A real-time PCR for SARS-coronavirus incorporating target gene pre-amplification

Lok Ting Lau; Yin Wan Wendy Fung; Freda Pui Fan Wong; Selma Sau Wah Lin; Chen Ran Wang; Hui Li Li; Natalie Dillon; Richard A. Collins; John Siu Lun Tam; Paul K.S. Chan; Chen G. Wang; Albert Cheung Hoi Yu

doi:10.1016/j.bbrc.2003.11.064

A real-time PCR for SARS-coronavirus incorporating target gene pre-amplification

Lok Ting Lau, Yin Wan Wendy Fung, Freda Pui Fan Wong, Selma Sau Wah Lin, Chen Ran Wang, Hui Li Li, Natalie Dillon, Richard A. Collins, John Siu Lun Tam, Paul K.S. Chan, Chen G. Wang, Albert Cheung Hoi Yu^*

^*Corresponding author for this work

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Abstract

An enhanced polymerase chain reaction (PCR) assay to detect the coronavirus associated with severe acute respiratory syndrome (SARS-CoV) was developed in which a target gene pre-amplification step preceded TaqMan real-time fluorescent PCR. Clinical samples were collected from 120 patients diagnosed as suspected or probable SARS cases and analyzed by conventional PCR followed by agarose gel electrophoresis, conventional TaqMan real-time PCR, and our enhanced TaqMan real-time PCR assays. An amplicon of the size expected from SARS-CoV was obtained from 28/120 samples using the enhanced real-time PCR method. Conventional PCR and real-time PCR alone identified fewer SARS-CoV positive cases. Results were confirmed by viral culture in 3/28 cases. The limit of detection of the enhanced real-time PCR method was 10²-fold higher than the standard real-time PCR assay and 10⁷-fold higher than conventional PCR methods. The increased sensitivity of the assay may help control the spread of the disease during future SARS outbreaks.

Original language	English
Pages (from-to)	1290-1296
Number of pages	7
Journal	Biochemical and Biophysical Research Communications
Volume	312
Issue number	4
DOIs	https://doi.org/10.1016/j.bbrc.2003.11.064
Publication status	Published - 26 Dec 2003

Scopus Subject Areas

Biophysics
Biochemistry
Molecular Biology
Cell Biology

User-Defined Keywords

Coronavirus
PCR
SARS
SARS-CoV
Severe acute respiratory syndrome
TaqMan real-time fluorescent polymerase chain reaction

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/j.bbrc.2003.11.064

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Lau, L. T., Fung, Y. W. W., Wong, F. P. F., Lin, S. S. W., Wang, C. R., Li, H. L., Dillon, N., Collins, R. A., Tam, J. S. L., Chan, P. K. S., Wang, C. G., & Yu, A. C. H. (2003). A real-time PCR for SARS-coronavirus incorporating target gene pre-amplification. Biochemical and Biophysical Research Communications, 312(4), 1290-1296. https://doi.org/10.1016/j.bbrc.2003.11.064

@article{dd1d5461ea424a429752728ba640b198,

title = "A real-time PCR for SARS-coronavirus incorporating target gene pre-amplification",

abstract = "An enhanced polymerase chain reaction (PCR) assay to detect the coronavirus associated with severe acute respiratory syndrome (SARS-CoV) was developed in which a target gene pre-amplification step preceded TaqMan real-time fluorescent PCR. Clinical samples were collected from 120 patients diagnosed as suspected or probable SARS cases and analyzed by conventional PCR followed by agarose gel electrophoresis, conventional TaqMan real-time PCR, and our enhanced TaqMan real-time PCR assays. An amplicon of the size expected from SARS-CoV was obtained from 28/120 samples using the enhanced real-time PCR method. Conventional PCR and real-time PCR alone identified fewer SARS-CoV positive cases. Results were confirmed by viral culture in 3/28 cases. The limit of detection of the enhanced real-time PCR method was 102-fold higher than the standard real-time PCR assay and 107-fold higher than conventional PCR methods. The increased sensitivity of the assay may help control the spread of the disease during future SARS outbreaks.",

keywords = "Coronavirus, PCR, SARS, SARS-CoV, Severe acute respiratory syndrome, TaqMan real-time fluorescent polymerase chain reaction",

author = "Lau, {Lok Ting} and Fung, {Yin Wan Wendy} and Wong, {Freda Pui Fan} and Lin, {Selma Sau Wah} and Wang, {Chen Ran} and Li, {Hui Li} and Natalie Dillon and Collins, {Richard A.} and Tam, {John Siu Lun} and Chan, {Paul K.S.} and Wang, {Chen G.} and Yu, {Albert Cheung Hoi}",

note = "Funding Information: The authors thank the Philip K.H. Wong Foundation, Kennedy Y.H. Wong, Pun-Hoi Yu, and the New Century Forum Foundation for financial support, Sino-i.com Ltd., Dr. Cecilia W.B. Pang (Biotechnology Director, Information Technology and Broadcasting Branch, Commerce, Industry and Technology Bureau, Hong Kong SAR), and Fung-Kwok Ma (New Century Forum) for facilitating this study. Chen G. Wang is the PI of the National Emergency Action on SARS Research (Beijing Group) supported by the Ministry of Public Health and the Ministry of Science and Technology of China. We acknowledge Professor K.-Y. Yuen (Department of Microbiology, University of Hong Kong) for providing clinical samples at the beginning of this study (data from these samples are not included in this report). We owe many thanks to Professor Po Tian (Member of Chinese Academy of Sciences, Institute of Microbiology) for his valuable suggestions and discussion. Publisher copyright: {\textcopyright} 2003 Elsevier Inc. All rights reserved.",

year = "2003",

month = dec,

day = "26",

doi = "10.1016/j.bbrc.2003.11.064",

language = "English",

volume = "312",

pages = "1290--1296",

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T1 - A real-time PCR for SARS-coronavirus incorporating target gene pre-amplification

AU - Lau, Lok Ting

AU - Fung, Yin Wan Wendy

AU - Wong, Freda Pui Fan

AU - Lin, Selma Sau Wah

AU - Wang, Chen Ran

AU - Li, Hui Li

AU - Dillon, Natalie

AU - Collins, Richard A.

AU - Tam, John Siu Lun

AU - Chan, Paul K.S.

AU - Wang, Chen G.

AU - Yu, Albert Cheung Hoi

N1 - Funding Information: The authors thank the Philip K.H. Wong Foundation, Kennedy Y.H. Wong, Pun-Hoi Yu, and the New Century Forum Foundation for financial support, Sino-i.com Ltd., Dr. Cecilia W.B. Pang (Biotechnology Director, Information Technology and Broadcasting Branch, Commerce, Industry and Technology Bureau, Hong Kong SAR), and Fung-Kwok Ma (New Century Forum) for facilitating this study. Chen G. Wang is the PI of the National Emergency Action on SARS Research (Beijing Group) supported by the Ministry of Public Health and the Ministry of Science and Technology of China. We acknowledge Professor K.-Y. Yuen (Department of Microbiology, University of Hong Kong) for providing clinical samples at the beginning of this study (data from these samples are not included in this report). We owe many thanks to Professor Po Tian (Member of Chinese Academy of Sciences, Institute of Microbiology) for his valuable suggestions and discussion. Publisher copyright: © 2003 Elsevier Inc. All rights reserved.

PY - 2003/12/26

Y1 - 2003/12/26

N2 - An enhanced polymerase chain reaction (PCR) assay to detect the coronavirus associated with severe acute respiratory syndrome (SARS-CoV) was developed in which a target gene pre-amplification step preceded TaqMan real-time fluorescent PCR. Clinical samples were collected from 120 patients diagnosed as suspected or probable SARS cases and analyzed by conventional PCR followed by agarose gel electrophoresis, conventional TaqMan real-time PCR, and our enhanced TaqMan real-time PCR assays. An amplicon of the size expected from SARS-CoV was obtained from 28/120 samples using the enhanced real-time PCR method. Conventional PCR and real-time PCR alone identified fewer SARS-CoV positive cases. Results were confirmed by viral culture in 3/28 cases. The limit of detection of the enhanced real-time PCR method was 102-fold higher than the standard real-time PCR assay and 107-fold higher than conventional PCR methods. The increased sensitivity of the assay may help control the spread of the disease during future SARS outbreaks.

AB - An enhanced polymerase chain reaction (PCR) assay to detect the coronavirus associated with severe acute respiratory syndrome (SARS-CoV) was developed in which a target gene pre-amplification step preceded TaqMan real-time fluorescent PCR. Clinical samples were collected from 120 patients diagnosed as suspected or probable SARS cases and analyzed by conventional PCR followed by agarose gel electrophoresis, conventional TaqMan real-time PCR, and our enhanced TaqMan real-time PCR assays. An amplicon of the size expected from SARS-CoV was obtained from 28/120 samples using the enhanced real-time PCR method. Conventional PCR and real-time PCR alone identified fewer SARS-CoV positive cases. Results were confirmed by viral culture in 3/28 cases. The limit of detection of the enhanced real-time PCR method was 102-fold higher than the standard real-time PCR assay and 107-fold higher than conventional PCR methods. The increased sensitivity of the assay may help control the spread of the disease during future SARS outbreaks.

KW - Coronavirus

KW - PCR

KW - SARS

KW - SARS-CoV

KW - Severe acute respiratory syndrome

KW - TaqMan real-time fluorescent polymerase chain reaction

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DO - 10.1016/j.bbrc.2003.11.064

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C2 - 14652014

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SN - 0006-291X

VL - 312

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EP - 1296

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

IS - 4

ER -

A real-time PCR for SARS-coronavirus incorporating target gene pre-amplification

Abstract

Scopus Subject Areas

User-Defined Keywords

UN SDGs

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