Development and Evaluation of a Novel Loop-Mediated Isothermal Amplification Method for Rapid Detection of Severe Acute Respiratory Syndrome Coronavirus

ABSTRACT The development and evaluation of a one-step single-tube accelerated real-time quantitative reverse transcription (RT) loop-mediated isothermal amplification (LAMP) assay is reported for rapid detection of the severe acute respiratory syndrome coronavirus (SARS-CoV) replicase gene. A total of 49 samples (15 throat washes, 13 throat swabs, and 21 combined throat and nasal swabs) collected from patients admitted to the Hanoi-French and Ninhbinh hospitals in Vietnam during the SARS epidemic were evaluated and compared to conventional RT-PCR. The RT-LAMP assay demonstrated 100-fold-greater sensitivity, with a detection limit of 0.01 PFU. The sensitivity and specificity of RT-LAMP assay for detecting viral RNA in clinical specimens with regard to RT-PCR were 100 and 87%, respectively. The specificity of the RT-LAMP assay was further validated by restriction analysis as well as nucleotide sequencing of the amplified product. The concentration of virus in most of the clinical samples was 0.1 PFU (0.1 to 102 PFU), as determined from the standard curve of SARS RT-LAMP and based on the time of positivity. The assay procedure is quite simple, wherein the amplification is carried out in a single tube under isothermal conditions at 63°C, and the result can be obtained in less than 1 h (as early as 11 min). Thus, the RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection and will be useful for rapid and reliable clinical diagnosis of SARS-CoV in developing countries.

[1]  Y. Mori,et al.  Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. , 2001, Biochemical and biophysical research communications.

[2]  M. Chan-yeung,et al.  A cluster of cases of severe acute respiratory syndrome in Hong Kong. , 2003, The New England journal of medicine.

[3]  Kozaburo Hayashi,et al.  Loop-Mediated Isothermal Amplification for Direct Detection of Mycobacterium tuberculosis Complex, M. avium, and M. intracellulare in Sputum Samples , 2003, Journal of Clinical Microbiology.

[4]  E. Lennette,et al.  Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections , 1989 .

[5]  C. Fraser,et al.  Epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in Hong Kong , 2003, The Lancet.

[6]  L. Poon,et al.  Clinical progression and viral load in a community outbreak of coronavirus-associated SARS pneumonia : a prospective study , 2003 .

[7]  T. Notomi,et al.  Loop-mediated isothermal amplification of DNA. , 2000, Nucleic acids research.

[8]  Arthur S Slutsky,et al.  Identification of severe acute respiratory syndrome in Canada. , 2003, The New England journal of medicine.

[9]  K. Stöhr.  A multicentre collaboration to investigate the cause of severe acute respiratory syndrome , 2003, The Lancet.

[10]  J. Gerberding,et al.  Update: Outbreak of severe acute respiratory syndrome--worldwide, 2003. , 2003, MMWR. Morbidity and mortality weekly report.

[11]  Christian Drosten,et al.  Identification of a novel coronavirus in patients with severe acute respiratory syndrome. , 2003, The New England journal of medicine.

[12]  Q. Nie,et al.  Advances in clinical diagnosis and treatment of severe acute respiratory syndrome. , 2003, World journal of gastroenterology.

[13]  Y. Guan,et al.  Evaluation of Reverse Transcription-PCR Assays for Rapid Diagnosis of Severe Acute Respiratory Syndrome Associated with a Novel Coronavirus , 2003, Journal of Clinical Microbiology.

[14]  S. Twu,et al.  Update: outbreak of severe acute respiratory syndrome--worldwide, 2003. , 2003, MMWR. Morbidity and mortality weekly report.

[15]  Peter Cameron,et al.  A major outbreak of severe acute respiratory syndrome in Hong Kong. , 2003, The New England journal of medicine.

[16]  T. Notomi,et al.  Accelerated reaction by loop-mediated isothermal amplification using loop primers. , 2002, Molecular and cellular probes.

[17]  Christian Drosten,et al.  Characterization of a Novel Coronavirus Associated with Severe Acute Respiratory Syndrome , 2003, Science.

[18]  J. Sung,et al.  Evaluation of WHO criteria for identifying patients with severe acute respiratory syndrome out of hospital: prospective observational study , 2003, BMJ : British Medical Journal.

[19]  P. Epstein,et al.  Emerging diseases threaten conservation. , 2003, Environmental health perspectives.

[20]  Y. Guan,et al.  Rapid Diagnosis of a Coronavirus Associated with Severe Acute Respiratory Syndrome (SARS) , 2003, Clinical chemistry.

[21]  Obi L. Griffith,et al.  The Genome Sequence of the SARS-Associated Coronavirus , 2003, Science.

[22]  J. A. Comer,et al.  A novel coronavirus associated with severe acute respiratory syndrome. , 2003, The New England journal of medicine.

[23]  Y. Guan,et al.  Coronavirus as a possible cause of severe acute respiratory syndrome , 2003, The Lancet.