malik@hku.hk
DOI: 10.1128/JCM.41.10.4521-4524.2003
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Tables

  • TABLE 1.

    RT-PCR protocols for rapid diagnosis of CoV associated with SARSa

    Characteristic or component of protocolWHO-HKUWHO-Hamburg
    RTPCRRT-PCRSecond PCR
    Primer sequences
        SenseTACACACCTCAGCGTTGATGAATTACCAAGTCAATGGTTACGAAGCTATTCGTCACGTTCG
        AntisenseCACGAACGTGACGAATCATAACCAGTCGGTACAGCTACCTGTAGAAAATCCTAGCTGGAG
    Reagent formulationSuperscript II RTA (Invitrogen)AmpliTaq Gold (Roche)Superscript II RT-PCR (Invitrogen)AmpliTaq Gold (Roche)
        (i) 4 μl of 5× first-strand buffer    (i) 5 μl of 10× reaction buffer    (i) 10 μl of 2× reaction buffer    (i) 5 μl of 10× reaction buffer
        (ii) 10 mM DTT    (ii) 200 μM dNTP    (ii) 2.45 mM MgSO4    (ii) 200 μM dNTP
        (iii) 500 μM dNTP    (iii) 2.5 mM MgSO4    (iii) 500 nM (each) primer    (iii) 2.5 mM MgSO4
        (iv) 0.15 μg of random primer    (iv) 250 nM (each) primer    (iv) 0.4 μl of RTA-Taq mixture    (iv) 200 nM (each) primer
        (v) 200 U of Superscript II    (v) 2 U of AmpliTaq Gold    (v) 2 μl of RNA extract    (v) 2 U of AmpliTaq Gold
        (vi) 12 μl of RNA extract    (vi) 2 μl of RT product    (vi) Make up to total volume of 20 μl    (vi) 1 μl of RT-PCR product
        (vii) Make up to total volume of 20 μl    (vii) Make up to total volume of 50 μl    (vii) Make up to total volume of 50 μl
    Thermal cycling profile(i) 25°C, 10 min(i) 94°C, 10 min(i) 45°C, 30 min(i) 95°C, 5 min
    (ii) 42°C, 50 min(ii) 40 cycles(ii) 95°C, 3 min(ii) 10 cycles
    (iii) 94°C, 3 min    (a) 94°C, 30 s(iii) 10 cycles    (a) 95°C, 10 s
        (b) 50°C, 40 s    (a) 95°C, 10 s    (b) 60°C, 10 s (decrease by 1°C/cycle)
        (c) 72°C, 15 s    (b) 60°C, 10 s (decrease by 1°C/cycle)    (c) 72°C, 20 s
    (iii) 72°C, 10 min    (c) 72°C, 30 s(iii) 20 cycles
    (iv) 40 cycles    (a) 95°C, 10 s
        (a) 95°C, 10 s    (b) 56°C, 10 s
        (b) 56°C, 10 s    (c) 72°C, 20 s
        (c) 72°C, 30 s
    Expected PCR product size (bp)182189108
    • ↵ a The RT-PCR protocols of two WHO SARS network laboratories, WHO-HKU (8) and WHO-Hamburg (4), are also available online (http://www.who.int/esr/sars/primers/en . Abbreviations: RTA, reverse transcriptase; DTT, dithiothreitol; dNTP, deoxynucleoside triphasphate.

  • TABLE 2.

    Performance of RT-PCR assays for rapid detection of CoV associated with SARS

    Specimens (no.)No. of specimens testedSeroconversionaNo. of specimens positive by RT-PCR assay
    WHO-HKUWHO-HamburgBoth WHO-HKU and WHO-Hamburg
    Clinically suspected SARS
        Nasopharygneal aspirate specimens (124)72+444943
    52−000
        Throat swab specimens (65)54+353933
    11−000
        Urine specimens (95)78+394239
    17−000
        Stool specimens (19)19+111211
    Controls
        Nasopharygneal aspirate specimens22bND000
        Stool specimens21cND000
    • ↵ a A fourfold rise or more in antibody titer against CoV was considered seroconversion (+). ND, not done.

    • ↵ b Samples positive for other viral pathogens included nine samples positive for influenza virus A, one sample positive for influenza virus B, six samples positive for adenovirus, and six samples positive for RSV by immunofluorescence (2).

    • ↵ c No intestinal pathogens detected.

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Evaluation of Reverse Transcription-PCR Assays for Rapid Diagnosis of Severe Acute Respiratory Syndrome Associated with a Novel Coronavirus
W. C. Yam, K. H. Chan, L. L. M. Poon, Y. Guan, K. Y. Yuen, W. H. Seto, J. S. M. Peiris
Journal of Clinical Microbiology Oct 2003, 41 (10) 4521-4524; DOI: 10.1128/JCM.41.10.4521-4524.2003

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Evaluation of Reverse Transcription-PCR Assays for Rapid Diagnosis of Severe Acute Respiratory Syndrome Associated with a Novel Coronavirus
W. C. Yam, K. H. Chan, L. L. M. Poon, Y. Guan, K. Y. Yuen, W. H. Seto, J. S. M. Peiris
Journal of Clinical Microbiology Oct 2003, 41 (10) 4521-4524; DOI: 10.1128/JCM.41.10.4521-4524.2003
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KEYWORDS

Reverse Transcriptase Polymerase Chain Reaction
Severe Acute Respiratory Syndrome

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